An L-pantolactone dehydrogenase mutant, encoding gene and application thereof

Abstract

The present invention relates to an L-pantolactone dehydrogenase mutant, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and their application in the microbial catalysis for preparing keto-pantolactone / D-pantolactone. The L-pantolactone dehydrogenase mutant is obtained by single mutation or multi-point combined mutation at the 156th, 224th, and 164th positions of the amino acid sequence shown in SEQ ID NO.1. The specific enzyme activity of the constructed RhoLPLDH mutant strain is significantly improved compared with the control group, and the co-expression of the RhoLPLDH mutant and the molecular chaperone pGro7 further improves the soluble expression of the target protein. The specific cell activity of the constructed co-expression engineering bacterium of the RhoLPLDH mutant and pGro7 is also significantly improved compared with the starting strain. The mutant specifically catalyzes LPL, and the substrate conversion rate can reach 94% in 30 hours. When the mutant catalyzes the D,L-PL substrate, the conversion rate can reach 91.8% in 48 hours. When the mutant and the engineering bacterium co-expressing conjugated polyketone reductase and glucose dehydrogenase use the "two-bacteria one-pot method" to catalyze the resolution of D,L-PL to prepare DPL, the DPL yield can reach 92.7%.

Description

(1) Technical Field

[0001] The present invention relates to L-pantolactone dehydrogenase, its encoding gene, a vector containing the encoding gene, a genetically engineered bacterium, and their application in the microbial catalysis for preparing keto-pantolactone / D-pantolactone. (2) Background Art

[0002] Calcium D-pantothenate, also known as vitamin B5, is a component of coenzyme A and has been widely used in industries such as food, feed, medicine, chemical engineering, and cosmetics. D-(-)-pantolactone, also known as (R)-pantolactone, has a chemical structure of the γ-lactone of D-(-)-pantothenic acid and is a key chiral intermediate for synthesizing D-(+)-pantothenic acid. Currently, the industrial synthesis of D-pantolactone adopts a technical route combining chemical methods and enzymatic hydrolysis resolution. Starting from isobutyraldehyde and formaldehyde as starting materials, DL-pantolactone is synthesized chemically. Among them, D-pantolactone can be stereoselectively hydrolyzed by D-pantolactone hydrolase to generate D-pantothenic acid, which is then lactonized to form D-pantolactone. The remaining L-pantolactone is chemically racemized to DL-pantolactone for recycling and resolution. The resolution of DL-pantolactone is a key step in the synthesis of D-pantolactone. The chiral resolution preparation process using hydrolase requires the racemization of L-pantolactone, the separation of D-pantothenic acid and L-pantolactone, and the acidification and cyclization of D-pantothenic acid to form D-pantolactone. Although the chiral resolution method catalyzed by hydrolase has a mature process, there are still problems such as complex processes, high energy and material consumption, and the need to consume a large amount of acids and bases. In view of this, developing a more direct, efficient, and environmentally friendly asymmetric synthesis method for D-pantolactone to replace the existing chiral resolution technology will have important application value.

[0003] D-pantolactone can be asymmetrically synthesized by the redox method, which can be achieved through two different routes. In the first route, L-pantolactone is first dehydrogenated by L-pantolactone dehydrogenase to generate ketopantolactone, and then ketopantolactone spontaneously hydrolyzes to form ketopantoic acid. Then, D-pantoic acid is generated under the action of D-ketopantoic acid reductase. Subsequently, D-pantoic acid forms D-pantolactone under the action of an acid. The more concise second route uses racemic DL-pantolactone as a substrate, and stereoselective and specific L-pantolactone dehydrogenase catalyzes the dehydrogenation of L-pantolactone to generate ketopantolactone. Then, ketopantolactone is asymmetrically generated into D-pantolactone under the catalysis of D-ketopantolactone reductase. Compared with the existing hydrolase catalytic pathway, the second route has a simpler process. The racemic substrate is directly converted into an optically pure product through biocatalysis, without the need for a racemization step or a separation step of lactone and acid. Therefore, the method of asymmetric synthesis of D-pantolactone by the redox enzyme in the second route is a very promising alternative to the biocatalysis method. The dehydrogenation of L-pantolactone in this route is one of the key steps, which is catalyzed by L-pantolactone dehydrogenase. At present, the number of known L-pantolactone dehydrogenases is small, and the lack of L-pantolactone dehydrogenases with excellent catalytic performance limits the application of the redox enzyme method in the asymmetric synthesis of D-pantolactone. The L-pantolactone dehydrogenases that have been studied more include the L-pantolactone dehydrogenase derived from Rhodococcus erythropolis and the L-pantolactone dehydrogenase derived from Nocardia asteroides. The L-pantolactone dehydrogenase derived from Rhodococcus erythropolis has poor soluble expression in the Escherichia coli system, which increases the difficulty of multi-enzyme combined catalysis. Using the genetically engineered bacterium AKU2103 with enhanced expression of the L-pantolactone dehydrogenase gene from Rhodococcus erythropolis in the same Rhodococcus erythropolis as a biocatalyst, the dehydrogenation reaction of 0.768 M L-pantolactone was catalyzed for 144 h, and the conversion rate of the reaction was 91.9%. Considering that the dehydrogenation product of L-pantolactone is ketopantolactone, and ketopantolactone is prone to spontaneous hydrolysis to ketopantoic acid. After 144 h of the above reaction, it is necessary to further add recombinant Escherichia coli expressing D-ketopantoic acid reductase as a biocatalyst to completely convert the generated ketopantoic acid into D-pantoic acid after 24 h of reduction reaction.Finally, D-pantothenic acid is further acidified to produce D-pantolactone (SiD, Urano N, Nozaki S, et al. L-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of L-pantoyl lactone. Applied Microbiology and Biotechnology, 2012, 95: 431-440). In addition, although the L-pantoyl lactone dehydrogenase derived from Nocardia asteroides has been studied in detail for its enzymatic properties (Kataoka M, Shimizu S, Yamada H. Purification and characterization of a novel FMN-dependent enzyme: membrane-bound L-(+)-pantoyl lactone dehydrogenase from Nocardia asteroides. European Journal of Biochemistry, 1992, 204, 799-806), its heterologous expression in Escherichia coli is poor and its catalytic activity is low, which hinders its further application in biocatalysis. At present, the L-pantoyl lactone dehydrogenase derived from Rhodococcus hoagii has been successfully expressed solubly in Escherichia coli, but the low membrane protease activity still limits its application in biocatalysis. (III) Summary of the Invention

[0004] The object of the present invention is to provide a mutant of L-pantoyl lactone dehydrogenase, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and their application in the microbial catalysis for the preparation of keto-pantolactone / D-pantolactone, aiming at the problems of low catalytic activity of the existing L-pantoyl lactone dehydrogenase RhoLPLDH towards L-pantoyl lactone and the large amount of catalyst (cells) used. L254I / V241I

[0005] The technical solution adopted by the present invention is as follows:

[0006] An L-pantolactone dehydrogenase RhoLPLDH mutant is obtained by single mutation or multi-point combined mutation at positions 156, 224, and 164 of the amino acid sequence shown in SEQ ID NO.1. The starting sequence of the L-pantolactone dehydrogenase amino acid mutant derived from Rhodococcus erythropolis in the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0007] Preferably, the mutation is one of the following or a combination of two or more of them: (1) isoleucine at position 156 is mutated to leucine; (2) phenylalanine at position 224 is mutated to glutamine; (3) asparagine at position 164 is mutated to lysine.

[0008] The RhoLPLDH mutant is preferably one of the following: (1) isoleucine at position 156 of the amino acid sequence shown in SEQ ID NO.1 is mutated to leucine (I156L); (2) phenylalanine at position 224 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamine, asparagine at position 164 is mutated to lysine, and isoleucine at position 156 is mutated to leucine (I156L / F224Q / N164K).

[0009] More preferably, the amino acid sequence of the mutant is shown in SEQ ID NO.3.

[0010] The present invention also encompasses proteins with at least 95% identity to the derived amino acid sequences obtained by substituting, deleting, or adding one or several amino acid residues in the amino acid sequences described herein and having transaminase activity.

[0011] The present invention also relates to a gene encoding the L-pantolactone dehydrogenase mutant.

[0012] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0013] The present invention also relates to a recombinant vector and a genetically engineered bacterium containing the gene encoding the L-pantolactone dehydrogenase mutant.

[0014] The present invention also relates to the use of the L-pantolactone dehydrogenase mutant in the microbial catalysis of L-pantolactone to prepare keto-pantolactone. The catalytic substrate can be L-pantolactone or racemic D,L-pantolactone. Specifically, the method of the use is as follows: using the wet cells obtained by induced culture of the engineered bacterium co-expressing the RhoLPLDH mutant gene and the molecular chaperone pGro7 as the catalyst, using L-pantolactone as the substrate, and using a PB buffer solution (0.05M Na2HPO4, 0.05M NaH2PO4) with pH 7.0 and 50 mM as the reaction medium to form a conversion system, reacting at 30-40 °C and 600-800 rpm (preferably 35 °C and 800 rpm), and adjusting the pH to 7.0-8.0 with a 2M NaOH solution, and regularly sampling to detect the concentrations of the substrate and the product.

[0015] The dehydrogenation reaction of L-pantolactone specifically catalyzed by L-pantolactone dehydrogenase for L-pantolactone or the L-pantolactone in the racemate is shown in Figure 1 as follows, generating keto-pantolactone.

[0016] The present invention also relates to the further use of the L-pantolactone dehydrogenase mutant in the microbial catalysis for preparing D-pantolactone. The synthesis pathway of D-pantolactone is shown in Figure 1 as follows. Specifically, the use is as follows: adopting a "two-bacteria one-pot" conversion system, adding the substrate to a final concentration of 500-1000 mM, adding glucose to a final concentration of 500-1250 mM, and the dosage of the catalyst is 45 g WCW / L / 100 mM LPL (WCW wet cell weight) based on the wet weight of the cells. The wet cells obtained by induced culture of the engineered bacterium co-expressing the RhoLPLDH mutant and the molecular chaperone pGro7 in the cell catalyst are mixed with the wet cells obtained by induced culture of the engineered bacterium co-expressing the conjugated polyketone reductase and glucose dehydrogenase at a wet weight ratio of 20:25. Specifically, the amino acid sequence of the RhoLPLDH mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4. The conjugated polyketone reductase gene (GenBank NO.CCG25060.1) is from Candida orthopsilosis, and the nucleotide sequence is shown in SEQ ID NO.5. The glucose dehydrogenase gene (GenBank NO.KM817194.1) is from Exiguobacterium sibirium DSM 17290, and the nucleotide sequence is shown in SEQ ID NO.6.

[0017] Further, the wet bacterial cells are prepared as follows: The engineered bacteria co-expressing the RhoLPLDH mutant vector and the molecular chaperone pGro7 are inoculated into an LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 25 μg / mL, and cultured at 37 °C for 10 h to obtain a seed solution; The seed solution is inoculated into a fresh LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 25 μg / mL at an inoculation amount of 1.0% (v / v). Meanwhile, 0.5 g / L of L-arabinose is added to induce the molecular chaperone protein, and the culture is carried out at 37 °C and 180 rpm for 2 h (OD 600 = 0.4 - 0.6). Isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM is added to the culture solution, and after culturing at 20 °C for 12 h, centrifugation is carried out at 4 °C and 8000 rpm for 10 min to obtain the wet bacterial cells containing the RhoLPLDH mutant protein and the molecular chaperone protein; In the method for preparing wet bacterial cells obtained by inducing and culturing the engineered bacteria co-expressing the conjugated polyketone reductase and glucose dehydrogenase, the antibiotic is 50 μg / mL kanamycin, L-arabinose does not need to be added, the induction temperature is 20 °C, and the others are the same as those of the wet bacterial cells co-expressing the RhoLPLDH mutant vector and the molecular chaperone.

[0018] The pure enzyme of the present invention can be prepared as follows: The wet bacterial cells containing L-pantolactone dehydrogenase RhoLPLDH and its mutants are resuspended in a 50 mM PB buffer solution with a pH of 7.0 at a concentration of 50 g / L, and ultrasonically disrupted on an ice-water mixture for 30 min. The ultrasonic disruption conditions are: amplitude 50%, disruption for 1 s and pause for 2 s. The disrupted mixture is taken, centrifuged at 8000 rpm and 4 °C for 10 min, and the supernatant is collected. After microfiltration through a 0.45 μm membrane, a crude enzyme solution is obtained. The mutant protein is purified using a nickel affinity column (1.6 × 10 cm, Bio-Rad, USA). The specific operations are as follows: ① Pre-equilibrate with buffer A (containing 300 mM NaCl, 20 mM NaH2PO4, pH 7.0, 0.1 mM FMN); ② Load the crude enzyme solution onto the nickel affinity column at a flow rate of 1.0 mL / min; ③ Wash away the unbound impurities with buffer A at a flow rate of 1.0 mL / min until the conductivity is stable; ③ Then wash away the non-specifically bound miscellaneous proteins with buffer B (containing 300 mM NaCl, 20 mM NaH2PO4, 50 mM imidazole, pH 7.0, 0.1 mM FMN); ③ Then elute and collect the target protein with buffer C (containing 300 mM NaCl, 20 mM NaH2PO4, 500 mM imidazole, pH 7.0, 0.1 mM FMN). The collected eluate is not dialyzed and stored at low temperature at 4 °C for a short term, which is the pure enzyme solution of L-pantolactone dehydrogenase.

[0019] The full-length base sequences of the RhoLPLDH and its mutants of the present invention are both 1203 bp, starting from the first base to the 1203rd base. The start codon is ATG and the stop codon is TAA.

[0020] The acquisition of the RhoLPLDH mutants of the present invention adopts site-directed mutagenesis technology and iterative saturation mutagenesis technology. Using this technology, the gene of RhoLPLDH (SEQ ID NO.2) is mutated. The obtained mutant plasmid is transformed into E. coli BL21(DE3) competent cells by heat shock. The obtained strain is inoculated, transferred, induced, and the cells are recovered. The resuspended cell suspension is used to catalyze L-pantolactone. The specific method is as follows: In the first step, the control bacterium E. coli BL21(DE3) / pET28b(+)-RhoLPLDH L254I / V241I is activated and the plasmid pET28b(+)-RhoLPLDH is extracted L254I / V241I , and stored at -20 °C. In the second step, homology modeling is carried out through SWISS-MODEL to obtain the three-dimensional structure of RhoLPLDH; then the active center and related amino acids of RhoLPLDH are predicted through HOTSPOT WIZARD. The key amino acid sites Ile 156, Phe224, and Asn164 that affect the binding of the substrate to RhoLPLDH are obtained. Using pET28b(+)-RhoLPLDH L254I / V241I as the template plasmid, site-directed saturation mutagenesis is carried out on Ile 156, Phe224, and Asn164 to obtain mutant plasmids, which are then transformed to obtain a mutant library. The redox indicator 2,6-dichlorophenolindophenol (DCPIP) is used to obtain H + in the catalytic reaction of dehydrogenase, changing from the blue oxidation state to the colorless reduction state. Based on the principle that the oxidation state has a characteristic absorption peak at a wavelength of 600 nm, a high-throughput screening method is established. Using the high-throughput method, the dominant mutant strains are screened from the site-directed saturation mutagenesis library of RhoLPLDH L254I / V241I , and the dominant mutants are obtained. Then, gas-phase re-screening is used to obtain the dominant mutant I156L, and the mutant strain E. coli BL21(DE3) / pET28b(+)-RhoLPLDH L254I / V241I / I156L (denoted as RhoLPLDH L254I / V241I / I156L ) is obtained. Then, using the recombinant plasmid pET28b(+)-RhoLPLDH of the mutant strain L254I / V241I / I156LUsing [Phe224 and Asn164] as templates, site-directed saturation mutagenesis was performed on Phe224 and Asn164 respectively to obtain mutant plasmids, which were then transformed. The above-mentioned high-throughput method was used to obtain dominant mutants, and then gas-phase rescreening was used to obtain dominant mutations and combine beneficial mutations to obtain the mutant strain E. coli BL21(DE3) / pET28b(+)-RhoLPLDH L254I / V241I / I156L / F224Q / N164K ). It was found that the mutant strain RhoLPLDH L254I / V241I / I156L and RhoLPLDH L254I / V241I / I156L / F224Q / N164K had 0.43-fold and 1.04-fold higher specific cell viabilities compared to their parental strains, respectively.

[0021] The engineered bacteria co-expressing the RhoLPLDH mutant of the present invention and the molecular chaperone pGro7 were prepared by introducing the recombinant plasmid of the RhoLPLDH mutant gene into the competent cells of the molecular chaperone bacterium E. coli BL21(DE3) / pGro7 and screening on a solid medium with double resistance to kanamycin and chloramphenicol. After inoculation, transfer, induction, and cell recovery of the engineered bacteria co-expressing the RhoLPLDH mutant and the molecular chaperone pGro7, the wet cells were used to test the specific cell viability. It was found that the co-expressing strains pGro7 / RhoLPLDH L254I / V241I / I156L and pGro7 / RhoLPLDH L254I / V241I / I156L / F224Q / N164K (CM5) had 0.16-fold and 0.68-fold higher specific cell viabilities compared to the parental strain pGro7 / RhoLPLDH L254I / V241I , respectively.

[0022] For the inoculation, transfer, induction, and cell recovery of the RhoLPLDH mutant, conjugated polyketone reductase, and glucose dehydrogenase genetic engineering bacteria of the present invention, the culture medium can be any medium in the art that can allow the growth of the bacteria and produce the present invention. Preferably, it is LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and the pH is adjusted to 7.0. There are no special restrictions on the culture method and culture conditions, and the culture method and conditions can be optimized according to factors such as the host type and culture method.

[0023] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that the specific enzyme activity of the RhoLPLDH mutant strain constructed in the present invention is significantly improved compared with the control group, and the co-expression of the RhoLPLDH mutant and the molecular chaperone pGro7 further improves the soluble expression of the target protein. The specific cell activity of the recombinant strain co-expressing the RhoLPLDH mutant and pGro7 constructed in the present invention is also significantly improved compared with the starting strain. The mutant specifically catalyzes LPL, and the substrate conversion rate can reach 94% in 30 hours. When the mutant catalyzes D,L-PL substrate, the conversion rate can reach 91.8% in 48 hours. When the mutant and the recombinant strain co-expressing conjugated polyketone reductase and glucose dehydrogenase use the "two-bacteria one-pot method" to catalyze the resolution of D,L-PL to prepare DPL, the yield of DPL can reach 92.7%. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a reaction schematic diagram of the three-enzyme coupling catalysis of L-pantolactone dehydrogenase, conjugated polyketone reductase and glucose dehydrogenase for the resolution of D,L-pantolactone to prepare D-pantolactone.

[0025] Figure 2 It is a standard curve of the GC signal value (pA) and the corresponding concentration (mM) of the intermediate product ketopantolactone.

[0026] Figure 3 It is a gas chromatogram of L-pantolactone, ketopantolactone and D-pantolactone.

[0027] Figure 4 It is the specific cell enzyme activity of the co-expression of the L-pantolactone dehydrogenase mutant and the molecular chaperone.

[0028] Figure 5 The recombinant strain co-expressing the L-pantolactone dehydrogenase mutant and the molecular chaperone catalyzes L-pantolactone.

[0029] Figure 6 The recombinant strain co-expressing the RhoLPLDH mutant and the molecular chaperone specifically catalyzes L-pantolactone in D,L-pantolactone.

[0030] Figure 7 The recombinant strain co-expressing the RhoLPLDH mutant and the molecular chaperone and the co-expressing strain pR-CorCPR / GDH use the "two-bacteria one-pot method" to catalyze the configuration resolution of D,L-pantolactone to prepare D-pantolactone;

[0031] 1: 20 g / L CM5 + 25 g / L pR, 1.5, 1 equiv glucose are added at 0 and 12 h respectively, and 10 g / L pR-CorCPR / GDH is added at 12 h;

[0032] 2: 20 g / L CM5 + 25 g / L pR, 1,1 equiv glucose was added at 0 and 12 h respectively, and 10 g / L CM5 and 12.5 g / L pR-CorCPR / GDH were added at 12 h;

[0033] 3: 20 g / L CM5 + 25 g / L pR, 1,1 equiv glucose was added at 0 and 12 h respectively;

[0034] 4: 20 g / L CM5 + 25 g / L pR, 1.25,1 equiv glucose was added at 0 and 12 h respectively, and 10 g / L pR-CorCPR / GDH was added at 12 h;

[0035] 5: 20 g / L CM5 + 25 g / L pR, 1.25,1 equiv glucose was added at 0 and 12 h respectively, and 10 g / L CM5 and 12.5 g / L pR-CorCPR / GDH were added at 12 h. (V) Specific implementation manners

[0036] The present invention will be further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments:

[0037] Example 1: Construction and screening of L-pantolactone dehydrogenase mutant library

[0038] 1. Starting strain:

[0039] Using the laboratory-preserved engineered strain E. coli BL21(DE3) / pET28b(+)-RhoLPLDH L254I / V241I (The construction method can be referred to CN 113564136A) as the original strain, activating and extracting the plasmid pET28b(+)-RhoLPLDH L254I / V241I , where the amino acid sequence of L-pantolactone dehydrogenase RhoLPLDH L254I / V241I is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2.

[0040] 2. Single mutation:

[0041] (1) Construction of mutant library

[0042] The preparation of the RhoLPLDH mutant library was achieved by site-directed mutagenesis, using the vector pET28b(+)-RhoLPLDH in the original strain L254I / V241IUsing it as a template, site-directed saturation mutation primers were designed and polymerase chain reaction (PCR) was carried out. The recombinant plasmid digested by DpnⅠ was transferred into Escherichia coli BL21(DE3) competent cells, and the clones were inoculated into 10 mL of LB plate medium and cultured at 37 °C for 12 - 16 h.

[0043] (2) Primary screening

[0044] Positive clones and the original strain on the plate were randomly selected and inoculated into a 96-well plate. 1000 μL of LB medium (containing 50 μg / mL kanamycin) was added, and the cells were cultured at 37 °C and 180 rpm for 10 h to obtain seed liquid. 50 μL of each seed liquid was transferred to another new 96-well plate (added with 1000 μL of LB medium containing 50 μg / mL kanamycin), and after culturing at 37 °C and 180 rpm for 4 h, IPTG (final concentration 0.10 mM) was added and the culture was transferred to 28 °C for 12 h. The obtained cells were centrifuged at 4000 rpm and 4 °C for 10 min by a 96-well plate centrifuge to obtain the wet cells of the mutants.

[0045] 300 μL of sodium phosphate buffer solution (50 mM, pH 7.0) was added to each well of the 96-well plate containing wet cells to resuspend the cells. Then, 100 μL of the cell suspension was added to the corresponding position of a 96-well microplate reader. 2,6-dichlorophenolindophenol (DCPIP) with a final concentration of 100 μM was added, and L-pantolactone with a final concentration of 200 μM was added to initiate the reaction. In the Kinetics mode of a microplate reader (MDSpectraMax M5, USA), the change in OD absorbance value was measured at 30 °C every 30 s for 5 min. 600 Correspondingly, the higher the enzyme activity of the mutant, the more the OD decreases, so as to screen out the mutants with relatively higher activity in the mutant library for further rescreening and sequencing verification. 600

[0046] (3) GC rescreening

[0047] The mutants obtained in step (2) were screened for dominant mutants. The dominant mutants were fermented in a shake flask to obtain wet cells, and the wet cells were used for the rescreening reaction. The rescreening conditions were as follows: The obtained wet cells of the mutants were resuspended in PB (50 mM) with a pH of 7.0 at a wet weight of 10 g / L, and then L-pantolactone with a final concentration of 10 mM was added. The reaction was carried out at 30 °C and 1200 rpm in a constant temperature shaker for 30 min. 200 μL of the reaction solution was taken and 50 μL of 6 M hydrochloric acid (acidification) was added, and then 200 μL of ethyl acetate was added for extraction 3 times. The ethyl acetate phases were combined. GC was used to detect the concentrations and conversion rates of L-pantolactone and ketopantolactone. Using the conversion rate of the product ketopantolactone as an index, the dominant strain was screened.

[0048] The standard curve of the GC signal value (pA) and the corresponding concentration (mM) of the intermediate product ketopantolactone is y = 63.277x + 1.2139, R 2 = 0.9994, and the standard curve is as Figure 2 shown.

[0049] Conversion rate = amount of substance of ketopantolactone / (amount of substance of ketopantolactone + amount of substance of L-pantolactone).

[0050] The obtained dominant strain was sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored in a -80 °C refrigerator. The finally screened dominant mutant is RhoLPLDH L254I / V241I / I156L .

[0051] 3. Iterative mutation

[0052] Using the vector pET28b(+)-RhoLPLDH in the strain E. coli BL21(DE3) / pET28b(+)-RhoLPLDH L254I / V241I / I156L as a template, site-directed saturation mutagenesis primers were designed and polymerase chain reaction (PCR) was carried out. According to the steps of high-throughput screening and gas-phase re-screening, further screening for dominant mutants was carried out on the basis of the RhoLPLDH L254I / V241I / I156L mutant. Finally, the dominant strain of the RhoLPLDH mutant E. coli BL21(DE3) / pET28b(+)-RhoLPLDH was further screened and obtained L254I / V241I / I156L . L254I / V241I / I156L / F224Q / N164K .

[0053] PCR reaction system (25 μL): 1 μL forward primer (100 μM), 1 μL reverse primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 1 μL plasmid template, 0.5 μL DNA polymerase Phanta (Novoprotein, China) and 8.5 μL ultrapure water.

[0054] The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual is as follows: pre-denaturation at 95 °C for 5 min, then 30 cycles (denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 4 min), final extension at 72 °C for 10 min, and incubation at 16 °C.

[0055] Gas detection conditions: Chromatographic column BGB174 (30m × 0.25mm, 0.25μm), carrier gas: helium, flow rate: 0.6 mL / min, inlet and detector temperatures: 250 °C; injection volume: 1 μL; split ratio: 30:1; program: 170 °C, 8 min. The retention times of D-pantolactone, L-pantolactone and keto-pantolactone are: 5.4 min, 5.6 min and 5.9 min respectively. The gas chromatograms of D-pantolactone, L-pantolactone and keto-pantolactone are as Figure 3 shown.

[0056] 4. Catalytic activity

[0057] The cell suspensions of the single mutant strain, iterative mutant strain and control strain were used as catalysts respectively, and L-pantolactone was used as the substrate to compare the specific cell activities of each mutant. The reaction system was selected as 1 mL, the catalyst dosage was 10 g / L of wet cell weight, the final concentration of the substrate was 10 mM, pH 7.0, 50 mM PB buffer was used as the reaction medium, and the reaction was carried out by vortex oscillation at 30 °C and 1200 rpm for 30 min. 200 μL of the reaction solution was taken and added with 50 μL of 6 M hydrochloric acid (acidification), and then extracted 3 times with 200 μL of ethyl acetate, and the ethyl acetate phases were combined. For the ethyl acetate sample, the concentrations of L-pantolactone and keto-pantolactone were detected by GC as described in Example 1.

[0058] The unit of cell enzyme activity (U) is defined as: the amount of enzyme required to generate 1 μmol of keto-pantolactone per minute under the conditions of 30 °C and pH 7.0 is defined as one enzyme activity unit U. The specific cell enzyme activity is defined as the number of activity units per gram of cell, U / g.

[0059] The specific cell enzyme activities of each mutant are shown in Figure 4 . The mutant strains RhoLPLDH L254I / V241I / I156L (M3) and RhoLPLDH L254I / V241I / I156L / F224Q / N164K (M5) showed 0.43-fold and 1.04-fold increases in specific cell activity compared to their parental strain (M2), respectively.

[0060] Example 2: Construction of strains co-expressing L-pantolactone dehydrogenase mutants and molecular chaperones

[0061] The recombinant plasmids pET28b(+)-RhoLPLDH L254I / V241I and pET28b(+)-RhoLPLDH L254I / V241I / I156L / F224Q / N164K were respectively transformed into pGro7 competent cells by heat shock, and positive clones co-expressing L-pantolactone dehydrogenase and molecular chaperones were screened on LB solid medium containing double resistance of 25 μg / mL chloramphenicol and 50 μg / mL kanamycin.

[0062] After shaking flask fermentation of the co-expression bacteria of RhoLPLDH mutant and molecular chaperone, the bacterial cells were collected. The specific cell enzyme activity of the recombinant bacteria co-expressing the L-pantolactone dehydrogenase mutant and the molecular chaperone was determined by the cell enzyme activity assay method described in Example 1, and the results are as Figure 4 shown. The mutant strains pGro7 / RhoLPLDH L254I / V241I / I156L (CM3) and pGro7 / RhoLPLDH L254I / V241I / I156L / F224Q / N164K (CM5) had 0.16-fold and 0.68-fold increases in specific cell viability compared to their parental strain (CM2), respectively.

[0063] Example 3: Induced expression of L-pantolactone dehydrogenase mutant, conjugated polyketone reductase and glucose dehydrogenase

[0064] 1. Engineering bacteria co-expressing conjugated polyketone reductase and glucose dehydrogenase: The glucose dehydrogenase gene EsGDH (GenBank NO.KM817194.1) from E. sibirium DSM 17290 was inserted into the multiple cloning site 2 of pRSFDuet, and the conjugated polyketone reductase gene CorCPR from Candida orthopsilosis (GenBank NO.CCG25060.1) was codon-optimized and inserted into the multiple cloning site 1 of pRSFDuet1 to construct a recombinant dual-expression vector; and this expression vector was transferred into E. coli BL21(DE3). A single colony was picked and inoculated into LB medium, and cultured at 37 °C for 12 h. Sequencing confirmed the successful construction of conjugated polyketone reductase and glucose dehydrogenase, and E. coli BL21(DE3) / pRSFDuet-CorCPR / GDH (denoted as pR-CorCPR / GDH) was obtained.

[0065] 2. Induced expression: E. coli BL21(DE3) / pRSFDuet-CorCPR / GDH was inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin at a final concentration, and cultured at 37 °C and 180 rpm for 10 h to obtain a seed solution. The seed solution was inoculated into a fresh 100 mL LB liquid medium shake flask containing 50 μg / mL kanamycin at a final concentration at an inoculation amount of 1.0% (v / v) by volume, and cultured at 37 °C and 180 rpm until the OD 600 was between 0.6 and 0.8, then 0.1 mM IPTG was added to the culture medium, and after culturing at 20 °C for 12 h, centrifuged at 4 °C and 8000 rpm for 10 min to obtain the corresponding wet bacterial cells.

[0066] The recombinant bacteria co-expressing the RhoLPLDH mutant of Example 2 and pGro7 were inoculated into 10 mL of LB liquid medium containing two antibiotics, kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 25 μg / mL, and cultured at 37 °C and 180 rpm for 10 h to obtain a seed solution. The seed solution was inoculated into a 100 mL LB liquid medium shake flask containing fresh kanamycin at a final concentration of 50 μg / mL, chloramphenicol at a final concentration of 25 μg / mL, and arabinose at a final concentration of 0.5 g / L at an inoculation amount of 1.0% (v / v) by volume, and cultured at 37 °C and 180 rpm until the OD 600 was between 0.6 and 0.8. Then, IPTG at a final concentration of 0.1 mM was added to the culture solution, and after culturing at 20 °C for 12 h, centrifuged at 4 °C and 8000 rpm for 10 min to obtain the corresponding wet cell mass.

[0067] The cells obtained above produced the corresponding protein, which can be used for the preparation of pure enzyme solution of the protein, or for the preparation of crude enzyme solution or whole cell to catalyze L-pantolactone.

[0068] Example 4: RhoLPLDH mutant catalyzes L-pantolactone

[0069] Using the wet cell mass of the recombinant bacteria co-expressing the RhoLPLDH mutant prepared by the method of Example 3 and the molecular chaperone as a catalyst to catalyze the synthesis of ketopantolactone from L-pantolactone.

[0070] The ratio of the addition amount of the wet cell mass of the recombinant bacteria co-expressing RhoLPLDH and the molecular chaperone prepared by the method of Example 3 to the substrate LPL was 20 g / L:100 mM LPL. In a 5 mL reaction system, the wet cell mass was first resuspended with PB buffer at pH 7.0 and 50 mM. The feeding amounts of the substrate L-pantolactone were 500, 750, and 1000 mM. Using PB buffer at pH 7.0 and 50 mM as the reaction medium to form a conversion system, reacting at 35 °C and 800 rpm, and adjusting the reaction pH with 2 M NaOH to maintain pH 7.0 - 7.5. Detected by the method of Example 1, the conversion rate during the reaction is shown in Figure 5 . RhoLPLDH L254I / V241I / I156L / F224Q / N164K catalyzed the conversion of 500 mM LPL substrate into the product ketopantolactone in 16 h, and the substrate conversion rate reached 95.9%. RhoLPLDH L254I / V241I / I156L / F224Q / N164K catalyzed the conversion of 750 mM LPL substrate into the product ketopantolactone in 20 h, and the substrate conversion rate reached 95.6%. RhoLPLDH L254I / V241I / I156L / F224Q / N164K catalyzed the conversion of 1000 mM LPL substrate into the product ketopantolactone in 24 h, and the substrate conversion rate reached 94.0%.

[0071] Example 5: Catalysis of D,L-pantolactone by RhoLPLDH mutant

[0072] The wet cells of the recombinant bacterium co-expressing the RhoLPLDH mutant prepared by the method of Example 3 and the molecular chaperone were used as a catalyst to specifically catalyze the synthesis of ketopantolactone from L-pantolactone in D,L-pantolactone.

[0073] The addition amount of the wet cells of the recombinant bacterium co-expressing the RhoLPLDH prepared by the method of Example 3 and the molecular chaperone and the ratio to the substrate D,L-PL were 20 g / L: 200 mM D,L-PL. In a 5 mL reaction system, the wet cells were first resuspended with PB buffer at pH 7.0 and 50 mM. The feeding amount of the substrate D,L-pantolactone was 1500 mM. A conversion system was constituted with PB buffer at pH 7.0 and 50 mM as the reaction medium. The reaction was carried out at 35 °C and 800 rpm, and the reaction pH was adjusted with 1 M NaOH to maintain pH 7.0 - 7.5. Detection was carried out by the method of Example 1, and the LPL conversion rate during the reaction is shown in Figure 6 . RhoLPLDH L254I / V241I / I156L / F224Q / N164K catalyzed the conversion of LPL in 1500 mM D,L-PL into the product ketopantolactone, and the conversion rate of the substrate reached 91.8% after 48 h.

[0074] Example 6: Resolution of D,L-pantolactone by RhoLPLDH mutant to prepare D-pantolactone

[0075] The wet cells of the recombinant bacterium co-expressing the RhoLPLDH mutant prepared by the method of Example 3 and the molecular chaperone and the wet cells of the recombinant bacterium co-expressing the conjugated polyketone reductase CorCPR and glucose dehydrogenase EsGDH were used to establish a dual-bacterium and three-enzyme coupling system to catalyze the resolution of D,L-pantolactone to prepare D-pantolactone in one pot.

[0076] The wet cells CM5 of the recombinant bacterium co-expressing the RhoLPLDH mutant prepared by the method of Example 3 and the molecular chaperone and the wet cells pR-CorCPR / GDH of the recombinant bacterium co-expressing the conjugated polyketone reductase CorCPR and glucose dehydrogenase EsGDH were mixed with the cells at a wet weight ratio of 20:25 g / L / 200 mM D,L-PL and added to the initial reaction system. In a 5 mL reaction system, the feeding amounts of the substrate D,L-PL were 500 and 1000 mM, and the glucose concentration was 500 - 1250 mM. A conversion system was constituted with PB buffer at pH 7.0 and 50 mM as the reaction medium. The reaction was carried out at 35 °C and 800 rpm, and the reaction pH was adjusted with 1 M NaOH to maintain pH 7.0 - 7.5. At 12 h of the reaction, an appropriate amount of catalyst was supplemented. Detection was carried out by the method of Example 1, and the conversion rate during the reaction is shown in Figure 7Reaction 5 catalyzes 500 mM D,L-PL, and the conversion yield of DPL after 36 h is 92.7%, with 3.85% of LPL remaining. Reaction 1 catalyzes 1000 mM D,L-PL, and the conversion yield of DPL after 48 h is 91.1%, with 1.59% of LPL remaining.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall 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 amino acid sequence of the mutant is shown in SEQ ID NO.

3.

2. A gene encoding the L-pantolactone dehydrogenase mutant according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. A recombinant vector containing the encoding gene of the L-pantolactone dehydrogenase mutant according to claim 2.

5. A genetically engineered bacterium containing the encoding gene of the L-pantolactone dehydrogenase mutant according to claim 2.

6. Use of the L-pantolactone dehydrogenase mutant according to claim 1 in the microbial catalysis of L-pantolactone to prepare keto-pantolactone.

7. Use of the L-pantolactone dehydrogenase mutant according to claim 1 in the microbial catalysis to prepare D-pantolactone.

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