L-pantolactone dehydrogenase, encoding gene and application thereof

Abstract

The present invention relates to an L-pantoic acid lactone dehydrogenase, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and its application in the preparation of keto-pantoic acid lactone by microbial catalysis. The amino acid sequence of the L-pantoic acid lactone dehydrogenase is shown in SEQ ID NO.1, and the coding gene is shown in SEQ ID NO.2. The L-pantoic acid lactone dehydrogenase derived from Pseudonocardia sp.73-21 excavated by the present invention has strict L-stereoselectivity, and the recombinant Escherichia coli expressed L-pantoic acid lactone dehydrogenase constructed by the present invention has good solubility, avoiding the problem that the heterologous expression of L-pantoic acid lactone dehydrogenase is easy to form a large number of inclusion bodies. The L-pantoic acid lactone dehydrogenase provided by the present invention has application value in the preparation of keto-pantoic acid lactone / D-pantoic acid lactone by biocatalysis.

Description

(I) Technical field

[0001] The present invention relates to an L-pantolactone dehydrogenase, a coding gene, a vector containing the coding gene, a genetic engineering bacterium, and application of the same in preparing ketopantolactone / D-pantolactone by microbial catalysis. (II) Background technology

[0002] Calcium pantothenate is a water-soluble vitamin B family and one of the nutrients necessary for normal growth of organisms. Calcium pantothenate has three forms, namely D-calcium pantothenate (dextrorotatory), DL-calcium pantothenate (mixed) and L-calcium pantothenate (levorotatory), of which only D-calcium pantothenate has biological activity. Pantothenic acid, as a precursor of coenzyme A, has a variety of physiological effects. Calcium pantothenate is widely used in feed, medicine, food and other industries due to its unique biochemical functions. At present, the industry synthesizes D-calcium pantothenate through calcium β-aminopropionic acid and D-pantothenate lactone.

[0003] D-(-)-pantoic acid lactone, also known as (R)-pantoic acid lactone, is the γ-lactone of D-(-)-pantoic acid and is a key chiral intermediate for the synthesis of D-(+)-pantoic acid. The industrial synthesis of D-pantoic acid lactone is a technical route that combines chemical method and chemical method-hydrolase resolution method. The generation pathway of the intermediate mixture DL-pantoic acid lactone in the two pathways is the same, both of which are synthesized chemically from isobutyraldehyde and formaldehyde as starting materials; the industrial complete conversion of DL-pantoic acid lactone into D-pantoic acid lactone includes chemical method and hydrolase resolution method. The chemical synthesis route is subject to many restrictions due to many environmental pollution problems and the use of hazardous reagents. The hydrolase resolution method selectively hydrolyzes D-pantoic acid lactone in the racemic pantoic acid lactone to generate D-pantoic acid, and then separates D-pantoic acid and L-pantoic acid lactone. The separated D-pantoic acid is acidified to form D-pantoic acid lactone, while L-pantoic acid lactone is racemized and reused. Therefore, although the hydrolase-catalyzed chiral resolution method is mature, it still has problems such as long steps and high acid and alkali consumption. In view of this, it will be of great application value to develop a more direct, efficient and environmentally friendly asymmetric synthesis method for D-pantoic acid lactone to replace the existing chiral resolution technology.

[0004] D-pantoic acid lactone can be synthesized asymmetrically by the redox method, which includes two pathways. The first pathway is to use the d-pantoic acid lactone as a substrate, and utilize the stereoselective and specific L-pantoic acid lactone dehydrogenase to catalyze the dehydrogenation of L-pantoic acid lactone to generate ketopantoic acid lactone, and then the ketopantoic acid lactone is asymmetrically generated into D-pantoic acid lactone under the catalysis of D-ketopantoic acid lactone reductase; the second pathway is also to firstly catalyze the dehydrogenation of L-pantoic acid lactone to generate ketopantoic acid lactone by L-pantoic acid lactone dehydrogenase, and then the ketopantoic acid lactone is spontaneously hydrolyzed to form ketopantoic acid, and then D-pantoic acid is generated under the action of D-ketopantoic acid reductase, and then D-pantoic acid is closed under the action of acid to form D-pantoic acid lactone. The first pathway of this process is more straightforward. Compared with the existing hydrolase catalytic pathway, the process is simpler. The mixed substrate is directly biocatalyzed to obtain an optically pure product, without the need for a racemization step or a separation step of lactone and acid. The coenzyme circulation system is constructed in the genetically engineered bacteria, so no external coenzyme is required. The use of genetically engineered bacteria as whole cell catalysts does not require enzyme separation and purification steps. Therefore, the oxidoreductase asymmetric synthesis of D-pantoic acid lactone is a very promising alternative to the biohydrolase method. The dehydrogenation of L-pantoic acid lactone is one of the key steps in the redox method, and L-pantoic acid lactone dehydrogenase is the key enzyme catalyzing the reaction.

[0005] At present, there are not many known L-pantolactone dehydrogenases, and the most studied ones are L-pantolactone dehydrogenases from Rhodococcus erythropolis and Nocardia asteroides. The L-pantolactone dehydrogenase from Rhodococcus erythropolis is less soluble in the Escherichia coli system, which limits the enzyme's catalytic ability for substrates. The genetically engineered bacteria AKU2103, in which the L-pantolactone dehydrogenase gene of Rhodococcus erythropolis is enhanced in the same Rhodococcus erythropolis, is used as a biocatalyst to catalyze the dehydrogenation reaction of 0.768M L-pantolactone for 144h, and the conversion rate of the reaction is 91.9%. Although the enzymatic properties of L-pantolactone dehydrogenase from Nocardia asteroides have been studied in detail, the protein is easy to form inclusion bodies when expressed in Escherichia coli, and the enzyme activity is low, which hinders its further application in biocatalysis. The lack of L-pantolactone dehydrogenase with excellent catalytic performance limits the application of redox enzyme method in the asymmetric synthesis of D-pantolactone. (III) Summary of the invention

[0006] The present invention aims to provide a novel L-pantolactone dehydrogenase and its encoding gene, a vector containing the encoding gene, a genetically engineered bacterium, and the use of the same in the microbial catalytic preparation of ketopantolactone / D-pantolactone.

[0007] The technical solution adopted by the present invention is:

[0008] An L-pantolactone dehydrogenase, the amino acid sequence of which is shown as SEQ ID NO.1.

[0009] After a lot of preliminary screening work, the present invention discovered a protein derived from the microorganism Pseudonocardia sp.73-21, which has L-pantolactone dehydrogenase activity, which is not found in the prior art. The protein can catalyze L-pantolactone to generate ketopantolactone. The protein with L-pantolactone dehydrogenase activity has good solubility, so it can be fully dissolved in the catalytic reaction liquid, and the catalytic activity can be fully exerted when applied to catalytic related reactions. The discovery of the protein has important practical significance for the fermentation production of L-pantolactone dehydrogenase in microorganisms and the production of related catalytic products.

[0010] Without changing the biochemical properties of L-pantolactone dehydrogenase, those skilled in the art may make certain modifications to its amino acid sequence, such as introducing deletion, addition and / or substitution of one or several amino acid residues, which are all within the scope of protection of the present invention.

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

[0012] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0013] The invention also relates to a recombinant vector and a genetic engineering bacterium containing the L-pantolactone dehydrogenase encoding gene.

[0014] The present invention also relates to the expression of a recombinant vector containing the L-pantoic acid lactone dehydrogenase encoding gene in Escherichia coli. Specifically, the L-pantoic acid lactone dehydrogenase is prepared as follows: the recombinant engineering bacteria containing the vector containing the L-pantoic acid lactone dehydrogenase encoding gene are inoculated into an LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C for 10 hours to obtain a seed solution; the seed solution is inoculated into a fresh LB liquid culture medium containing kanamycin at a final concentration of 50 μg / mL at an inoculum concentration of 1.0% by volume, and cultured at 37°C and 180 rpm for 2 hours (OD 600 =0.4-0.6), add isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM into the culture medium, culture at 20°C for 12 h, and centrifuge at 4°C and 8000 rpm for 10 min to obtain wet cells containing L-pantolactone dehydrogenase.

[0015] Further, the solubility of the recombinant Escherichia coli expressing L-pantolactone dehydrogenase was studied and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The wet bacteria containing L-pantolactone dehydrogenase were resuspended in pH 7.0, 50mM PB buffer at an amount of 10g / L, and ultrasonically broken for 30min on an ice-water mixture. The ultrasonic breaking conditions were: amplitude 50%, breaking for 1s, pause for 2s, taking the broken mixed solution, centrifuging at 8000rpm, 4°C for 10min, collecting the supernatant, and adding an equal volume of PB buffer to the precipitate for resuspending. Take 60μL of the supernatant and the precipitate solution to a 1.5mL EP tube, and then add 20μL of 4× protein loading buffer to mix. After boiling the protein sample for 10min, take 10μL of the sample and load it into a protein precast gel (GenScript, Nanjing), and electrophoresed at 180V for 30min. After staining and decolorization with a protein stainer (eStain L1, GenScript), the expression of L-pantolactone dehydrogenase was analyzed by a protein imaging system. L-pantolactone dehydrogenase from Pseudonocardia sp.73-21 has good solubility in E. coli and is almost completely dissolved in the supernatant.

[0016] The present invention also relates to the substrate specificity of the L-pantoic acid lactone dehydrogenase. The L-pantoic acid lactone dehydrogenase can specifically catalyze the L-isomers in L-pantoic acid lactone or racemate to generate ketopantoic acid lactone. The substrates D-pantoic acid lactone, D-pantoic acid and L-pantoic acid cannot be recognized, indicating that the L-pantoic acid lactone dehydrogenase derived from Pseudonocardia sp.73-21 has strict L-selectivity.

[0017] The beneficial effects of the present invention are mainly reflected in that the L-pantolactone dehydrogenase derived from Pseudonocardia sp.73-21 mined by the present invention has strict L-stereoselectivity, and the recombinant Escherichia coli constructed by the present invention to express the L-pantolactone dehydrogenase has good solubility, avoiding the problem that a large number of inclusion bodies are easily formed when the L-pantolactone dehydrogenase is heterologously expressed. The L-pantolactone dehydrogenase provided by the present invention has application value in the biocatalytic preparation of ketopantolactone / D-pantolactone. (IV) Description of the drawings

[0018] Figure 1 This is a gas phase spectrum of PseLPLDH catalyzing the substrate L-pantoic acid lactone to generate the product ketopantoic acid lactone according to one embodiment of the present invention.

[0019] Figure 2 A gas phase spectrum of the retention time of L-pantolactone according to an embodiment of the present invention;

[0020] Figure 3A gas phase spectrum of the retention time of ketopantoic acid lactone according to an embodiment of the present invention;

[0021] Figure 4 The standard curve of L-pantolactone according to one embodiment of the present invention;

[0022] Figure 5 is a standard curve of ketopantolactone according to an embodiment of the present invention;

[0023] Figure 6 This is a comparison chart of the solubility of L-pantolactone dehydrogenases of different sequences according to an embodiment of the present invention. (V) Specific implementation methods

[0024] The present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments:

[0025] Example 1: Bacterial cell construction

[0026] The codon-optimized gene SEQ ID NO:2 was constructed into the expression vector pET 28a, and a 6*his tag was added to the C-terminus for subsequent protein expression. The construction process is as follows:

[0027] 1. Use F-1 and R-1 in Table 1 as primers and PseLPLDH as template to extend gene fragment 1;

[0028] 2. Use F-2 and R-2 in Table 1 as primers and pET 28a as template to amplify vector fragment 2;

[0029] 3. Purify fragment 1 and fragment 2, and digest the purified fragment 1 and fragment 2 with NcoI and XhoI;

[0030] 4. Use T4 ligase to connect the purified fragment 1 and fragment 2, and transform the enzyme-linked product into E. coli BL21 competent cells;

[0031] 5. Pick a single colony and culture it in liquid culture medium containing 50 μg / μl kanamycin, and send it for sequencing.

[0032] Table 1: Amplification primers

[0033]

[0034] Example 2: Substrate catalysis

[0035] A single colony of the engineered strain constructed in Example 1 was inoculated into LB medium containing 50 μg / μL kanamycin and cultured overnight at 37°C and 180 rpm in a shaker. The seed solution was transferred to 100 mL LB liquid medium containing 50 μg / μL kanamycin at a 1% inoculum and cultured in a shaker at 37°C and 180 rpm for 2-2.5 h until the OD of the bacterial solution reached 0. 600 Reach between 0.6-0.8. Add isopropylβ-D-thiogalactoside (IPTG) to the culture medium at a final concentration of 0.1 mM and induce for 12 hours at 28°C and 180 rpm. Then centrifuge at 4°C and 8000 rpm for 10 minutes to obtain wet cells containing L-pantolactone dehydrogenase active protein PseLPLDH.

[0036] Prepare two portions of PB buffer (0.2M Na2HPO4, 0.2M NaH2PO4) with pH 7.0 and 50mM as the reaction medium to resuspend 10g / L of wet cells, add the substrate L-pantolactone with a final concentration of 1mM to one portion, and add the substrate D-pantolactone with a final concentration of 1mM to the other portion, and react the two samples in a constant temperature oscillator at 30°C and 800rpm for 30min. Take 200uL of the reaction solution and terminate the reaction with the same volume of 6M HCl, add 200μL of ethyl acetate to extract 3 times, and combine the ethyl acetate phase. After centrifugation of the extract, draw the upper organic phase into a centrifuge tube, add anhydrous sodium sulfate to remove water, and then centrifuge to take the supernatant and transfer it to a gas phase sample bottle for gas chromatography detection. GC is used to detect the concentration and conversion rate of L-pantolactone and L-pantolactone. Gas phase detection conditions: chromatographic column Agilent CycloSil-B (30m×0.25mm, 0.25μm), carrier gas: helium, flow rate: 0.5mL / min, injection port and detector temperature: 250℃; injection volume: 1μL; split ratio: 30:1; program: 100℃, 10℃ / min to 140℃, hold for 5min. The retention times of L-pantolactone and ketopantolactone are 9.2min and 6.6min, respectively. Figures 1 to 5 The gas phase spectrum of the retention time of L-pantoic acid lactone and ketopantoic acid lactone and their respective standard curves are shown. The results show that SEQ ID NO: 2 of the strain constructed in this example can encode a protein with L-pantoic acid lactone dehydrogenase activity, thereby catalyzing the reaction of L-pantoic acid lactone. After 30 minutes of reaction, the conversion rate of the substrate L-pantoic acid lactone reaches more than 40%.

[0037] Example 3: Protein induction expression

[0038] The wet cells of PseLPLDH expressing a protein with L-pantolactone dehydrogenase activity and NsLPLDH from Nocardia asteroides expressed in Escherichia coli BL21 (DE3) in Example 2 were washed twice with 0.9 g / mL saline. The total amount of wet cells was 10 g / L and resuspended in pH 7.0, 100 mM PB buffer, and ultrasonically disrupted on an ice-water mixture for 5 minutes. The ultrasonic disruption conditions were: amplitude 10%, disruption for 1 second, and pause for 2 seconds. The disrupted mixture was taken and centrifuged at 8000 rpm and 4°C for 10 minutes. The supernatant and sediment were collected, and the size of the protein and the solubility of the expression were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows. Figure 6 As shown, the results show that the protein expressing L-pantolactone dehydrogenase activity in Example 1 has good solubility and is almost completely dissolved in the supernatant; while the L-pantolactone dehydrogenase from Nocardia asteroides has poor solubility and is mostly distributed in the precipitate.

[0039] Example 4: Investigation of substrate specificity of L-pantolactone dehydrogenase

[0040] The wet bacteria of PseLPLDH expressing the protein with L-pantolactone dehydrogenase activity in Example 2 was expressed in Escherichia coli BL21 (DE3) and used as a biocatalyst. The substrate specificity of L-pantolactone dehydrogenase from Pseudonocardia sp.73-21 was investigated by whole-cell catalysis using 10mM D-pantolactone, L-pantolactone and DL-pantolactone as substrates, respectively. The reaction system of L-pantolactone dehydrogenase catalyzing the dehydrogenation of L-pantolactone was 1mL, containing: 10g / L wet bacteria, 10mM substrate and 200mM phosphate buffer (pH 7.0), respectively. The reaction solution was added to an EP tube, and the reaction conditions were maintained at 30°C and 1200rpm on a constant temperature oscillator. After 30min of reaction, 200μL of the reaction solution was added to an equal volume of 6M hydrochloric acid, and an appropriate amount of anhydrous sodium sulfate was added. 200 μL of ethyl acetate was added and fully extracted three times. After centrifugation of the extract, the upper organic phase was drawn into a centrifuge tube, anhydrous sodium sulfate was added to remove water, and then centrifuged to take the supernatant and transferred into a gas phase sample bottle for gas chromatography detection. The substrate specificity results are shown in Table 2. The results show that the L-pantoic acid dehydrogenase active protein derived from Pseudonocardia sp.73-21 cannot catalyze D-pantoic acid lactone, D-pantoic acid and L-pantoic acid, but can catalyze L-pantoic acid lactone and DL-pantoic acid lactone. The above results show that the L-pantoic acid dehydrogenase active protein derived from Pseudonocardia sp.73-21 specifically acts on the dehydrogenation reaction of L-pantoic acid lactone.

[0041] Table 2: Substrate specificity of L-pantoate dehydrogenase activity proteins from Pseudonocardia sp. 73-21

[0042]

[0043] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An L-pantolactone dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

1.

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

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

2.

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

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

6. Use of the L-pantolactone dehydrogenase according to claim 1 in the preparation of ketopantolactone from L-pantolactone catalyzed by microorganisms.

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