L-pantoic acid lactone dehydrogenase, engineered bacteria and application thereof in preparation of d-pantoic acid lactone

Abstract

The present application relates to the technical field of biological catalysis, and discloses application of L-pantoic acid lactone dehydrogenase and a mutant thereof in preparation of D-pantoic acid lactone, wherein the L-pantoic acid lactone dehydrogenase and the mutant thereof with improved activity are screened, and ketopantoic acid lactone reductase is co-expressed to catalyze production of D-pantoic acid lactone.The present application adopts a method for co-expressing L-pantoic acid lactone dehydrogenase mutants and ketopantoic acid lactone reductase to catalyze synthesis of D-pantoic acid lactone, and has the advantages of simple process, high enzymatic reaction selectivity, no by-product, simple product extraction and refining, and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological catalysis. Specifically relates to the application of L-pantoic acid lactone dehydrogenase mutant in the preparation of D-pantoic acid lactone. BACKGROUND

[0002] D-pantothenic acid, also known as vitamin B5, is widely present in the biological world, is the precursor of coenzyme A in animal body, and its main role is to participate in the metabolism of proteins, fats and sugars in animal body. Because D-pantothenic acid is not stable under high temperature, acid and alkali, its derivative D-pantothenic acid calcium with better stability is more widely used in food, medicine, feed and other industries.

[0003] The industrial production of D-pantothenic acid calcium takes D-pantoic acid lactone (D-PL) as a precursor, that is, D-pantoic acid lactone is condensed with calcium β-aminopropionate to obtain D-pantothenic acid calcium. Therefore, D-pantoic acid lactone is a key factor for the large-scale production of D-pantothenic acid calcium. There are two main methods for the synthesis of D-pantoic acid lactone: 1) chemical synthesis method, DL-pantoic acid lactone is obtained by a series of steps of isobutyraldehyde-formaldehyde-hydrocyanic acid, and then D-pantoic acid lactone is obtained by chiral resolving agent, and L-pantoic acid lactone is separated by extraction of racemate and then resolved again. The disadvantage of this method is the use of toxic compound hydrocyanic acid in the synthesis route; 2) microbial hydrolytic enzyme method, which is currently adopted by most enterprises to produce D-pantoic acid lactone. This method uses DL-pantoic acid lactone as raw material, and uses D-pantoic acid lactone hydrolytic enzyme to hydrolyze D-pantoic acid lactone in the raw material, and then separates D-pantoic acid and L-pantoic acid lactone, the separated D-pantoic acid is acidified to form a ring to form D-pantoic acid lactone, and the L-pantoic acid lactone is repeated after racemization to participate in hydrolysis. This process avoids the use of hydrocyanic acid in chemical synthesis, and has high enzyme activity, but also has problems such as repeated extraction of racemization, high acid and alkali consumption, etc. Therefore, it is of important application value to develop a more direct, efficient and environmentally friendly asymmetric synthesis method of D-pantoic acid lactone to replace the existing chiral resolution technology. SUMMARY

[0004] Therefore, the present application provides an L-pantoic acid lactone dehydrogenase, including wild type and mutant; the wild type includes wild type Nf, wild type Rea, wild type Cp; the amino acid sequence of the wild type Nf is shown as SEQ ID NO. 2; the amino acid sequence of the wild type Rea is shown as SEQ ID NO. 6; the amino acid sequence of the wild type Cp is shown as SEQ ID NO. 10; the mutant includes R164K Nf , N283K Rea , G81S Cp : the R164K Nfthe amino acid sequence of which is shown as SEQ ID NO. 4; the N283K Rea the amino acid sequence of which is shown as SEQ ID NO. 8; the G81S Cp the amino acid sequence of which is shown as SEQ ID NO. 12.

[0005] The present application also provides an engineered bacterium containing the L-protelolate dehydrogenase.

[0006] In the detailed description of the present application, the engineered bacterium also contains ketoprotelolate reductase.

[0007] The third object of the present application provides the use of the engineered bacterium in the preparation of D-protelolate.

[0008] The fourth object of the present application provides a method for preparing D-protelolate, comprising: mixing the engineered bacterium with DL-protelolate, and then reacting for 10-24 h at pH 7.0 and 30℃ to obtain D-protelolate.

[0009] The present application adopts the method of co-expressing L-protelolate dehydrogenase mutant and ketoprotelolate reductase to catalyze the synthesis of D-protelolate, which has the advantages of simple process, high selectivity of enzymatic reaction, no by-products, and simple product extraction and refining. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The present application uses L-protelolate dehydrogenase mutant to catalyze the production of D-protelolate.

[0011] Figure 2 The gas chromatogram of D-protelolate produced by the method of the present application. DETAILED DESCRIPTION

[0012] The present application will be further described in detail below in combination with examples, but the present application is not limited in the scope of the examples. The experimental steps not specifically described are operated according to the reagent instructions.

[0013] DL-protelolate (DL-PL) was purchased from Beijing Inokai Technology Co., Ltd.

[0014] Escherichia coli BL21 (DE3) and pET30a vector were purchased from Beijing Zhuangmeng International Biogene Co., Ltd.

[0015] L-pantoic acid lactone dehydrogenase is derived from Nocardia farcinica IFM mycofactocin biosynthesis FMN-dependent deaminase MftD, the gene sequence is shown as Seq ID NO: 1, and the amino acid sequence is shown as Seq ID NO: 2.

[0016] The L-pantoic acid lactone dehydrogenase mutant is mutant R164K obtained by screening of site-directed saturation mutation based on molecular docking Nf , the gene sequence is shown as Seq ID NO: 3, and the amino acid sequence is shown as Seq ID NO: 4.

[0017] Mutant N283K Rea , the gene sequence is shown as Seq ID NO: 7, and the amino acid sequence is shown as Seq ID NO: 8.

[0018] Mutant G81S Cp , the gene sequence is shown as Seq ID NO: 11, and the amino acid sequence is shown as Seq ID NO: 12.

[0019] Rhodococcus erythropolis AKU2103, the gene sequence is shown as Seq ID NO: 5, and the amino acid sequence is shown as Seq ID NO: 6.

[0020] Cnuibacter physcomitrellae, the gene sequence is shown as Seq ID NO: 9, and the amino acid sequence is shown as Seq ID NO: 10.

[0021] The ketopantoate reductase is derived from Candida parapsilosis CDC31, the gene sequence is shown as Seq NO: 13, and the amino acid sequence is shown as Seq NO: 14.

[0022] Example 1

[0023] 1. Molecular docking determines L-pantoic acid lactone dehydrogenase residue site to be mutated

[0024] The substrate molecule L-pantoic acid lactone is implanted into the active center of L-pantoic acid lactone dehydrogenase for analysis by using molecular docking software LeDock (https: / / lephar.com), and bioinformatics operations such as protein structure comparison and image analysis and processing are completed by PyMOL software.

[0025] Molecular docking shows that some amino acid residue sites may have potential effects on the oxidation activity of the enzyme. Specifically as follows:

[0026] Table 1. Residue sites to be mutated of L-camigrenate dehydrogenase from different sources

[0027]

[0028] According to the transformation procedure, the plasmids pET30a-Nf, pET30a-Rea, pET30a-Cp linked to pET30a were respectively transformed into E. coli BL21(DE3) to obtain wild-type L-camigrenate dehydrogenase strains pET30a-Nf-BL21(DE3), pET30a-Rea-BL21(DE3), pET30a-Cp-BL21(DE3) and the strains were preserved in the form of glycerol tube.

[0029] Using the QuickChange site-directed saturation mutagenesis method, the preserved plasmids pET30a-Nf, pET30a-Rea, pET30a-Cp were used as templates, and when designing primers, the codons of the target mutation sites were replaced with NNK (N: A / G / C / T; K: G / T), so as to ensure that 20 natural amino acids randomly appeared at the mutation sites.

[0030] After the sequences of each mutant gene were verified to be correct by sequencing, they were transformed into the BL21(DE3) chassis strain according to the transformation procedure. Then 50 ml of LB liquid medium containing 100 μg / ml kanamycin resistance was added, and after overnight culture at 37°C and 200 rpm / min, each mutant strain was preserved in the form of glycerol tube.

[0031] The seed liquid of each remaining mutant was inoculated into 200 ml of LB medium (100 μg / ml kanamycin resistance) at an inoculation amount of 1%, and was cultured at 37°C until the OD 600 was about 0.6. After adding IPTG at a final concentration of 0.5 mM, the cells were collected by centrifugation after induction culture at 30°C for 8-10 h. The cells were suspended in 1 / 10 of the fermentation liquid volume of 50 mM phosphate buffer (pH 7.0) and were ultrasonically broken. After centrifugation, the crude enzyme liquid of each mutant was obtained. In a 96-well plate, 200 μL of the above lysed enzyme liquid and 200 μL of 100 mM DL-camigrenate were added, and the reaction was carried out at pH 7.0 and 30°C for 3 h. After acidification to pH 3.5 with 6M HCl and heating at 80°C for 15 min, an equal volume of ethyl acetate was extracted, dried with anhydrous Na2SO4, and then detected by gas chromatography. Through screening, three mutants with significantly improved activity were obtained, namely mutant pET30a-R164K Nf -BL21(DE3) (Seq ID NO: 3 and 4), mutant pET30a-N283K Rea- BL21(DE3) (Seq ID NO: 7 and 8), mutant pET30a-G81S Cp - BL21(DE3) (Seq ID NO: 11 and 12).

[0032] Table 2. Comparison of amino acid residue changes and conversion rates of L- pantoic acid lactone dehydrogenase mutants

[0033] Strain name Amino acid residue change Substrate Conversion rate Wild type Nf (Seq ID NO: 1 and 2) None DL-PL 35 [R164K Nf (Seq ID NO:3and 4) Arg→Lys DL-PL 40 Wild type Rea (Seq ID NO: 5 and 6) None DL-PL 33 N283K Rea (Seq ID NO:7and 8) Asn→Lys DL-PL 39 Wild type Cp (Seq ID NO: 9 and 10) None DL-PL 37 G81S Cp (Seq ID NO:11and 12) Gly→Ser DL-PL 45

[0034] Example 2

[0035] 1. L-pantoic acid lactone dehydrogenase Nf, abbreviated as Nf, derived from Nocardia farcinica IFM mycofactocin biosynthesis FMN-dependent deaminase MftD, the nucleotide sequence of the Nf gene is shown in SEQ ID NO. 1 and the amino acid sequence is shown in SEQ ID NO. 2.

[0036] 2. Construction of recombinant ketopantoate reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0037] The gene sequence of ketopantoate reductase (abbreviated as DCR, Genebank ID: HE605209.1) derived from Candida parapsilosis CDC31 (Seq ID NO: 13 and 14) was synthesized and ligated to the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR and stored, and then pET30a-DCR was transformed into Escherichia coli BL21(DE3) according to the operation procedure, to obtain recombinant ketopantoate reductase pET30a-DCR-BL21(DE3), abbreviated as DCR. After 37°C overnight culture, a single colony was picked into a 5mL LB test tube containing 100μg / mL kanamycin, and the strain pET30a-DCR-BL21(DE3) was stored in a glycerol tube.

[0038] 3. Construction of co-expression strain Nf-DCR-BL21(DE3)

[0039] Nf gene primer pair was designed:

[0040] 5'-ggtgctcgagtgcggccgcaatgaacgcgtggtttgaaacggtcgca-3',

[0041] 5'-gtgcctcatgtctttctctagattaagcggtagcagcagtggcaacag-3';

[0042] DCR gene primer pairs:

[0043] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0044] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0045] PCR amplification was performed with plasmid pET30a-Nf and pET30a-DCR as templates, respectively. The Nf gene fragment obtained after PCR amplification had a HindIII enzyme cutting site at the upstream, and the DCR gene fragment had a NcoI enzyme cutting site at the downstream. The PCR products were purified by agarose gel electrophoresis, and the target fragments were recovered using an agarose gel DNA recovery kit. The pET30a vector was cut by HindIII and NcoI, and the Nf and DCR fragments were connected to the linear pET30a vector using Gibson ligase. The product was transformed into DH5a, and then plated on a kanamycin-resistant plate. After overnight culture, a single colony was selected and cultured in a test tube containing 5 mL of LB. The plasmid was extracted using a plasmid extraction kit, and the plasmid with the correct size of the inserted gene band was determined by PCR amplification and sent for sequencing. The plasmid with correct sequencing was saved and named pET30a-Nf-DCR. According to the operation procedure, the plasmid was transformed into competent cells BL21(DE3), plated on a kanamycin-resistant LB solid plate, and incubated at 37°C overnight. A single colony was selected and cultured in a test tube containing 5 mL of LB for 5 h, and the glycerol strain was saved. The co-expressed recombinant engineering bacteria Nf-DCR-BL21(DE3) were obtained.

[0046] 4. Culture of engineering bacteria Nf-DCR-BL21(DE3)

[0047] The strain Nf-DCR-BL21(DE3) saved in the glycerol strain tube was taken into 5 mL of LB liquid medium containing 100 μg / mL of kanamycin, and cultured at 37°C and 200 rpm for 5 h. Then, it was transferred into 800 mL of LB medium containing 100 μg / mL of kanamycin at a ratio of 2%, and cultured at 37°C and 200 rpm until the OD 600 was about 0.6. The final concentration of IPTG was 0.5 mM, and the induction culture was performed at 25°C and 200 rpm for 8-10 h. The cells were collected by centrifugation, and the engineering bacteria Nf-DCR-BL21(DE3) were obtained.

[0048] 5. The engineered bacteria Nf-DCR-BL21(DE3) catalyze DL-pantholactone to produce D-pantholactone

[0049] In 50 mM, 100 mL pH 7.0 PB buffer solution, add prepared Nf-DCR-BL21(DE3) wet bacteria 2.5 g, add 4.0 g of DL-pantholactone, control pH at 7.0, 30℃ for 10-24 h. Then acidified with 6M HCl to pH = 3.5 or so, heated at 80℃ for 15 min, added 0.5% H2O2 for 30 min, then extracted with equal volume of ethyl acetate three times, combined organic phase, concentrated by distillation to get D-pantholactone crude product 3.92 g. Recrystallized with ethyl acetate / petroleum ether (60-90℃) to get pure product 3.80 g, ee% = 97.8%, D-pantholactone gas phase retention time reference Figure 2 .

[0050] The detection and analysis results of D-pantholactone are shown in Figure 2 , and the detection and analysis conditions are as follows: take 300ul of the above sample solution, acidify with 6M HCl to pH about 4.0, then heat at 80℃ for 15 min, cool to room temperature, then add equal volume of chromatographically pure ethyl acetate, mix well, centrifuge at 12000 rpm for 2 min, then dry the organic phase with anhydrous Na2SO4 for 2 h, then use Agilent 7890 gas chromatograph for analysis, the chromatographic column used is Astec CHIRALDEX B-PN gas capillary chromatographic column, nitrogen flow rate is 6.5 ml / min, injection port and detector temperature are both 200℃, programmed temperature is 100℃ (hold for 2 min), 0.5℃ / min to 110℃, a total of 12.6 min.

[0051] Example 3.

[0052] 1. L-pantholactone dehydrogenase mutant R164K Nf , referred to as R164K Nf , the R164K Nf The nucleotide sequence of the R164K gene is shown in SEQ ID NO. 3 and the amino acid sequence is shown in SEQ ID NO. 4.

[0053] 2. Construction of recombinant ketopantholactone reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0054] The gene sequence of ketopantoyl lactone reductase (abbreviation: DCR, Genebank ID: HE605209.1) derived from Candida parapsilosis CDC31 (Seq ID NO: 13 and 14) was synthesized, ligated to the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR and stored, and then pET30a-DCR was transformed into Escherichia coli BL21 (DE3) according to the operation procedure, to obtain the recombinant ketopantoyl lactone reductase pET30a-DCR-BL21 (DE3), abbreviated as DCR. After 37°C overnight culture, a single colony was picked into a 5 mL LB test tube containing 100 μg / mL kanamycin, and the strain pET30a-DCR-BL21 (DE3) was stored in a glycerol tube.

[0055] 3. Co-expression of strain R164K Nf Construction of DCR-BL21 (DE3)

[0056] Design of R164K Nf Gene primer pairs:

[0057] 5'-ggtgctcgagtgcggccgcaatgaacgcgtggtttgaaacggtcgca-3',

[0058] 5'-gtgcctcatgtctttctctagattaagcggtagcagcagtggcaacag-3';

[0059] DCR gene primer pairs:

[0060] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0061] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0062] PCR amplification was performed with plasmids pET30a-R164K Nf and pET30a-DCR as templates, respectively. The R164K Nf gene fragment obtained after PCR amplification had a HindIII enzyme cutting site at the upstream, and the DCR gene fragment had a NcoI enzyme cutting site at the downstream. The PCR product was purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The pET30a vector was cut by HindIII and NcoI, and the R164KNf , DCR fragment and pET30a linear vector, the ligation product was transformed into DH5a, and plated on kanamycin-resistant plates. After overnight culture, single colonies were picked and inoculated into 5 mL LB for culture. The plasmid was extracted using a plasmid extraction kit, and PCR amplification was performed to determine the size of the inserted gene. The plasmid with the correct band size was sent for sequencing, and the correctly sequenced plasmid was named pET30a-R164K. Nf -DCR, according to the transformation operation procedure, the competent cells BL21(DE3) were transformed, plated on kanamycin-resistant LB solid plates, and incubated at 37°C overnight. Single colonies were picked and inoculated into 5 mL LB for 5 h, and the glycerol stock was stored. The co-expression recombinant engineering bacteria R164K were obtained. Nf -DCR-BL21(DE3).

[0063] 4. Engineering bacteria R164K Nf -DCR-BL21(DE3) culture

[0064] The R164K strain stored in the glycerol stock tube was inoculated. Nf -DCR-BL21(DE3) was cultured in 5 mL LB liquid medium containing 100 μg / mL kanamycin at 37°C and 200 rpm for 5 h. Then, it was transferred into 800 mL LB medium containing 100 μg / mL kanamycin at a ratio of 2%, and incubated at 37°C and 200 rpm until the OD 600 was about 0.6. The final concentration of IPTG was 0.5 mM, and the induction culture was performed at 25°C and 200 rpm for 8-10 h. The cells were collected by centrifugation, and the engineering bacteria R164K Nf -DCR-BL21(DE3).

[0065] 5. Engineering bacteria R164K Nf -DCR-BL21(DE3) catalyzing DL-pantoic acid lactone to produce D-pantoic acid lactone

[0066] In 50 mM, 100 mL pH 7.0 PB buffer solution, the prepared R164K Nf -DCR-BL21(DE3) wet bacteria 2.5 g, 4.5 g DL-pantoic acid lactone was added, and the reaction was controlled at pH 7.0 and 30°C. Then, it was acidified with 6M HCl to pH=3.5, heated at 80°C for 15 min, and decolorized with 0.5% H2O2 for 30 min. Then, it was extracted with an equal volume of ethyl acetate three times, and the organic phase was combined and concentrated by distillation to obtain 4.38 g of D-pantoic acid lactone crude product. Recrystallization with ethyl acetate / petroleum ether (60-90°C) obtained a total of 4.29 g of pure product, with ee%=97.4%.

[0067] D-pantoic acid lactone detection analysis: take 300ul of the above sample solution, add 6M HC1 to pH about 4.0, then heat at 80℃ for 15min, cool to room temperature, then add the same volume of chromatographically pure ethyl acetate, high speed centrifugation for 2min at 12000rpm, dry the organic phase with anhydrous Na2SO4 for 2h, then use Agilent 7890 gas chromatograph for analysis, the chromatographic column used is Astec CHIRALDEX B-PN gas capillary chromatographic column, nitrogen flow rate is 6.5ml / min, sample inlet and detector temperature are both 200℃, programmed temperature is 100℃ (hold for 2min), 0.5℃ / min to 110℃, a total of 12.6min.

[0068] Example 4.

[0069] 1. L-pantoic acid lactone dehydrogenase Rea derived from Rhodococcus erythropolis AKU2103, referred to as Rea, the nucleotide sequence of the Rea gene is shown in SEQ ID NO. 5 and the amino acid sequence is shown in SEQ ID NO. 6.

[0070] 2. Construction of recombinant ketopantoate reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0071] Synthetic ketopantoate reductase (referred to as: DCR, Genebank ID: HE605209.1) gene sequence (as Seq ID NO: 13 and 14) derived from Candida parapsilosis CDC31 is connected to the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR and stored, then according to the operation procedure, pET30a-DCR is transformed into Escherichia coli BL21(DE3) to obtain recombinant ketopantoate reductase pET30a-DCR-BL21(DE3), referred to as DCR. After 37℃ overnight culture, single colony is picked into 5mL LB test tube containing 100μg / mL kanamycin, and the strain pET30a-DCR-BL21(DE3) is stored in the form of glycerol tube.

[0072] 3. Construction of co-expression strain Rea-DCR-BL21(DE3)

[0073] Design Rea gene primer pair:

[0074] 5'-gtggtgctcgagtgcggccgcaatggctaagaatgctttcttcgag-3',

[0075] 5'-gtgcctcatgtctttctctagattagctggcacccaatgtgcgtgtgaa-3';

[0076] DCR gene primer pair:

[0077] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0078] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0079] PCR amplification was performed with plasmid pET30a-Rea and pET30a-DCR as templates, respectively. The upstream of the Rea gene fragment obtained after PCR amplification had a HindIII enzyme cutting site, and the downstream of the DCR gene fragment had a Ncol enzyme cutting site. The PCR product was purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The pET30a vector was cut with HindIII and Ncol, and the Rea and DCR fragments and the linear pET30a vector were connected using Gibson ligase. The product was transformed into DH5a, and then plated on a kanamycin-resistant plate. After overnight culture, a single colony was selected and cultured in a test tube containing 5 mL of LB. The plasmid was extracted using a plasmid extraction kit, and PCR amplification was performed to determine the size of the inserted gene band. The plasmid with the correct size was sent for sequencing, and the correct sequence was saved and named pET30a-Rea-DCR. According to the transformation procedure, the plasmid was transformed into competent cells BL21(DE3), plated on a kanamycin-resistant LB solid plate, and incubated at 37°C overnight. A single colony was selected and cultured in a test tube containing 5 mL of LB for 5 h, and the glycerol strain was saved. The co-expressed recombinant engineering strain Rea-DCR-BL21(DE3) was obtained.

[0080] 4. Culture of engineering strain Rea-DCR-BL21(DE3)

[0081] The strain Rea-DCR-BL21(DE3) saved in the glycerol strain tube was taken into 5 mL of LB liquid medium containing 100 μg / mL of kanamycin, and cultured at 37°C and 200 rpm for 5 h. Then, it was transferred into 800 mL of LB medium containing 100 μg / mL of kanamycin at a ratio of 2%, and cultured at 37°C and 200 rpm until the OD 600 was about 0.6. The final concentration of IPTG was 0.5 mM, and the induction culture was performed at 25°C and 200 rpm for 8-10 h. The cells were collected by centrifugation, and the engineering strain Rea-DCR-BL21(DE3) was obtained.

[0082] 5. The engineered bacteria Rea-DCR-BL21(DE3) catalyze the production of D- camphyolactone from DL-camphyolactone

[0083] In 50 mM, 100 mL pH 7.0 PB buffer solution, add prepared Rea-DCR-BL21(DE3) wet bacteria 2.5 g, add 4.0 g of DL-camphyolactone, control pH at 7.0, 30°C reaction. After acidification with 6M HCl to pH = 3.5 or so, 80°C heating reaction solution for 15 min, add 0.5% H2O2 decolorization 30 min, then extracted with equal volume of ethyl acetate three times, combined organic phase, distillation concentration to get D-camphyolactone crude product 3.90 g. Recrystallized with ethyl acetate / petroleum ether (60-90°C) to get pure product 3.85 g, ee% = 97.1%.

[0084] D-camphyolactone detection and analysis conditions: take the above sample solution 300ul, add 6M HCl acidification to pH about 4.0, then heat at 80°C for 15 min, cool to room temperature, then add the same volume of chromatographically pure ethyl acetate, high speed centrifugation for 2 min, then dry the organic phase with anhydrous Na2SO4 for 2h, then use Agilent 7890 gas chromatograph for analysis, the used chromatographic column is Astec CHIRALDEX B-PN gas capillary chromatographic column, nitrogen flow rate is 6.5ml / min, the temperature of injection port and detector is 200°C, the program temperature is 100°C (hold for 2 min), 0.5°C / min to 110°C, a total of 12.6 min.

[0085] Example 5

[0086] 1. L-camphyolactone dehydrogenase mutant N283K Rea , abbreviated as N283K Rea , the N283K Rea The nucleotide sequence of the gene is shown in SEQ ID NO. 7 and the amino acid sequence is shown in SEQ ID NO. 8.

[0087] 2. Construction of recombinant ketocamphyolactone reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0088] The gene sequence (e.g., Seq ID NO: 13 and 14) of ketopantolactone reductase (DCR, Genebank ID: HE605209.1) derived from *Candida parapsilosis* CDC31 was synthesized and ligated into the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR, which was then preserved. Subsequently, pET30a-DCR was transformed into *Escherichia coli* BL21(DE3) according to the chemical transformation procedure to obtain the recombinant ketopantolactone reductase pET30a-DCR-BL21(DE3), abbreviated as DCR. After overnight culture at 37°C, a single colony was picked and cultured in a 5 mL LB tube containing 100 μg / mL kanamycin. The strain pET30a-DCR-BL21(DE3) was preserved in glycerol tubes.

[0089] 3. Co-expression strain N283K Rea Construction of -DCR-BL21(DE3)

[0090] Design N283K Rea Gene primer pairs:

[0091] 5'-gtggtgctcgagtgcggccgcaatggctaagaatgctttcttcgag-3',

[0092] 5'-gtgcctcatgtctttctctagattagctggcacccaatgtgcgtgtgaa-3';

[0093] DCR gene primer pair:

[0094] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0095] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0096] Using plasmid pET30a-N283K Rea Using pET30a-DCR as a template, PCR amplification was performed, and the N283K obtained after PCR amplification was... Rea The gene fragment contains a HindIII restriction site upstream, and the DCR gene fragment contains an NcoI restriction site downstream. The PCR products were purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The pET30a vector was digested with HindIII and NcoI, and then ligated to N283K using Gibson ligase.Rea The DCR fragment and pET30a linear vector were ligated, transformed into DH5α, plated on kanamycin-resistant plates, and cultured overnight. Single colonies were picked and cultured in 5 mL LB tubes. Plasmids were extracted using a plasmid extraction kit. PCR amplification was performed to confirm the correct size of the inserted gene band, and the plasmid was sent for sequencing. The correctly sequenced plasmid was saved and named pET30a-N283K. Rea -DCR, following the chemical transformation procedure, was transformed into competent BL21(DE3) cells, plated on kanamycin-resistant LB agar plates, and incubated statically overnight at 37°C. Single colonies were picked and cultured in test tubes containing 5 mL LB for 5 hours, and then the glycerol culture was preserved to obtain the co-expressing recombinant engineered strain N283K. Rea -DCR-BL21(DE3).

[0097] 4. Engineered bacteria N283K Rea Culture of -DCR-BL21(DE3)

[0098] The N283K bacterial strain preserved by aspirating glycerol inoculum tubes Rea -DCR-BL21(DE3) was added to 5 mL of LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 5 h. Then, it was transferred at a 2% ratio to 800 mL of LB medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD reached. 600 Approximately 0.6. Induction culture was performed at 25℃ and 200 rpm with 0.5 M mIPTG added to the cells for 8-10 hours. Cells were then collected by centrifugation to obtain the engineered strain N283K. Rea -DCR-BL21(DE3).

[0099] 5. Engineered bacteria N283K Rea -DCR-BL21(DE3) catalyzes the production of D-pantolactone from DL-pantolactone.

[0100] The prepared N283K was added to a 50 mM, 100 mL pH 7.0 PB buffer solution. Rea 2.5 g of wet DCR-BL21(DE3) bacterial cells were added to 4.5 g of DL-pantolactone, and the reaction was carried out at pH 7.0 and 30 °C. The solution was then acidified with 6 M HCl to approximately pH 3.5, heated to 80 °C for 15 min, and decolorized with 0.5% H₂O₂ for 30 min. The solution was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain 4.35 g of crude D-pantolactone. Recrystallization from ethyl acetate / petroleum ether (60-90 °C) yielded 4.27 g of pure product, ee% = 97.0%.

[0101] The detection and analysis conditions of D-pantoic acid lactone: 300ul of the above sample solution was acidified to pH about 4.0 by adding 6M HCl, and then heated at 80℃ for 15 minutes. After cooling to room temperature, an equal volume of chromatographically pure ethyl acetate was added, mixed, and then centrifuged at 12000rpm for 2 minutes. The organic phase was dried with anhydrous Na2SO4 for 2 hours, and then analyzed using an Agilent 7890 gas chromatograph. The chromatographic column used was Astec CHIRALDEX B-PN gas capillary chromatographic column, the nitrogen flow rate was 6.5ml / min, the injection port and detector temperature were both 200℃, and the programmed temperature was 100℃ (maintained for 2min), 0.5℃ / min to 110℃, a total of 12.6min.

[0102] Example 6

[0103] 1. L-pantoic acid lactone dehydrogenase Cp derived from Cnuibacter physcomitrellae, referred to as Cp, the nucleotide sequence of the Cp gene is shown in SEQ ID NO. 9 and the amino acid sequence is shown in SEQ ID NO. 10.

[0104] 2. Construction of recombinant ketopantoate reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0105] The gene sequence of ketopantoate reductase (referred to as: DCR, Genebank ID: HE605209.1) derived from Candida parapsilosis CDC31 (as Seq ID NO: 13 and 14) was synthesized, ligated to the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR and stored. Subsequently, according to the operation procedure, pET30a-DCR was transformed into Escherichia coli BL21(DE3) to obtain recombinant ketopantoate reductase pET30a-DCR-BL21(DE3), referred to as DCR. After 37℃ overnight culture, a single colony was picked into a 5mL LB test tube containing 100ug / mL kanamycin, and the strain pET30a-DCR-BL21(DE3) was stored in a glycerol tube.

[0106] 3. Construction of co-expression strain Cp-DCR-BL21(DE3)

[0107] Cp gene primer pair:

[0108] 5'-tggtgctcgagtgcggccgcaatgaacccgtggttcgagacggtggc-3',

[0109] 5'-gtgcctcatgtctttctctagatcaggcgctctccgcctcggccttgg-3';

[0110] DCR gene primer pair:

[0111] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0112] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0113] PCR amplification was performed with plasmids pET30a-Cp and pET30a-DCR as templates, respectively. The Cp gene fragment obtained after PCR amplification had a HindIII enzyme cutting site at the upstream, and the DCR gene fragment had a Ncol enzyme cutting site at the downstream. The PCR products were purified by agarose gel electrophoresis, and the target fragments were recovered using an agarose gel DNA recovery kit. The pET30a vector was cut by HindIII and Ncol, and the Cp and DCR fragments were connected to the linear pET30a vector using Gibson ligase. The connection product was transformed into DH5a, and then plated on a kanamycin-resistant plate. After overnight culture, a single colony was selected and cultured in a test tube containing 5 mL of LB. The plasmid was extracted using a plasmid extraction kit, and the plasmid with the correct size of the inserted gene band was determined by PCR amplification and sent for sequencing. The plasmid with correct sequencing was saved and named pET30a-Cp-DCR. According to the operation process, the competent cells BL21(DE3) were transformed, plated on a kanamycin-resistant LB solid plate, and incubated at 37°C overnight. A single colony was selected and cultured in a test tube containing 5 mL of LB for 5 h, and the glycerol strain was saved. The co-expressed recombinant engineering bacteria Cp-DCR-BL21(DE3) were obtained.

[0114] 4. Culture of engineering bacteria Cp-DCR-BL21(DE3)

[0115] The glycerol strain tube saved strain Cp-DCR-BL21(DE3) was taken into 5 mL of LB liquid medium containing 100 μg / mL of kanamycin, and cultured at 37°C and 200 rpm for 5 h. Then, it was transferred into 800 mL of LB medium containing 100 μg / mL of kanamycin at a ratio of 2%, and cultured at 37°C and 200 rpm until the OD 600 was about 0.6. The final concentration of IPTG was 0.5 Mm, and the induction culture was performed at 25°C and 200 rpm for 8-10 h. The cells were collected by centrifugation, and the engineering bacteria Cp-DCR-BL21(DE3) were obtained.

[0116] 5. The engineered bacteria Cp-DCR-BL21(DE3) catalyze DL-panolactone to produce D-panolactone

[0117] In 50 mM, 100 mL pH 7.0 PB buffer solution, add prepared Cp-DCR-BL21(DE3) wet bacteria 2.5 g, add 4.0 g of DL-panolactone, control pH at 7.0, 30℃ reaction. After acidification with 6M HCl to pH = 3.5 or so, 80℃ heating reaction solution for 15 min, add 0.5% H2O2 decolorization 30 min, then extracted with equal volume of ethyl acetate three times, combined organic phase, distillation concentration to get D-panolactone crude product 3.96 g. Recrystallized with ethyl acetate / petroleum ether (60-90℃) to get pure product 3.89 g, ee% = 97.7%.

[0118] D-panolactone detection and analysis conditions: take the above sample solution 300ul, add 6M HCl acidification to pH about 4.0, then heat at 80℃ for 15 min, cool to room temperature, then add the same volume of chromatographically pure ethyl acetate, high speed centrifugation for 2 min, then dry the organic phase with anhydrous Na2SO4 for 2h, then use Agilent 7890 gas chromatograph for analysis, the used chromatographic column is Astec CHIRALDEX B-PN gas capillary chromatographic column, nitrogen flow rate is 6.5ml / min, the temperature of injection port and detector is 200℃, the program temperature is 100℃(hold for 2 min), 0.5℃ / min to 110℃, a total of 12.6 min.

[0119] Example 7

[0120] 1. L-panolactone dehydrogenase mutant G81S Cp , referred to as G81S Cp , the G81S Cp The nucleotide sequence of the gene is shown in SEQ ID NO. 11 and the amino acid sequence is shown in SEQ ID NO. 12.

[0121] 2. Construction of recombinant ketopanolactone reductase pET30a-DCR-BL21(DE3) derived from Candida parapsilosis CDC31

[0122] The gene sequence of ketopantoyl lactone reductase (abbreviation: DCR, Genebank ID: HE605209.1) derived from Candida parapsilosis CDC31 (Seq ID NO: 13 and 14) was synthesized, ligated to the NdeI and EcoRI sites of the expression vector pET30a to obtain the plasmid pET30a-DCR and stored, and then pET30a-DCR was transformed into Escherichia coli BL21 (DE3) according to the operation procedure, to obtain the recombinant ketopantoyl lactone reductase pET30a-DCR-BL21 (DE3), abbreviated as DCR. After 37°C overnight culture, a single colony was picked into a 5 mL LB test tube containing 100 μg / mL kanamycin, and the strain pET30a-DCR-BL21 (DE3) was stored in a glycerol tube.

[0123] 3. Co-expression of strain G81S Cp Construction of DCR-BL21 (DE3)

[0124] Design of G81S Cp Gene primer pairs:

[0125] 5'-tggtgctcgagtgcggccgcaatgaacccgtggttcgagacggtggc-3',

[0126] 5'-gtgcctcatgtctttctctagatcaggcgctctccgcctcggccttgg-3';

[0127] DCR gene primer pairs:

[0128] 5'-gagaaagacatgaggcactagatgactcaaagtaacttactaccaaaaa-3',

[0129] 5'-acgacgacgacaaggccatgctacaaatctttaaattgctcatggaa-3'

[0130] PCR amplification was performed with plasmids pET30a-G81S Cp and pET30a-DCR as templates, respectively. The G81S Cp gene fragment obtained after PCR amplification had a HindIII enzyme cutting site at the upstream, and the DCR gene fragment had a NcoI enzyme cutting site at the downstream. The PCR product was purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. The pET30a vector was cut by HindIII and NcoI, and the G81S CpThe DCR fragment and pET30a linear vector were ligated, transformed into DH5α, plated on kanamycin-resistant plates, and cultured overnight. Single colonies were then picked and cultured in 5 mL LB tubes. Plasmids were extracted using a plasmid extraction kit. PCR amplification was performed to confirm the correct size of the inserted gene band, and the plasmid was sent for sequencing. The correctly sequenced plasmid was saved and named pET30a-G81S. Cp -DCR, following the chemical transformation procedure, was transformed into competent BL21(DE3) cells, plated on kanamycin-resistant LB agar plates, and incubated overnight at 37°C. Single colonies were picked and cultured in test tubes containing 5 mL LB for 5 hours, and then the glycerol culture was preserved to obtain the co-expressing recombinant engineered strain G81S. Cp -DCR-BL21(DE3).

[0131] 4. Engineered bacteria G81S Cp Culture of -DCR-BL21(DE3)

[0132] G81S strain preserved by aspirating glycerol culture tube Cp -DCR-BL21(DE3) was added to 5 mL of LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 5 h. Then, it was transferred at a 2% ratio to 800 mL of LB medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD reached. 600 Approximately 0.6. Induction culture was performed at 25℃ and 200 rpm with 0.5 M mIPTG added for 8-10 h. Cells were then collected by centrifugation to obtain the engineered strain G81S. Cp -DCR-BL21(DE3).

[0133] 5. Engineered bacteria G81K Cp -DCR-BL21(DE3) catalyzes the production of D-pantolactone from DL-pantolactone.

[0134] Add the prepared G81K to 50 mM, 100 mL pH 7.0 PB buffer solution. Cp 2.5 g of wet DCR-BL21(DE3) bacterial cells were added to 4.5 g of DL-pantolactone, and the reaction was carried out at pH 7.0 and 30 °C. The solution was then acidified with 6 M HCl to approximately pH 3.5, heated to 80 °C for 15 min, and decolorized with 0.5% H₂O₂ for 30 min. The solution was then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain 3.90 g of crude D-pantolactone. Recrystallization from ethyl acetate / petroleum ether (60-90 °C) yielded 3.81 g of pure product, ee% = 97.2%.

[0135] The detection and analysis conditions of D-pantoic acid lactone are as follows: 300ul of the sample solution is taken, acidified to pH about 4.0 by adding 6M HCl, heated at 80℃ for 15 minutes, cooled to room temperature, mixed with the same volume of chromatographically pure ethyl acetate, centrifuged at 12000rpm for 2 minutes, and then the organic phase is dried with anhydrous Na2SO4 for 2 hours, and then analyzed by using Agilent 7890 gas chromatograph, the chromatographic column used is Astec CHIRALDEX B-PN gas capillary chromatographic column, the nitrogen flow rate is 6.5ml / min, the temperature of the injection port and detector is 200℃, the programmed temperature is 100℃ (maintained for 2 minutes), 0.5℃ / min to 110℃, a total of 12.6 minutes.

[0136] The present application has the following beneficial effects:

[0137] 1. The present application uses L-pantoic acid lactone dehydrogenase mutant and ketopantoic acid lactone reductase co-expression to catalyze the synthesis of D-pantoic acid lactone, which is simple in process, does not need repeated extraction, racemization and other steps, and is suitable for industrial production in terms of production cost and product quality.

[0138] 2. The enzymatic reaction has high selectivity and no by-products, and the product extraction and purification are simple.

[0139] The above is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above-mentioned embodiments, and any technical solution falling within the idea of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principle of the present application shall also be considered as the protection scope of the present application.

Claims

1. An L-pantolactone dehydrogenase mutant, characterized in that, The mutant is R164K. Nf N283K Rea or G81S Cp : The R164K Nf The amino acid sequence is shown in SEQ ID NO. 4; The N283K Rea The amino acid sequence is shown in SEQ ID NO. 8; The G81S Cp The amino acid sequence is shown in SEQ ID NO.

12.

2. Engineered bacteria, characterized in that, Contains the L-indohydrin dehydrogenase mutant as described in claim 1.

3. The engineered bacteria according to claim 2, characterized in that, It also contains keto-indohydrin reductase.

4. The use of the engineered bacteria described in claim 2 in the preparation of D-pantolactone.

5. A method for preparing D-pantolytic lactone, characterized in that, include: The engineered bacteria described in claim 3 were mixed with DL-indospirulina lactone and reacted at pH 7.0 and temperature 30°C for 10-24 hours to obtain D-indospirulina lactone.

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