CN114934061A - Engineering bacteria and their application in whole-cell catalyzed production of D-pantolactone from ketopantolactone - Google Patents

Abstract

The invention belongs to the technical field of biological catalysis. The invention discloses engineering bacteria and application thereof in producing D-pantolactone by catalyzing keto-pantolactone in whole cells. The engineering bacteria of the invention can realize the catalytic synthesis of D-pantolactone in a coenzyme regeneration system by ketopantolactone reductase mutant, the substrate concentration is as high as 100g/L, the conversion rate is more than or equal to 99.0 percent, and the weight yield is as high as 95 percent.

Description

Engineering bacteria and their application in whole-cell catalyzed production of D-pantolactone from ketopantolactone

technical field

The invention belongs to the technical field of biocatalysis.

Background technique

Pantothenic acid, also known as pantothenic acid and vitamin B ₅ , is widely present in the biological world and is the precursor for the synthesis of coenzyme A in animals. Its main function is to participate in the metabolism of protein, fat and sugar in animals. The active ingredient of pantothenic acid is D-type, but D-pantothenic acid is unstable under high temperature, acid and alkali, so compared with D-pantothenic acid, its more stable derivatives have been widely used in food, medicine, feed and other industries. application. Currently widely used pantothenic acid derivatives mainly include D-pantothenate calcium, D-panthenol and D-pantethamine, among which D-pantothenate calcium is the main one.

The industrial production of D-pantothenic acid and its derivatives uses D-pantolactone (D-PL) as the precursor material. Pantolactone exists in three forms due to its chiral carbon atom: DL-pantolactone (racemic pantolactone), D-pantolactone and L-pantolactone , of which only D-pantolactone has biological activity and can be used as an important precursor for the synthesis of D-pantothenic acid and its derivatives.

There are two main methods for the synthesis of D-pantolactone: 1) chemical synthesis method, using isobutyraldehyde-formaldehyde-hydrocyanic acid to obtain DL-pantolactone through a series of steps, and then by enantiomeric excess D-pantolactone is obtained by crystallization or a chiral resolving agent, while L-pantolactone is re-resolved after racemization by extraction. The method involves the use of the toxic compound hydrocyanic acid in the synthetic route, which leads to increased environmental protection pressure on the production enterprise; 2) the microbial hydrolase method is currently the main channel for obtaining D-pantolactone. The method uses chemically synthesized racemic pantolactone as raw material, utilizes lactone hydrolase to selectively hydrolyze D-pantolactone in the racemate, and then separates D-pantoic acid and L-pantolactone. Acid lactone, the separated D-pantoic acid is acidified into a ring to form D-pantoic acid lactone, while L-pantoic acid lactone is repeatedly involved in hydrolysis after racemization. This process does not involve the use of toxic substances, but there are also problems such as repeated extraction racemization and acid-base consumption. Therefore, it is of great research significance to develop a simple, efficient and environmentally friendly asymmetric reduction route.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a plasmid, the plasmid pBAD-DCR is obtained by inserting the DCR gene into the expression vector pBAD; the amino acid sequence of the DCR gene is shown in SEQ ID NO. 2; and it is used for cofactor recycling The plasmid pBAD-GDH is obtained by inserting the GDH gene into the expression vector pBAD; the amino acid sequence of the GDH gene is shown in SEQ ID NO.8.

The invention also provides a recombinant plasmid, including: plasmid pBAD-DCR-GDH, plasmid pBAD-E224G-GDH and plasmid pBAD-E224Q-GDH; the plasmid pBAD-DCR-GDH is cut from the plasmid pBAD by T4 ligase ligase -The DCR fragment after DCR, the GDH fragment after digestion of the plasmid pBAD-GDH of claim 1 and the linear vector fragment after digestion of pBAD are obtained; the plasmid pBAD-E224G-GDH is cut from the plasmid pBAD by T4 ligase ligase -The E224G fragment after E224G, the GDH fragment after the plasmid pBAD-GDH according to claim 1 was digested and the linear vector fragment after the digestion of pBAD were obtained; the plasmid pBAD-E224G was obtained by inserting the E224G gene into the expression vector pBAD; the The amino acid sequence of the E224G gene is shown in SEQ ID NO.4; the plasmid pBAD-E224Q-GDH is cut by the T4 ligase ligase to the E224Q fragment after the plasmid pBAD-E224Q, and the plasmid pBAD of claim 1 is cut by restriction enzymes -The GDH fragment after GDH and the linear vector fragment after digestion of pBAD are obtained; the plasmid pBAD-E224Q is obtained by inserting the E224Q gene into the expression vector pBAD; the amino acid sequence of the E224Q gene is shown in SEQID NO.6.

The present invention further provides an engineering bacterium, the engineering bacterium pBAD-DCR-GDH BW25113 is transformed into the competent cell Escherichia coli BW25113 by transforming the plasmid pBAD-DCR-GDH of claim 2 to obtain the engineering bacterium.

In a specific embodiment of the present invention, the engineered strain pBAD-E224G-GDH BW25113 is transformed from the plasmid pBAD-E224G-GDH described in claim 2 into a competent cell E. coli BW25113 to obtain the engineered strain pBAD-E224G-GDHBW25113.

In a specific embodiment of the present invention, the engineered strain pBAD-E224Q-GDH BW25113 is transformed from the plasmid pBAD-E224Q-GDH described in claim 2 into competent cells E. coli BW25113 to obtain the engineered strain pBAD-E224Q-GDHBW25113.

The invention finally provides the application of engineering bacteria in whole cell catalyzing ketopantolactone to produce D-pantolactone.

The present invention has the following beneficial effects:

1. Using efficient ketopantolactone reductase and coenzyme regeneration system to catalyze the synthesis of D-pantolactone, the substrate concentration is as high as 100g/L, the conversion rate is ≥99.0%, and the weight yield is as high as 95% %. The production cost and product quality are better than those of chemical synthesis and microbial hydrolase, suitable for industrial production;

2. The enzymatic reaction has high selectivity, no by-products, and simple extraction and purification after the product.

Description of drawings

Figure 1. Roadmap for efficient catalytic production of D-pantolactone using mutants of ketopantolactone reductase.

Figure 2. Gas phase analysis of D-pantolactone.

Figure 3. Gas phase analysis of D-pantolactone.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to the scope of the described embodiments.

Ketopantolactone was purchased from Beijing Inokay Technology Co., Ltd.

Escherichia coli BW25113 strain was purchased from Shanghai Zeye Biotechnology Co., Ltd.

The pBAD vector was purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.

Unspecified experimental steps were operated in accordance with the reagent instructions.

Example 1.

Engineering bacteria pBAD-DCR-GDH BW25113 efficiently catalyzes the production of D-pantolactone from ketopantolactone

Add 1.0 g of the prepared engineering bacteria pBAD-DCR-GDH BW25113 wet cell to 50 mM, pH 7.0, 10 mL of PB buffer solution, add 0.5 g of ketopantolactone (KPL), and then add 1 times the amount of KPL The feeding amount of glucose (molar ratio) is used to promote the recycling of cofactors through GDH, and the pH is controlled to react under the conditions of 7.0 and 30 ° C. The process is monitored by gas chromatography until the KPL reaction is complete. After acidification to pH=3.5 or so with 6M HCl, the reaction solution was heated at 80°C for 15 min, 0.5% H ₂ O ₂ was added for decolorization for 30 min, and then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain D-pantolysis. Crude acid lactone 0.526g. It was recrystallized with ethyl acetate/petroleum ether (60-90°C) to obtain a total of 0.483 g of pure product.

Detection and analysis conditions of D-pantolactone: Take 300ul of the above sample solution, add 1/3 volume of 6M HCl for acidification, heat at 80°C for 15 minutes, cool to room temperature and add the same volume of chromatographically pure ethyl acetate to mix Evenly, take the organic phase solution after high-speed centrifugation at 12000rpm for 2min, use an Agilent 7890 gas chromatograph to analyze after drying with anhydrous Na ₂ SO ₄ , the chromatographic column used is an Astec CHIRALDEX B-PN gas capillary chromatographic column, and the nitrogen flow rate is 6.5ml/min, The temperature of the injection port and the detector were both 200°C, the temperature was programmed to be 100°C (maintained for 2 min), and the temperature was 0.5°C/min to 110°C for a total of 12.6 min.

Example 2.

Whole-cell catalyzed production of D-pantolactone by engineering bacteria pBAD-E224G-GDH BW25113

Add 1.0 g of the prepared engineering bacteria pBAD-E224G-GDH BW25113 wet cell to 50 mM, pH 7.0, 10 mL of PB buffer solution, add 0.70 g of ketopantolactone (KPL), and then add 1 times the amount of KPL The feeding amount of glucose (molar ratio) is used to promote the recycling of cofactors through GDH, and the pH is controlled to react under the conditions of 7.0 and 30 ° C. The process is monitored by gas chromatography until the KPL reaction is complete. After acidification to pH=3.5 or so with 6M HCl, the reaction solution was heated at 80°C for 15 min, 0.5% H ₂ O ₂ was added for decolorization for 30 min, and then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain D-pantolysis. Crude acid lactone 0.73g. It was recrystallized with ethyl acetate/petroleum ether (60-90°C) to obtain a total of 0.68 g of pure product.

Example 3.

Whole-cell catalyzed production of D-pantolactone by engineering bacteria pBAD-E224G-GDH BW25113

Add 1.5 g of the prepared engineering bacteria pBAD-E224G-GDH BW25113 wet cell to 50 mM, pH 6.5, 10 mL PB buffer solution, add 1 g of ketopantolactone (KPL), and then add 1 times the amount of KPL feeding Amount of glucose (molar ratio) and promote the recycling of cofactors through GDH, control pH to react under the conditions of 6.5 and 30 °C, and monitor the progress by gas chromatography until the KPL reaction is complete. After acidification to pH=3.5 or so with 6M HCl, the reaction solution was heated at 80°C for 15 min, 0.5% H ₂ O ₂ was added for decolorization for 30 min, and then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain D-pantolysis. Crude acid lactone 1.05g. It was recrystallized with ethyl acetate/petroleum ether (60-90°C) to obtain a total of 0.97 g of pure product.

Example 4.

Whole-cell catalyzed production of D-pantolactone by engineering bacteria pBAD-E224Q-GDH BW25113

Add 1.5 g of the prepared pBAD-E224Q-GDH BW25113 wet cells to 50 mM, pH 7.0, 10 mL of PB buffer solution, add 0.75 g of ketopantolactone (KPL), and then add 1 times the substrate KPL The feeding amount of glucose (molar ratio) is used to promote the recycling of cofactors through GDH, and the pH is controlled to react under the conditions of 7.0 and 30 ° C. The process is monitored by gas chromatography until the KPL reaction is complete. After acidification to pH=3.5 or so with 6M HCl, the reaction solution was heated at 80°C for 15 min, 0.5% H ₂ O ₂ was added for decolorization for 30 min, and then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain D-pantolysis. Crude acid lactone 0.78g. It was recrystallized with ethyl acetate/petroleum ether (60-90°C) to obtain a total of 0.73 g of pure product.

Example 5.

Whole-cell catalyzed production of D-pantolactone by engineering bacteria pBAD-E224Q-GDH BW25113

Add 2.0 g of prepared pBAD-E224Q-GDH BW25113 wet cells to 50 mM, pH 6.5, 10 mL of PB buffer solution, add 0.95 g of ketopantolactone (KPL), and then add 1 times the substrate KPL The feeding amount of glucose (molar ratio) is used to promote the recycling of cofactors through GDH, and the pH is controlled to react under the conditions of 6.5 and 30 ° C. The process is monitored by gas chromatography until the KPL reaction is complete. After acidification to pH=3.5 or so with 6M HCl, the reaction solution was heated at 80°C for 15 min, 0.5% H ₂ O ₂ was added for decolorization for 30 min, and then extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated by distillation to obtain D-pantolysis. Crude acid lactone 0.98g. It was recrystallized with ethyl acetate/petroleum ether (60-90°C) to obtain a total of 0.92 g of pure product.

Example 6

1. Construction of a new recombinant ketopantolactone reductase pBAD-DCR-BW25113 derived from Candida parapsilosis CDC31

The ketopantolactone reductase gene derived from Candida parapsilosis CDC31 was synthesized, abbreviation: DCR, Genebank ID: HE605209.1, the nucleotide sequence of DCR gene is shown in SEQ ID NO.1 and the amino acid The sequence is shown in SEQ ID NO. 2, and the DCR gene is inserted into the NdeI and EcoRI sites of the expression vector pBAD to obtain the plasmid pBAD-DCR. Plasmid pBAD-DCR was subsequently transformed into E. coli host BW25113, resulting in strain pBAD-DCR-BW25113. After overnight culture at 37°C, single colonies were picked and cultured in test tubes containing 5 mL of LB (100 μg/mL ampicillin resistance), and the strains were stored in glycerol tubes. The remaining seed solution was inoculated into 800 mL of LB medium containing 100 μg/mL ampicillin resistance at a ratio of 2%, and cultured at 37 °C and 200 rpm until OD600 = about 0.6. At 30 °C and 200 rpm, a final concentration of 1% L-arabinose was added. Induction culture was performed for 8-10 h, and cells were collected by centrifugation. The cells were suspended in 50 mM phosphate buffer (pH 7.0) at 1/20 the volume of the fermentation broth and sonicated, and the recombinant wild-type DCR crude enzyme solution was obtained after centrifugation.

2. Construction and acquisition of highly active ketopantolactone reductase mutants

Using the molecular docking software LeDock (https://lephar.com), the substrate molecule ketopantolactone KPL was inserted into the active center of DCR for analysis; protein structure comparison, image analysis, processing and other biological information All learning operations are completed by PyMOL software. Molecular docking indicated that Thr27, Lys28, Glu224 and Thr225 residues of ketopantolactone reductase DCR may have potential effects on its reducing activity.

Using the pBAD-DCR plasmid as a template, the saturation mutants of the above amino acid residues were constructed by the QuickChange site-directed saturation mutagenesis method. After the gene sequences of the mutants were verified by sequencing, they were transferred into BW25113. Then, 50 ml of LB liquid medium containing 100 μg/ml ampicillin resistance was added, and after overnight incubation at 37° C., 200 rpm/min, the bacteria were stored in a glycerol tube.

Taking the wild-type pBAD-DCR-BW25113 strain as a control, the remaining mutant seed liquids were inserted into 800 ml LB medium (100 μg/ml ampicillin resistance) according to 2% of the inoculum and continued to be shaken at 37 ° C to OD600 = 0.6 After adding L-arabinose with a final concentration of 1%, the cells were collected by centrifugation after induction and culture at 30°C for 8-10 hours. The cells were suspended in 50 mM phosphate buffer (pH 7.0) at 1/20 the volume of the fermentation broth and sonicated, and the recombinant wild-type DCR crude enzyme solution was obtained after centrifugation.

Add 300 μL of the above lyase solution and 0.078 mol of ketopantolactone to the EP tube, react at pH 7.0 at 30 °C for 5 h, acidify with 6M HCl to pH 3.5, and heat at 80 °C for 15 min, etc. After extraction with volumetric ethyl acetate, it was detected by gas chromatography.

After four rounds of screening, two mutants E224G and E224Q with significantly improved activity after the mutation of the 224th residue were obtained. The nucleotide sequence of the mutant E224G is shown in SEQ ID NO. 3 and the amino acid sequence is shown in SEQ ID NO. 3. As shown in ID NO.4, the nucleotide sequence of the mutant E224Q is shown as SEQ ID NO.5 and the amino acid sequence is shown as SEQ ID NO.6.

Table 1. Comparison of amino acid residue changes and transformation rates of DCR high-efficiency mutants

strain name Amino acid residue changes substrate Conversion rate Wild type (Seq ID NO: 1 and 2) none KPL 25 E224G (Seq ID NO: 3 and 4) Glu→Gly KPL 32.4 E224Q (Seq ID NO: 5 and 6) Glu→Gln KPL 31.5

3. Construction of engineering strain pBAD-DCR-GDHBW25113 co-expressing ketopantolactone reductase DCR and glucose dehydrogenase GDH

Ketopantolactone reductase DCR of Candida parapsilosis CDC31, the nucleotide sequence of the DCR gene is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.

The GDH gene of Bacillus megateriu (ATCC 14581), the nucleotide sequence of the GDH gene is shown in SEQ ID NO.7 and the amino acid sequence is shown in SEQ ID NO.8.

The DCR gene primer pairs were designed: 5'-ctagccatggccactcaaagtaacttactaccaaa-3' and 5'-ccctcgagctacaaatctttaaattgctcatggaa-3'.

GDH gene primer pair: 5'-ccctcgagtctagagaaagaggggacaaactagatgtatacagatttaaaagataaagta-3' and 5'-ggggtaccttagcctcttcctgcttggaaagaagggtacagcgtcataccaccatcagca-3'.

The GDH gene of Bacillus megateriu (ATCC 14581) was inserted into the NdeI and EcoRI sites of the expression vector pBAD to obtain the plasmid pBAD-GDH. The nucleotide sequence of the GDH gene is shown in SEQ ID NO.7 and the amino acid sequence is shown in SEQ ID NO.8.

The plasmids pBAD-DCR and pBAD-GDH were used as templates for PCR amplification, and NcoI, XhoI and XhoI, KpnI were used for digestion, and the two ends of the gene sequence obtained by PCR were respectively cut with NcoI, XhoI and XhoI, KpnI. The PCR product was purified by agarose gel electrophoresis, and the target fragment was recovered using the agarose gel DNA recovery kit. The pBAD vector was digested with NcoI and KpnI, and the DCR fragment, GDH fragment and pBAD vector fragment were ligated with T4 ligase. The ligated product was transformed into DH5α and then coated with ampicillin-resistant plates. The plasmid was extracted by the plasmid extraction kit, and the plasmid with the correct size of the inserted gene band was confirmed by PCR amplification and sent for sequencing. The correctly sequenced plasmid was saved and named pBAD-DCR-GDH. According to the transformation operation process, it was transformed into competent cells E. coli BW25113 , coated with ampicillin-resistant LB solid plate, and cultured overnight at 37°C. Single colonies were picked to a test tube containing 5 mL of LB for 5 hours, and then the glycerol strains were stored to obtain the co-expression engineering strain pBAD-DCR-GDH BW25113.

4. Construction of engineering strain pBAD-E224G-GDH co-expressing mutant E224G and glucose dehydrogenase GDH

The plasmids pBAD-E224G and pBAD-GDH were used as templates for PCR amplification. The primers, restriction sites and operation procedures were the same as the construction of the co-expression recombinant strain pBAD-DCR-GDH BW25113, thereby obtaining the engineering bacteria pBAD-E224G. - GDH BW25113.

5. Construction of engineering strain pBAD-E224Q-GDH co-expressing mutant E224Q and glucose dehydrogenase GDH

The plasmids pBAD-E224Q and pBAD-GDH were used as templates for PCR amplification, and the primers, restriction sites and operation procedures were the same as the construction of the co-expression recombinant strain pBAD-DCR-GDH BW25113, thereby obtaining the engineering bacteria pBAD-E224Q - GDH BW25113.

6. Cultivation of engineering bacteria

1) Engineering bacteria pBAD-DCR-GDH BW25113:

Pipette the strain pBAD-DCR-GDH BW25113 stored in the glycerol strain tube into 5 mL of LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37° C. and 200 rpm for 5 h. Then, it was transferred to 800 mL of LB medium containing 100 μg/mL kanamycin at a ratio of 2%, and cultured at 37° C. and 200 rpm to an OD600 of about 0.6. L-arabinose with a final concentration of 1% was added at 25° C. and 200 rpm for induction and culture for 8-10 h, and the cells were collected by centrifugation.

2) Engineering bacteria pBAD-E224G-GDH BW25113:

Pipette the strain pBAD-E224G-GDH BW25113 stored in a glycerol strain tube into 5 mL of LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37° C. and 200 rpm for 5 h. Then, it was inoculated into 800 mL of LB medium containing 100 μg/mL kanamycin at a ratio of 2%, and cultured at 37° C. and 200 rpm to an OD600 of about 0.6. L-arabinose with a final concentration of 1% was added at 25° C. and 200 rpm for induction and culture for 8-10 h, and the cells were collected by centrifugation.

3) Engineering bacteria pBAD-E224Q-GDH BW25113:

Pipette the strain pBAD-E224Q-GDH BW25113 stored in a glycerol strain tube into 5 mL of LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37° C. and 200 rpm for 5 h. Then, it was inoculated into 800 mL of LB fermentation medium containing 100 μg/mL kanamycin at a ratio of 2%, and cultured at 37° C. and 200 rpm to an OD600 of about 0.6. L-arabinose with a final concentration of 1% was added at 25° C. and 200 rpm for induction and culture for 8-10 h, and the cells were collected by centrifugation.

The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions under the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

sequence listing

<110> Institute of Microbiology, Chinese Academy of Sciences

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Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn

275 280 285

Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe

290 295 300

Lys Asp Leu

305

<210> 5

<211> 924

<212> DNA

<213> Artificial Sequence

<400> 5

atgactcaaa gtaacttact accaaaaaca tttcgtacca aatctgggaa ggagatatca 60

attgcacttg gaacagggac caaatggaaa caagcccaaa caattaacga tgttagtact 120

gagttggtgg acaatatcct tttggggtta aagttgggtt ttagacacat tgatactgct 180

gaagcttaca acacgcaaaa ggaagttggt gaagccctca aaagaaccga tgttccaaga 240

gaggatattt gggttaccac aaaatatagt ccaggttggg gttcaatcaa ggcatacagt 300

aaatcgccaa gtgattcaat tgataaagct ttggcacagc ttggtgttga ctacgttgat 360

ttatttttga ttcactcccc attcttcacc actgagcaaa ctcatggata tacattagag 420

caagcttggg aagctttggt tgaagcaaag aaggcgggaa aggttagaga aattggtatc 480

tcaaatgctg ctattccaca cttggaaaaa ctctttgctg cctccccgag tcctgagtac 540

taccctgttg tcaaccaaat tgaattccat ccattcttgc aaaaccaatc taaaaacatt 600

gttagatttt gtcaagagca tgggatcttg gtcgaagctt tttcgccatt ggcgccattg 660

gcaagagttc agactaatgc tctcgctgag acattaaaga gattggcgga aaagtacaaa 720

aagaccgaag ctcaagtttt attgaggtat actttgcaaa gaggtatttt gccagtgaca 780

acatcgtcaa aggaaagcag attaaaagag tctttgaatt tgtttgactt tgaattgact 840

gacgaggagg ttaatgaaat caacaagatt ggcgatgcta atccctatag agctttcttc 900

catgagcaat ttaaagattt gtag 924

<210> 6

<211> 307

<212> PRT

<213> Artificial Sequence

<400> 6

Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly

1 5 10 15

Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala

20 25 30

Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu

35 40 45

Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn

50 55 60

Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg

65 70 75 80

Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile

85 90 95

Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala

100 105 110

Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe

115 120 125

Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu

130 135 140

Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile

145 150 155 160

Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro

165 170 175

Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe

180 185 190

Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly

195 200 205

Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Gln

210 215 220

Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys

225 230 235 240

Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile

245 250 255

Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu

260 265 270

Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn

275 280 285

Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe

290 295 300

Lys Asp Leu

305

<210> 7

<211> 786

<212> DNA

<213> Bacillus megateriu

<400> 7

atgtatacag atttaaaaga taaagtagtt gtaattacag gtggatcaac aggtttagga 60

cgcgcaatgg ctgttcgttt cggtcaagaa gaagcaaaag ttgttattaa ctattacaac 120

aatgaagaag aagctttaga tgcgaaaaaa gaagtagaag aagcaggcgg acaagcaatc 180

atcgttcaag gcgacgtaac aaaagaagaa gatgttgtaa accttgttca aacagctatt 240

aaagaattcg gtacattaga cgttatgatt aataacgctg gtgttgaaaa cccagttcct 300

tctcatgagt tatctttaga caactggaat aaagtaatcg atacaaactt aacgggcgca 360

tttttaggaa gccgcgaagc gattaaatat tttgttgaaa acgacattaa aggaaacgtt 420

attaacatgt ctagtgttca tgaaatgatt ccttggccat tatttgttca ttacgcagca 480

agtaaaggcg gtatgaaact aatgacgaaa acattggctc ttgaatatgc gccaaaaggt 540

atccgcgtaa ataacattgg accaggtgcg atgaacacac caattaacgc agagaaattt 600

gcagatcctg tacaacgtgc agacgtagaa agcatgattc caatgggtta catcggtaaa 660

ccagaagaag tagcagcagt tgcagcattc ttagcatcat cacaagcaag ctatgtaaca 720

ggtattacat tatttgctga tggtggtatg acgctgtacc cttctttcca agcaggaaga 780

ggctaa 786

<210> 8

<211> 261

<212> PRT

<213> Bacillus megateriu

<400> 8

Met Tyr Thr Asp Leu Lys Asp Lys Val Val Val Ile Thr Gly Gly Ser

1 5 10 15

Thr Gly Leu Gly Arg Ala Met Ala Val Arg Phe Gly Gln Glu Glu Ala

20 25 30

Lys Val Val Ile Asn Tyr Tyr Asn Asn Glu Glu Glu Ala Leu Asp Ala

35 40 45

Lys Lys Glu Val Glu Glu Ala Gly Gly Gln Ala Ile Ile Val Gln Gly

50 55 60

Asp Val Thr Lys Glu Glu Asp Val Val Asn Leu Val Gln Thr Ala Ile

65 70 75 80

Lys Glu Phe Gly Thr Leu Asp Val Met Ile Asn Asn Ala Gly Val Glu

85 90 95

Asn Pro Val Pro Ser His Glu Leu Ser Leu Asp Asn Trp Asn Lys Val

100 105 110

Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Met Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Met Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Val Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Lys Pro Glu Glu Val

210 215 220

Ala Ala Val Ala Ala Phe Leu Ala Ser Ser Gln Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

Claims (7)

1. Plasmid pBAD-GDH, characterized in that, the plasmid pBAD-GDH is obtained by inserting the GDH gene into the expression vector pBAD; the amino acid sequence of the GDH gene is shown in SEQ ID NO.8. 2. a recombinant plasmid, comprising: plasmid pBAD-DCR-GDH, plasmid pBAD-E224G-GDH and plasmid pBAD-E224Q-GDH; Described plasmid pBAD-DCR-GDH is obtained by the DCR fragment after T4 ligase ligase cuts the plasmid pBAD-DCR, the GDH fragment after restriction enzyme cuts the plasmid pBAD-GDH of claim 1 and the linear vector fragment after restriction enzyme cuts pBAD; The plasmid pBAD-DCR is obtained by inserting the DCR gene into the expression vector pBAD; the amino acid sequence of the DCR gene is shown in SEQ ID NO.2; The plasmid pBAD-E224G-GDH is obtained from the E224G fragment after the plasmid pBAD-E224G is cut by T4 ligase ligase, the GDH fragment after the plasmid pBAD-GDH according to claim 1 is cut, and the linear vector fragment after the enzyme cut pBAD; The plasmid pBAD-E224G is obtained by inserting the E224G gene into the expression vector pBAD; the amino acid sequence of the E224G gene is shown in SEQ ID NO.4; The plasmid pBAD-E224Q-GDH is obtained from the E224Q fragment after the T4 ligase ligase cut the plasmid pBAD-E224Q, the GDH fragment after the plasmid pBAD-GDH according to claim 1 is cut, and the linear vector fragment after the enzyme cut pBAD. ; The plasmid pBAD-E224Q is obtained by inserting the E224Q gene into the expression vector pBAD; the amino acid sequence of the E224Q gene is shown in SEQ ID NO.6. 3. Engineering bacteria, characterized in that, the engineering bacteria pBAD-DCR-GDH BW25113 is transformed into competent cells Escherichia coli BW25113 by transforming the plasmid pBAD-DCR-GDH described in claim 2 into the engineering bacteria. 4. Engineering bacteria, characterized in that, the engineering bacteria pBAD-E224G-GDH BW25113 is transformed into competent cells E. coli BW25113 by transforming the plasmid pBAD-E224G-GDH described in claim 2 into the engineering bacteria pBAD-E224G-GDHBW25113. 5. Engineering bacteria, characterized in that, the engineering bacteria pBAD-E224Q-GDH BW25113 is transformed into competent cells Escherichia coli BW25113 by transforming the plasmid pBAD-E224Q-GDH described in claim 2 into the engineering bacteria pBAD-E224Q-GDHBW25113. 6. The application of any one of the engineering bacteria of claims 3 to 5 in the whole-cell catalysis of ketopantolactone to produce D-pantolactone. 7. according to the application of engineering bacteria described in claim 6 in whole cell catalysis ketopantolactone to produce D-pantolactone, it is characterized in that, described engineering bacteria and ketopantolactone add The mass ratio of quantity is 2-4:1-2.

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