Engineering bacteria and their application in whole-cell catalysis of ketopantolactone to produce D-pantolactone

Abstract

The present invention belongs to the field of biocatalysis technology. The present invention discloses an engineering bacterium and its application in whole-cell catalysis of ketopantoic acid lactone to produce D-pantoic acid lactone. The engineering bacterium of the present invention realizes the catalytic synthesis of D-pantoic acid lactone by ketopantoic acid lactone reductase mutant in a coenzyme regeneration system, with a substrate concentration of up to 100g / L, a conversion rate of ≥99.0%, and a weight yield of up to 95%.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis. Background Art

[0002] Pantothenic acid, also known as pantothenic acid, vitamin B 5 , widely present in the biological world, is a precursor for the synthesis of coenzyme A in animals, and 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 at high temperature and acid and alkali conditions. Therefore, compared with D-pantothenic acid, its derivatives with better stability have been more widely used in food, medicine, feed and other industries. Currently, the widely used pantothenic acid derivatives mainly include D-calcium pantothenate, D-panthenol and D-pantethine, among which D-calcium pantothenate is the main one.

[0003] The industrial production of D-pantolic acid and its derivatives uses D-pantolactone (D-PL) as a precursor. Pantolactone has three forms due to its chiral carbon atom: DL-pantolactone (racemic pantolactone), D-pantolactone and L-pantolactone. Among them, only D-pantolactone has biological activity and can be used as an important precursor for the synthesis of D-pantolic acid and its derivatives.

[0004] There are two main methods for the synthesis of D-pantoic acid lactone: 1) Chemical synthesis method, using isobutyraldehyde-formaldehyde-hydrocyanic acid through a series of steps to obtain DL-pantoic acid lactone, and then obtaining D-pantoic acid lactone through enantiomeric excess crystallization method or chiral resolving agent, while L-pantoic acid lactone is extracted and racemized and then split again. This method involves the use of toxic compound hydrocyanic acid in the synthesis route, which leads to increased environmental pressure on production enterprises; 2) Microbial hydrolase method, which is currently the main channel for obtaining D-pantoic acid lactone. This method uses chemically synthesized racemic pantoic acid lactone as raw material, uses lactone hydrolase to selectively hydrolyze D-pantoic acid lactone in the racemate, and then separates D-pantoic acid and L-pantoic acid lactone. The separated D-pantoic acid is acidified to form D-pantoic acid lactone, and L-pantoic acid lactone is repeatedly hydrolyzed after racemization. This process does not involve the use of toxic substances, but there are also problems such as repeated extraction and racemization and high acid and alkali consumption. Therefore, it is of great research significance to develop asymmetric reduction routes that are simple, efficient and environmentally friendly. Summary of the invention

[0005] In view of this, the present invention provides a plasmid, wherein 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 a plasmid pBAD-GDH for cofactor cycle, wherein 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.

[0006] The present 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 obtained by ligating the DCR fragment after enzyme cutting of plasmid pBAD-DCR with T4 ligase, the GDH fragment after enzyme cutting of plasmid pBAD-GDH with enzyme cutting and the linear vector fragment after enzyme cutting of pBAD with T4 ligase; the plasmid pBAD-E224G-GDH is obtained by ligating the E224G fragment after enzyme cutting of plasmid pBAD-E224G with T4 ligase, the GDH fragment after enzyme cutting of plasmid pBAD-GDH with enzyme cutting and the linear vector fragment after enzyme cutting of pBAD with T4 ligase; 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 as shown in SEQ ID NO.4; the plasmid pBAD-E224Q-GDH is obtained by ligating the E224Q fragment after enzyme digestion of plasmid pBAD-E224Q with T4 ligase, the GDH fragment after enzyme digestion of the plasmid pBAD-GDH, and the linear vector fragment after enzyme digestion of 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.

[0007] The present invention also provides an engineering bacterium. The engineering bacterium pBAD-DCR-GDH BW25113 is obtained by transferring the plasmid pBAD-DCR-GDH into competent cell Escherichia coli BW25113.

[0008] In a specific embodiment of the present invention, the engineered bacteria pBAD-E224G-GDH BW25113 is obtained by transforming the plasmid pBAD-E224G-GDH into competent cells of Escherichia coli BW25113 to obtain the engineered bacteria pBAD-E224G-GDHBW25113.

[0009] In a specific embodiment of the present invention, the engineered bacteria pBAD-E224Q-GDH BW25113 is obtained by transforming the plasmid pBAD-E224Q-GDH into competent cells of Escherichia coli BW25113 to obtain the engineered bacteria pBAD-E224Q-GDHBW25113.

[0010] Finally, the present invention provides the application of the engineering bacteria in whole-cell catalysis of ketopantolactone to produce D-pantolactone.

[0011] The present invention has the following beneficial effects:

[0012] 1. Use efficient ketopantolactone reductase and coenzyme regeneration system to catalyze the synthesis of D-pantolactone, with a substrate concentration of up to 100g / L, a conversion rate of ≥99.0%, and a weight yield of up to 95%. The production cost and product quality are better than chemical synthesis and microbial hydrolase methods, and are suitable for industrial production;

[0013] 2. The enzymatic reaction has high selectivity, no by-products, and simple post-extraction and purification of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .Route map for efficient catalytic production of D-pantolactone using ketopantolactone reductase mutants.

[0015] Figure 2 .Gas phase analysis of D-pantolactone.

[0016] Figure 3 .Gas phase analysis of D-pantolactone. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with the embodiments, but the present invention is not limited to the scope of the embodiments.

[0018] Ketopantolactone was purchased from Beijing Inokai Technology Co., Ltd.

[0019] Escherichia coli BW25113 strain was purchased from Shanghai Zeye Biotechnology Co., Ltd.

[0020] The pBAD vector was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0021] All experimental steps not otherwise specified were performed according to the reagent instructions.

[0022] Example 1.

[0023] The engineered bacterium pBAD-DCR-GDH BW25113 efficiently catalyzes the production of D-pantolactone from ketopantolactone

[0024] 1.0 g of the prepared engineered bacteria pBAD-DCR-GDH BW25113 wet cells were added to 10 mL of 50 mM, pH 7.0 PB buffer solution, and 0.5 g of ketopantolactone (KPL) was added. Then, glucose (molar ratio) of 1 times the amount of KPL was added and the cofactor was circulated and regenerated through GDH. The reaction was controlled at pH 7.0 and 30°C. The process was monitored by gas chromatography until the KPL reaction was complete. Then, 6 M HCl was used to acidify to pH = 3.5, and the reaction solution was heated at 80°C for 15 min. 0.5% H 2 O 2 After decolorization for 30 minutes, the mixture was extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and concentrated by distillation to obtain 0.526 g of crude D-pantoic acid lactone, which was then recrystallized from ethyl acetate / petroleum ether (60-90°C) to obtain a total of 0.483 g of pure product.

[0025] Detection and analysis conditions of D-pantolactone: Take 300ul of the above sample solution, add 1 / 3 volume of 6M HCl to acidify, heat at 80℃ for 15 minutes, cool to room temperature, add an equal volume of chromatographic pure ethyl acetate and mix, centrifuge at 12000rpm for 2min, take the organic phase solution, anhydrous Na 2 SO 4 After drying, an Agilent 7890 gas chromatograph was used for analysis. The chromatographic column used was an Astec CHIRALDEX B-PN gas capillary column. The nitrogen flow rate was 6.5 ml / min, the injection port and detector temperatures were both 200°C, the program temperature was 100°C (held for 2 min), 0.5°C / min to 110°C, and the total operation time was 12.6 min.

[0026] Example 2.

[0027] Production of D-pantolactone from ketopantolactone catalyzed by whole cells of engineered bacteria pBAD-E224G-GDH BW25113

[0028] 1.0 g of the prepared engineered bacteria pBAD-E224G-GDH BW25113 wet cells were added to 50 mM, pH 7.0, 10 mL of PB buffer solution, and 0.70 g of ketopantolactone (KPL) was added. Then, glucose (molar ratio) of 1 times the amount of KPL was added and the cofactor was circulated and regenerated through GDH. The pH was controlled at 7.0 and 30°C to react. The process was monitored by gas chromatography until the KPL reaction was complete. Then, 6 M HCl was used to acidify to pH = 3.5, and the reaction solution was heated at 80°C for 15 min. 0.5% HCl was added. 2 O 2After decolorization for 30 minutes, the mixture was extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and concentrated by distillation to obtain 0.73 g of crude D-pantoic acid lactone, which was then recrystallized from ethyl acetate / petroleum ether (60-90°C) to obtain a total of 0.68 g of pure product.

[0029] Example 3.

[0030] Production of D-pantolactone from ketopantolactone catalyzed by whole cells of engineered bacteria pBAD-E224G-GDH BW25113

[0031] 1.5 g of the prepared engineered bacteria pBAD-E224G-GDH BW25113 wet cells were added to 10 mL of 50 mM, pH 6.5 PB buffer solution, and 1 g of ketopantolactone (KPL) was added. Then, glucose (molar ratio) of 1 times the amount of KPL was added and the cofactor cycle was promoted by GDH. The reaction was controlled at pH 6.5 and 30°C. The process was monitored by gas chromatography until the KPL reaction was complete. Then, 6 M HCl was used to acidify to pH = 3.5, and the reaction solution was heated at 80°C for 15 min. 0.5% H 2 O 2 After decolorization for 30 minutes, the mixture was extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and concentrated by distillation to obtain 1.05 g of crude D-pantoic acid lactone, which was then recrystallized from ethyl acetate / petroleum ether (60-90°C) to obtain a total of 0.97 g of pure product.

[0032] Example 4.

[0033] Production of D-pantolactone from ketopantolactone catalyzed by whole cells of engineered bacteria pBAD-E224Q-GDH BW25113

[0034] 1.5 g of the prepared pBAD-E224Q-GDH BW25113 wet cells were added to 10 mL of 50 mM, pH 7.0 PB buffer solution, and 0.75 g of ketopantolactone (KPL) was added. Then, glucose (molar ratio) was added at 1 times the amount of substrate KPL and the cofactor was circulated and regenerated through GDH. The reaction was controlled at pH 7.0 and 30°C. The process was monitored by gas chromatography until the KPL reaction was complete. Then, 6 M HCl was used to acidify to pH = 3.5, and the reaction solution was heated at 80°C for 15 min. 0.5% H 2 O 2 After decolorization for 30 minutes, the mixture was extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and concentrated by distillation to obtain 0.78 g of crude D-pantoic acid lactone, which was then recrystallized from ethyl acetate / petroleum ether (60-90°C) to obtain a total of 0.73 g of pure product.

[0035] Example 5.

[0036] Production of D-pantolactone from ketopantolactone catalyzed by whole cells of engineered bacteria pBAD-E224Q-GDH BW25113

[0037] 2.0 g of the prepared pBAD-E224Q-GDH BW25113 wet cells were added to 10 mL of 50 mM, pH 6.5 PB buffer solution, 0.95 g of ketopantolactone (KPL), and then glucose (molar ratio) was added at 1 times the amount of substrate KPL, and the cofactor cycle was promoted by GDH. The reaction was controlled at pH 6.5 and 30 ° C. The process was monitored by gas chromatography until the KPL reaction was complete. Then, 6 M HCl was used to acidify to pH = 3.5, and the reaction solution was heated at 80 ° C for 15 min. 0.5% H 2 O 2 After decolorization for 30 minutes, the mixture was extracted three times with an equal volume of ethyl acetate, and the organic phases were combined and concentrated by distillation to obtain 0.98 g of crude D-pantoic acid lactone, which was then recrystallized from ethyl acetate / petroleum ether (60-90°C) to obtain a total of 0.92 g of pure product.

[0038] Example 6

[0039] 1. Construction of a new recombinant ketopantolactone reductase pBAD-DCR-BW25113 from Candida parapsilosis CDC31

[0040] The ketopantoic acid lactone reductase gene derived from Candida parapsilosis CDC31 was synthesized, referred to as DCR, Genebank ID: HE605209.1, 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, and the DCR gene is inserted into the NdeI and EcoRI sites of the expression vector pBAD to obtain the plasmid pBAD-DCR. The plasmid pBAD-DCR was then transformed into the Escherichia coli host BW25113 to obtain the pBAD-DCR-BW25113 strain. After overnight culture at 37°C, a single colony was picked and cultured in a test tube containing 5mL LB (100μg / mL ampicillin resistance), and the strain was stored in a glycerol tube. The remaining seed liquid 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, and L-arabinose with a final concentration of 1% was added at 30°C and 200 rpm for induction culture for 8-10 hours, and the cells were collected by centrifugation. The cells were suspended in 50 mM phosphate buffer (pH 7.0) with a volume of 1 / 20 of the fermentation liquid and ultrasonically disrupted, and the recombinant wild-type DCR crude enzyme solution was obtained after centrifugation.

[0041] 2. Construction and acquisition of highly active ketopantolactone reductase mutants

[0042] The molecular docking software LeDock (https: / / lephar.com) was used to insert the substrate molecule ketopantolactone KPL into the active center of DCR for analysis; protein structure alignment and image analysis and processing and other bioinformatics operations were completed using PyMOL software. Molecular docking showed that the Thr27, Lys28, Glu224 and Thr225 residues of ketopantolactone reductase DCR may have potential effects on its reducing activity.

[0043] Using the pBAD-DCR plasmid as a template, the QuickChange site-directed saturation mutagenesis method was used to construct saturation mutants of the above amino acid residues. After the gene sequences of each mutant were verified to be correct by sequencing, they were transferred into BW25113. Then 50 ml of LB liquid medium containing 100 μg / ml ampicillin resistance was inoculated, and after overnight culture at 37°C and 200 rpm / min, the strains were preserved in glycerol tubes.

[0044] The wild-type pBAD-DCR-BW25113 strain was used as a control, and the remaining mutant seed liquid was inoculated into 800 ml LB medium (100 μg / ml ampicillin resistance) at a 2% inoculation amount and continued to be shaken and cultured at 37°C until OD600 = about 0.6, and then L-arabinose with a final concentration of 1% was added, and the cells were centrifuged and collected after induction culture at 30°C for 8-10 hours. The cells were suspended in 50 mM phosphate buffer (pH 7.0) with a volume of 1 / 20 of the fermentation liquid and ultrasonically disrupted, and the recombinant wild-type DCR crude enzyme solution was obtained after centrifugation.

[0045] Add 300 μL of the above lysing enzyme solution and 0.078 mol of ketopantolactone to an EP tube, react at pH 7.0 and 30°C for 5 h, acidify to pH 3.5 with 6 M HCl, heat at 80°C for 15 min, extract with an equal volume of ethyl acetate, and detect by gas chromatography.

[0046] After four rounds of screening, two mutants E224G and E224Q were obtained, whose activities were significantly improved after the mutation of the residue at position 224. 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.4, and the nucleotide sequence of the mutant E224Q is shown in SEQ ID NO.5 and the amino acid sequence is shown in SEQ ID NO.6.

[0047] Table 1. Comparison of amino acid residue changes and conversion rates of DCR efficient mutants

[0048] Strain name Amino acid residue changes Substrate Conversion rate Wild type (Seq ID NO:1and 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

[0049] 3. Construction of the engineering bacterium pBAD-DCR-GDHBW25113 for co-expression of ketopantolactone reductase DCR and glucose dehydrogenase GDH

[0050] The ketopantolactone reductase DCR of Candida parapsilosis CDC31, the nucleotide sequence of the DCR gene is shown as SEQ ID NO.1 and the amino acid sequence is shown as SEQ ID NO.2.

[0051] The GDH gene of Bacillus megateriu (ATCC 14581), the nucleotide sequence of the GDH gene is shown as SEQ ID NO.7 and the amino acid sequence is shown as SEQ ID NO.8.

[0052] Design the primer pair for DCR gene: 5'-ctagccatggccactcaaagtaacttactaccaaa-3' and 5'-ccctcgagctacaaatctttaaattgctcatggaa-3'.

[0053] Primer pair for GDH gene: 5'-ccctcgagtctagagaaagaggggacaaactagatgtatacagatttaaaagataaagta-3' and 5'-ggggtaccttagcctcttcctgcttggaaagaagggtacagcgtcataccaccatcagca-3'.

[0054] 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.

[0055] PCR amplification was performed using plasmids pBAD-DCR and pBAD-GDH as templates, respectively. The products were digested with NcoI, XhoI and XhoI, KpnI. The gene sequences obtained by PCR had two restriction sites, NcoI, XhoI and XhoI, KpnI, at both ends. The PCR products were purified by agarose gel electrophoresis, and the target fragments were recovered using an 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 connected with T4 ligase. The ligation product was transformed into DH5α and then coated with ampicillin-resistant plates. After overnight culture, a single colony was picked and cultured in a test tube containing 5 mL LB. The plasmid was extracted with a 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 chemical transformation operation process, it was transferred into competent cells Escherichia coli BW25113, coated with ampicillin-resistant LB solid plates, and cultured at 37 degrees overnight. A single colony was picked and cultured in a test tube containing 5 mL LB for 5 hours, and the glycerol strain was saved to obtain the co-expression engineering bacteria pBAD-DCR-GDH BW25113.

[0056] 4. Construction of the engineering bacterium pBAD-E224G-GDH for co-expression of mutant E224G and glucose dehydrogenase GDH

[0057] PCR amplification was performed using plasmids pBAD-E224G and pBAD-GDH as templates, respectively. The primers, restriction sites and operation procedures used were consistent with the construction of the co-expression recombinant strain pBAD-DCR-GDH BW25113, thereby obtaining the engineered bacteria pBAD-E224G-GDH BW25113.

[0058] 5. Construction of the engineering bacterium pBAD-E224Q-GDH for co-expression of mutant E224Q and glucose dehydrogenase GDH

[0059] PCR amplification was performed using plasmids pBAD-E224Q and pBAD-GDH as templates, respectively. The primers, restriction sites and operation procedures used were consistent with the construction of the co-expression recombinant strain pBAD-DCR-GDH BW25113, thereby obtaining the engineered bacteria pBAD-E224Q-GDH BW25113.

[0060] 6. Cultivation of engineered bacteria

[0061] 1) Engineering bacteria pBAD-DCR-GDH BW25113:

[0062] The strain pBAD-DCR-GDH BW25113 stored in the glycerol tube was taken into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm for 5 hours. Then, it was transferred into 800 mL of LB medium containing 100 μg / mL kanamycin at a ratio of 2%, and cultured at 37°C and 200 rpm until OD600 was about 0.6. L-arabinose with a final concentration of 1% was added for induction culture at 25°C and 200 rpm for 8-10 hours, and the cells were collected by centrifugation.

[0063] 2) Engineering bacteria pBAD-E224G-GDH BW25113:

[0064] The strain pBAD-E224G-GDH BW25113 stored in the glycerol tube was taken out and added to 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm for 5 hours. 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 until OD600 was about 0.6. L-arabinose with a final concentration of 1% was added for induction culture at 25°C and 200 rpm for 8-10 hours, and the cells were collected by centrifugation.

[0065] 3) Engineering bacteria pBAD-E224Q-GDH BW25113:

[0066] The strain pBAD-E224Q-GDH BW25113 stored in the glycerol tube was taken out and added to 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm for 5 hours. 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 until OD600 was about 0.6. L-arabinose with a final concentration of 1% was added for induction culture at 25°C and 200 rpm for 8-10 hours, and the cells were collected by centrifugation.

[0067] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, 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 <120> Engineered bacteria and their application in whole-cell catalysis of ketopantolactone to produce D-pantolactone <130> IM2022057I <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 924 <212> DNA <213> Candida parapsilosis (Candida parapsilosis) <400> 1 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 aaatatagt ccaggttggg gttcaatcaa ggcatacagt 300 aaatcgccaa gtgattcaat tgataaagct ttggcacagc ttggtgttga ctacgttgat 360 ttattttga ttcactcccc attcttcacc actgagcaaa ctcatggata tacattagag 420 caagcttggg aagctttggt tgaagcaaag aaggcgggaa aggttagaga aattggtatc 480 tcaaatgctg ctattccaca cttggaaaa ctctttgctg cctccccgag tcctgagtac 540 taccctgttg tcaaccaaat tgaattccat ccattcttgc aaaaccaatc taaaacatt 600 gttagattt gtcaagagca tgggatcttg gtcgaagctt ttcgccatt ggcgccattg 660 gcaagagttg aaactaatgc 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> 2 <211> 307 <212> PRT <213> Candida parapsilosis (Candida parapsilosis) <400> 2 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 Glu 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> 3 <211> 924 <212> DNA <213> Artificial Sequence <400> 3 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 gcaagagttg gtactaatgc tctcgctgag acattaaaga gattggcgga aaagtacaaa 720 aagaccgaag ctcaagttt attgaggtat actttgcaaa gaggtatttt gccagtgaca 780 acatcgtcaa aggaaagcag attaaaagag tctttgaatt tgtttgactt tgaattgact 840 gacgaggagg ttaatgaaat caacaagatt ggcgatgcta atccctatag agctttcttc 900 catgagcaat ttaaagattt gtag 924 <210> 4 <211> 307 <212> PRT <213> Artificial Sequence <400> 4 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 Gly 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> 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 to Leu to Glu Thr to Lys Arg to Glu to Lys Tyr Lys 225 230 235 240 Lys Thr Glu Ala Gln Val To Arg Tyr Thr To 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 Lion 305 <210> 7 <211> 786 <212> DNA <213> Bacillus megaterium <400> 7 atgtatacag atttaaaaga taaagtagtt gtattacag gtggatcaac aggtttagga 60 cgcgcaatgg ctgttcgttt cggtcaagaa gagcaaag ttgttattaa ctattacaac 120 aatgagaag aagctttaga tgcgaaaaaa gaagtagaag aagcaggcgg acaagcaatc 180 atcgttcaag gcgacgtaac aaagaagaa gatgttgtaa accttgttca aacagctatt 240 aaagaattcg gtacattaga cgttatgatt aataacgctg gtgttgaaaa cccagttcct 300 tctcatgagt tatctttaga caactggaat aaagtaatcg atacaaactt aacgggcgca 360 ttttaggaa 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

1. An engineered bacterium, characterized in that: The engineered bacteria pBAD-E224G-GDH BW25113 was transformed into competent cells of Escherichia coli BW25113 by plasmid pBAD-E224G-GDH to obtain the engineered bacteria pBAD-E224G-GDH BW25113; The plasmid pBAD-E224G-GDH is obtained by ligating the E224G fragment after restriction digestion of the plasmid pBAD-E224G, the GDH fragment after restriction digestion of the plasmid pBAD-GDH, and the linear vector fragment after restriction digestion of pBAD with T4 ligase; 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-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. Use of the engineered bacteria according to claim 1 in whole-cell catalysis of ketopantolactone to produce D-pantolactone.

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