A mutant of meso-diaminopimelate dehydrogenase from Proteus vulgaris and its application

Abstract

The present invention discloses a meso-diaminopimelate dehydrogenase mutant of Proteus vulgaris and its application, belonging to the technical field of bioengineering. The present invention constructs a PvDAPDH mutant, which, compared with the wild enzyme, can catalyze the synthesis of D-amino acids from a variety of aromatic α-ketoacids, and the stereoselectivity can reach 95%. The triple mutant W121I / H227I / R181S of pvDAPDH constructed in the present invention shows good compatibility with different large-volume aromatic α-ketoacid substrates, can simultaneously catalyze the conversion of most aromatic α-ketoacid substrates into D-amino acids, shows high catalytic activity towards 2-oxo-4-phenylbutyric acid, and the yield of the product D-homophenylalanine reaches 2.1 g / L. The present invention effectively expands the toolbox for the biosynthesis of aromatic D-amino acids.

Description

Technical Field

[0001] The present invention relates to a meso-diaminopimelate dehydrogenase mutant of Proteus vulgaris and its application, belonging to the technical field of bioengineering. Background Art

[0002] As an important class of organic small molecules, D-amino acids are key intermediates constituting important components of various important pharmaceuticals and pesticides, and have a wide range of applications in the fields of medicine, pesticides, food, fine chemicals, etc. For example, D-2,4,5-trifluorophenylacetic acid is a precursor substance for the diabetes drug sitagliptin.

[0003] The methods for asymmetric synthesis of D-amino acids mainly include biological methods and non-biological methods. Among them, non-biological asymmetric catalysis for C-N bond synthesis of chiral amines has been widely used in industry. For example, the use of transition metal complexes (such as Ru, Pd, Pt) or inorganic metal catalysts (such as Fe, Cu, Co) can catalyze the asymmetric synthesis of D-amino acids from α-keto acids and ammonia. However, due to the relatively harsh and complex catalytic reaction conditions, the toxic waste generated during the synthesis process is likely to cause environmental pollution and safety problems. At present, enzyme-mediated chemo-biosynthesis is considered a reliable alternative to traditional synthesis because the enzyme-catalyzed process has mild reaction conditions, high stereoselectivity, regioselectivity, and environmental friendliness, and the organic synthesis route is shorter. The biological method for asymmetric synthesis of D-amino acids shows considerable application advantages compared with non-biological catalysis.

[0004] The biological methods for synthesizing D-amino acids mainly include the hydantoinase method, the transaminase method, and the asymmetric resolution method. However, these methods have a long conversion process and low production efficiency, and cannot meet industrial applications. Preparing D-phenylalanine by the keto acid reductive amination method has the advantages of simple production process, mild process conditions, short time consumption, high production rate, and can reduce environmental and resource pressure. Therefore, there is an urgent need for a biological method for efficiently preparing D-phenylalanine. Since NAD(P)H-dependent meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16) can catalyze the asymmetric reductive amination of α-keto acids and ammonia donors to synthesize D-amino acids in one step, and the theoretical maximum yield (Yield) and product optical purity (e.e.) of the reaction can both reach 100%, it has become one of the key methods for synthesizing D-amino acids. However, meso-DAPDH has low activity towards non-natural substrates, especially has no catalytic activity towards large-volume aromatic α-keto acid substrates.

[0005] In recent decades, protein engineering has become an effective directed evolution strategy for improving enzyme properties at the molecular level. Rational directed evolution by combining the crystal structure of enzyme molecules and their catalytic mechanisms is the most effective method to effectively expand the catalytic range of enzymes for substrates, improve enzyme activity, and enhance enzyme stability. Currently, the modified meso-DAPDH has good catalytic activity towards aliphatic α-keto acids, but has low or no catalytic activity towards most aromatic α-keto acids. Summary of the Invention

[0006] In view of the narrow catalytic substrate spectrum of meso-DAPDH and its lack of catalytic activity towards most aromatic α-keto acids, the present invention provides a PvDAPDH mutant that can expand the asymmetric synthesis of D-amino acids from aromatic α-keto acids and ammonia donors, showing catalytic activity towards a variety of difficult-to-catalyze or non-catalyze aromatic α-keto acids, and expanding the substrate spectrum range for the synthesis of D-amino acids.

[0007] The present invention provides a meso-diaminopimelic acid dehydrogenase PvDAPDH mutant, in which one or more amino acids at positions 121, 171, 181, and 227 of PvDAPDH are mutated on the basis of the parental sequence shown in SEQ ID NO.1.

[0008] In one embodiment, the nucleic acid sequence encoding the PvDAPD gene is as shown in SEQ ID NO.2.

[0009] In one embodiment, relative to the PvDAPDH parent, the tryptophan at position 121 is mutated to isoleucine or glycine in the mutant to obtain mutants W121I or W121G.

[0010] In one embodiment, relative to the PvDAPDH parent, the arginine at position 181 is mutated to serine or methionine or alanine in the mutant to obtain mutants W181S or W181M or W181A.

[0011] In one embodiment, relative to the PvDAPDH parent, the histidine at position 227 is mutated to glycine, isoleucine, lysine, or threonine in the mutant to obtain mutants H227G, H227I, H227L, or H227T.

[0012] In one embodiment, relative to the PvDAPDH parent, the tryptophan at position 121 is mutated to isoleucine and the histidine at position 227 is mutated to isoleucine in the mutant to obtain mutant W121I / H227I.

[0013] In one embodiment, relative to the PvDAPDH parent, the mutant has the tryptophan at position 121 mutated to isoleucine, the threonine at position 171 mutated to leucine, and the histidine at position 227 mutated to isoleucine, to obtain the mutant W121I / H227I / T171L.

[0014] In one embodiment, relative to the PvDAPDH parent, the mutant has the tryptophan at position 121 mutated to isoleucine, the arginine at position 181 mutated to isoleucine, and the histidine at position 227 mutated to serine, to obtain the mutant W121I / H227I / R181S.

[0015] In one embodiment, relative to the PvDAPDH parent, the mutant has the tryptophan at position 121 mutated to isoleucine, the arginine at position 180 mutated to alanine, and the histidine at position 227 mutated to serine, to obtain the mutant W121I / H227I / R180A.

[0016] In one embodiment, relative to the PvDAPDH parent, the mutant has the tryptophan at position 121 mutated to isoleucine, the arginine at position 181 mutated to methionine, and the histidine at position 227 mutated to serine, to obtain the mutant W121I / H227I / R181M.

[0017] In one embodiment, relative to the PvDAPDH parent, the mutant has the tryptophan at position 121 mutated to isoleucine, the arginine at position 181 mutated to serine, and the histidine at position 227 mutated to serine, to obtain the mutant W121I / H227I / R181S.

[0018] The present invention provides a method for obtaining the PvDAPDH mutant, the method comprising the following steps:

[0019] (1) Determine the mutation sites based on the PvDAPDH amino acid sequence; design primers for site-directed mutagenesis, using the vector carrying the PvDAPDH gene as the template for gene site-directed mutagenesis; construct a plasmid vector containing the mutant;

[0020] (2) Transform the mutant plasmid into a host cell;

[0021] (3) Select positive monoclonal bodies for fermentation culture, induce the expression of the meso-diaminopimelic acid dehydrogenase mutant PvDAPDH by IPTG for 12 - 15 h, and collect the cells by centrifugation at 4°C and 12,000 rpm.

[0022] In one embodiment, the host is Escherichia coli BL21(DE3) as the starting strain.

[0023] In one embodiment, the PvDAPDH enzyme is expressed in Escherichia coli BL21(DE3) using pET28a as the expression vector.

[0024] The present invention also provides recombinant microbial cells expressing the mutant.

[0025] The present invention also provides the application of the mutant in catalyzing the synthesis of D-amino acids from aromatic α-keto acids.

[0026] In one embodiment, the application includes, but is not limited to, catalyzing the formation of 4-nitro-D-phenylalanine from 4-nitrophenyl-pyruvic acid; catalyzing the formation of D-tryptophan from 3-(3-indolyl)-2-oxopropionic acid; catalyzing the formation of (R)-2-amino-3-(3-hydroxyphenyl)propionic acid from m-hydroxy-2-oxopropionic acid; catalyzing the formation of 3-hydroxy-D-tyrosine from 3,4-dihydroxybenzene pyruvic acid; catalyzing the formation of D-p-hydroxyphenylglycine (D-Hpg) from 4-hydroxyphenylglyoxylic acid (HBF); catalyzing the formation of D-tyrosine (D-Tyr) from 4-hydroxyphenylpyruvic acid (HPPA); catalyzing the formation of D-phenylglycine from benzoylformic acid; catalyzing the formation of D-phenylalanine from phenylpyruvic acid; catalyzing the formation of D-3-(2-naphthyl)-alanine from 3-(naphthalen-2-yl)-2-oxopropionic acid.

[0027] In one embodiment, the aqueous phase of the catalytic reaction system is Tris-hydrochloric acid buffer.

[0028] In one embodiment, the pH of the reaction system is 8.0 - 9.5.

[0029] In one embodiment, the reaction temperature is 30 - 37 °C.

[0030] The present invention also provides the application of the PvDAPDH mutant in the production of aromatic amino acid intermediates in the fields of medicine, pesticides, food, or fine chemicals.

[0031] Beneficial effects: The present invention constructs a PvDAPDH mutant, which can catalyze the synthesis of D-amino acids from a variety of aromatic α-keto acids, with a stereoselectivity of up to 95%, effectively expanding the biological method for synthesizing aromatic D-amino acids toolbox. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 For PvDAPDH M1 Conversion effect on the aromatic substrate spectrum.

[0033] Figure 2 For PvDAPDH M2 Conversion effect on the aromatic substrate spectrum.

[0034] Figure 3 For PvDAPDH M3 The conversion effect on the aromatic substrate spectrum. Detailed implementation manners

[0035] (1) Enzyme activity assay method:

[0036] The enzyme activity of pvDAPDH was determined by spectrophotometry. One unit of the enzyme activity of pvDAPDH is defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute (U).

[0037] (2) The specific enzyme activity is defined as the number of enzyme activity units per milligram of protein (U / mg protein).

[0038]

[0039] (2) Determination of the content of D-AA products by HPLC method:

[0040] Sample treatment: The reaction of the conversion solution was terminated with 1 m NaOH solution, and the termination solution was diluted with ultrapure water to 5 times the initial volume. The diluted conversion solution was centrifuged at 12000 rpm for 10 min, and then the centrifuged supernatant was filtered through a 0.22-μm filter membrane. The filtrate was used for HPLC detection and analysis.

[0041] Mobile phase preparation: An aqueous perchloric acid solution with pH 1.5 and chromatographically pure acetonitrile were evenly mixed at a ratio of 8:2 (v / v), filtered through a 0.22-μm organic filter membrane by suction filtration, and then placed in an ultrasonic bath for ultrasonic degassing for 30 min.

[0042] (3) HPLC method for D-AA detection conditions: Dionex high-performance liquid chromatograph (equipped with an ultraviolet-visible light detector), using a Daicel CrownPak CR(+) column (4.6 × 150 mm, 5 μm) chromatographic column. The mobile phase was perchloric acid:acetonitrile = 8:2 (v / v); the flow rate was 0.2 mL / min; the column temperature of the chromatographic column was 25 °C; the detection wavelength was 200 - 230 nm.

[0043] Determination of the enantiomeric excess percentage (e.e.%) of D-AA by HPLC method

[0044] Using the above method (2) to separately determine the contents of [R] and [S] in the product, the calculation formula for the e.e.% value is as follows:

[0045] e.e.% = ([R] - [S] / [R] + [S]) * 100%.

[0046] Example 1: Construction and screening of single mutants

[0047] Method for constructing single mutants: Design PvDAPDHW121 , PvDAPDH H227 and PvDAPDH R181 Primers for the mutation sites, and the amino acid residues at the above sites were respectively mutated into nine small-volume amino acid residues (GAVLIMCST), and mutants were constructed by whole plasmid PCR.

[0048] PCR amplification system for the construction reaction: 0.5 μL of PrimSTAR enzyme, 10 μL of 5×PrimeSTAR Buffer, 4 μL of dNTP, 1 μL of each of the two primers for each mutation site, 4 μL of template (PvDAPDH WT ), 32.5 μL of water; the reaction conditions were: ① 94°C for 3 min; ② 98°C for 10 s; ③ 55°C for 30 s; ④ 72°C for 3 min; ⑤ Cycle steps ② - ④ 29 times; ⑥ 72°C for 5 min; ⑦ Incubate at 12°C.

[0049] Incubate the above reaction system at 37°C for 3 h to digest the plasmid template (the digestion system was: 0.5 μL of DpnI, 45 μL of the above reaction PCR product, 5 μL of 10×T Buffer). After digestion, the digested product was introduced into Escherichia coli BL21 competent cells by chemical transformation method. The specific steps of the chemical transformation method are as follows:

[0050] (1) Introduce 10 μl of the homologous recombination product into 100 μl of Escherichia coli BL21 competent cells;

[0051] (2) Ice bath for 15 - 30 min;

[0052] (3) Heat shock in a 42°C water bath for 90 s, and quickly place it on ice and let it stand in an ice bath for 3 - 5 min after taking it out;

[0053] (4) Add 800 μl of antibiotic-free LB medium, mix well, and culture at 37°C and 200 rpm for 1 h;

[0054] (5) Centrifuge at 5000 rpm for 2 min to collect bacteria;

[0055] (6) Remove the supernatant, and resuspend the remaining 100 - 200 μl by pipetting and spreading it on a kanamycin-resistant plate containing 0.05 mg / mL, and incubate at 37°C for about 12 h.

[0056] (7) Pick monoclonal colonies into kanamycin-resistant LB containing 0.05 mg / mL, culture at 200 rpm and 37°C for 12 h, and then send them to the company for sequencing. Those with correct sequencing are positive transformants.

[0057] Example 2: Construction of double mutants and triple mutants

[0058] (1) Construction of double mutants: In the mutant PvDAPDH H227I On the basis of, W121 was mutated into LSI residue and R181 was mutated into GALMS residue, respectively, and double mutants were constructed by whole plasmid PCR using mutation primers W121-F, W121-R and R181-F, R181-R. For specific implementation methods, see the steps in Example 1.

[0059] Table 1 Mutant primer sequences

[0060]

[0061] (2) Construction of triple mutants: In the mutant PvDAPDH H227I and PvDAPDH H227I / R181S On the basis of, using mutation primers W121-F and W121-R, triple mutants were constructed by whole plasmid PCR. For specific implementation methods, see the steps in Example 1.

[0062] Table 2 Mutant primer sequences

[0063]

[0064]

[0065] Example 3: Expression and purification of PvDAPDH

[0066] The positive transformants of the mutant recombinant strains prepared in Examples 1 and 2 were inoculated into LB medium and cultured at 37°C until OD 600 When the pH is 0.6-1.0, add 5g / L lactose at a final concentration to induce the expression of the enzyme, the induction temperature is 25°C, the induction time is 12h, and the fermentation broth is obtained. The fermentation broth is centrifuged at 4°C and 6000rpm for 10min to obtain the bacteria. Add 10mL of binding solution A (20mM sodium phosphate, 0.5mM NaCl, 20mM imidazole, 1% glycerol according to the final concentration, and adjust the pH to 7.4 with HCl) to fully resuspend the bacteria, then place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The conditions for ultrasonic disruption are: working time 4s, interval time 4s, a total of 10min. The obtained disrupted liquid is subjected to low-temperature high-speed centrifugation, centrifuged at 4°C and 8000rpm for 30min to obtain a crude enzyme solution. Filter with a 0.22μm microporous filter membrane and set aside.

[0067] Prepare a nickel ion affinity chromatography column. First, use a constant flow pump to pump ultrapure water into the column to wash the column (about 6 - 12 times the column volume) at 4°C, and then balance the column environment with 10 mL of binding solution A. When the pH value of the effluent at the lower end of the column is the same as that of the low-salt concentration buffer solution pumped into the column (about 5 times the column volume of buffer solution is required), add the obtained crude enzyme solution after passing through the membrane to the column. First, wash the miscellaneous proteins with binding solution A until baseline balance, and then elute with elution solution B (20 mM sodium phosphate, 0.5 mM NaCl, 500 mM imidazole). Collect the eluate of the absorption peak, measure the enzyme activity, and obtain the target protein that reaches electrophoresis purity.

[0068] Example 4: PvDAPDH WT Determination of enzymatic parameters for aromatic substrates

[0069] To evaluate PvDAPDH WT for its catalytic effect on aromatic α-keto acid substrates, the present invention determined the kinetic parameters of PvDAPDH WT for different aromatic α-keto acid substrates.

[0070] Table 3 PvDAPDH WT Reaction conditions for the determination of enzymatic parameters of PvDAPDH for aromatic substrates

[0071]

[0072] Monitor the increase or decrease in the absorbance of NADP(H) at 340 nm by spectrophotometry. Use Origin to plot graphs to separately obtain the K m value and V max value of the enzyme, and the results are shown in Table 4.

[0073] Table 4 PvDAPDH WT Enzymatic parameters for catalyzing different aromatic α-keto acid substrates

[0074]

[0075] By determining the enzymatic parameters for different aromatic α-keto acid substrates, PvDAPDH WT has catalytic activity only for substrates 7a, 8a, 11a, and 12a, generating the corresponding D-amino acids (7, 8, 11, 12), among which PvDAPDH WT has a k cat / k m (mmol L -1 S -1 ) of 17.01, showing relatively high catalytic activity. However, PvDAPDH WT has no catalytic activity for the remaining 8 substrates.

[0076] Table 5 Different Substrate Structures and Product Structures Catalyzed by Pv-DAPDH

[0077]

[0078]

[0079] Example 5: Transformation Assay of Aromatic Substrates by PvDAPDH Single / Double Mutants

[0080] The single mutants constructed in Example 1 and the double mutant strains constructed in Example 2 with correct sequencing on the plate were respectively inoculated into LB containing 0.05 mg / mL kanamycin resistance, cultured at 200 rpm and 37 °C for 10 - 12 h, and then inoculated into TB medium at an inoculation amount of 5% by volume, and cultured at 200 rpm and 37 °C until the OD 600 When it reached 0.8, lactose was added for induction at a final concentration of 5 g / L, the induction temperature was 25 °C, and after 15 h of induction, the cells were collected by centrifugation at 6,000×g for 15 min at 4 °C.

[0081] Table 6 PvDAPDH Mut Catalytic Reaction Conditions for Aromatic α-Keto Acid Substrates

[0082]

[0083]

[0084] Weigh the centrifuged mutant strain cells (50 g / L) and break them by ultrasonic wave. Then, centrifuge the cell lysate at 12,000 rpm and 4 °C for 10 min. Take the prepared supernatant crude enzyme solution and add different α-keto acid substrates HBF and HPPA according to the conditions in Table 6, place it in a shaking water bath at 37 °C and 400 rpm for 24 h for reductive amination reaction. Ultrasonic disruption conditions: working time 4 s, interval time 4 s, for a total of 10 min. Centrifuge the obtained disrupted solution at low temperature and high speed at 4 °C and 8,000 rpm for 30 min to obtain the crude enzyme solution. Filter it with a 0.22 μm microporous filter membrane for standby. After 24 hours of reaction, terminate the reaction of the conversion solution with 1 mM NaOH solution, and dilute the termination solution with ultrapure water to 5 times the initial volume. Centrifuge the diluted conversion solution at 12,000 rpm for 10 min, and then filter the centrifuged supernatant through a 0.22 μm filter membrane. The filtrate is used for HPLC detection and analysis.

[0085] Compared with other single mutants, H227T and H227I have better transformation effects on HBF and HPPA, and can respectively produce 0.35 g / L D-Hpg and 2.45 g / L D-Tyr( Figure 1)。By combinatorial mutation, compared with other M2s, the mutant H227I / W121I can simultaneously catalyze the α-keto acid substrates HBF and HPPA to generate the corresponding products D-Hpg and D-Tyr, which are 1.7 g / L and 2.9 g / L respectively( Figure 2 )。

[0086] Example 6: Conversion of the aromatic substrate spectrum by PvDAPDH triple mutant

[0087] The triple mutant strains constructed in Example 2 with correct sequencing on the plate were respectively inoculated into LB containing 0.05 mg / mL kanamycin resistance, cultured at 200 rpm and 37 °C for 10 - 12 h, inoculated into TB medium at an inoculation amount of 5% by volume, and cultured at 200 rpm and 37 °C until OD 600 When it reached 0.8, lactose was added for induction at a final concentration of 5 g / L, the induction temperature was 25 °C, and after 15 h of induction, the cells were collected by centrifugation at 6,000×g and 4 °C for 15 min. The cells were disrupted according to the method of Example 5, the enzyme solution was collected, and the catalytic reaction was carried out according to the method of Example 5. The aromatic keto acid substrates used are shown in Table 5, items 1 - 12.

[0088] It can be seen from Figure 3 that the pvDAPDH triple mutant W121I / H227I / R181S shows good compatibility with different large-volume aromatic α-keto acid substrates and can simultaneously catalyze most aromatic α-keto acid substrates to be converted into D-amino acids (100 mg / L - 1800 mg / L). In addition, the triple mutant W121L / H227I / T171L shows relatively high catalytic activity towards 2-oxo-4-phenylbutyric acid (12a), reaching 2.1 g / L( Figure 3 ) The mutant library constructed above greatly enriches the biosynthetic aromatic D-amino acid toolbox. At the same time, it provides a basic template for the development of more efficient biocatalysts in the future.

[0089] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. meso-diaminopimelic acid dehydrogenase Pv a DAPDH mutant, characterized in that Based on the parental sequence shown in SEQ ID NO.1, the tryptophan at position 121 was mutated to isoleucine.

2. meso-Diaminopimelate dehydrogenase Pv a DAPDH mutant, characterized in that Based on the parental sequence shown in SEQ ID NO.1, the tryptophan at position 121 was mutated to isoleucine, and the histidine at position 227 was mutated to isoleucine.

3. meso - Diaminopimelic acid dehydrogenase Pv a DAPDH mutant, characterized in that Based on the parental sequence shown in SEQ ID NO.1, the tryptophan at position 121 was mutated to isoleucine, the histidine at position 227 was mutated to isoleucine, and the arginine at position 181 was mutated to serine.

4. A recombinant microbial cell expressing the mutant according to any one of claims 1 to 3.

5. The recombinant microbial cell according to claim 4, wherein The host is Escherichia coli ( Escherichia coli ) BL21(DE3).

6. A recombinant Escherichia coli, characterized in that, Using pET28a as the expression vector, the mutant according to any one of claims 1 to 3 was expressed in Escherichia coli BL21(DE3).

7. Use of the mutant according to claim 3 in the synthesis of D-amino acids by catalyzing aromatic α-keto acids, characterized in that, The application is: catalyzing 4-nitrophenyl-pyruvic acid to generate 4-nitro-D-phenylalanine, or catalyzing 3-(3-indolyl)-2-oxopropionic acid to generate D-tryptophan, or catalyzing m-hydroxy-2-oxopropionic acid to generate (R)-2-amino-3-(3-hydroxyphenyl)propionic acid, or catalyzing 3,4-dihydroxybenzene pyruvic acid to generate 3-hydroxy-D-tyrosine, or catalyzing 4-hydroxyphenylglyoxylic acid to generate D-p-hydroxyphenylglycine, or catalyzing 4-hydroxyphenylpyruvic acid to generate D-tyrosine, or catalyzing benzoylformic acid to generate D-phenylglycine, or catalyzing phenylpyruvic acid to generate D-phenylalanine, or catalyzing 3-(naphthalen-2-yl)-2-oxopropionic acid to generate D-3-(2-naphthyl)-alanine.

8. The application according to claim 7, wherein The application uses α-keto acid as the substrate, the mutant according to claim 3 as the catalyst, and reacts at 35 - 37 °C for at least 24 h.

9. The application according to claim 7 or 8, characterized in that, The reaction system also contains NADPH.

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