4-hydroxyphenylacetic acid-3-hydroxylase mutant with improved activity and application thereof

Abstract

The invention discloses a 4-hydroxyphenylacetic acid-3-hydroxylase mutant with improved activity and application thereof, the 4-hydroxyphenylacetic acid-3-hydroxylase mutant is obtained by mutating wild type 4HPA3H enzyme from Escherichia coli, the amino acid sequence of the wild type 4HPA3H enzyme is as shown in SEQ ID No.1, and the amino acid sequence is specifically mutated into any one of the following: (1) M205F; and (2) T398S. According to the present invention, the whole-cell catalysis activity of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant, the M205F mutant and the T398S mutant on the p-hydroxyphenyllactic acid is improved by 1.70 times and 1.24 times compared with the WT;

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a 4-hydroxyphenylacetic acid-3-hydroxylase mutant with enhanced activity and application thereof. Background Art

[0002] Danshensu is a naturally occurring catechol compound that can be produced by the ortho-hydroxylation of p-hydroxyphenyllactic acid. It has physiological functions such as anti-thrombosis, hypolipidemic and anti-atherosclerotic effects, and has important application value in the fields of food and medicine.

[0003] In nature, danshensu is mainly found in the Chinese herbal medicine Danshen, but the content of danshensu in natural sources is relatively low and it is relatively difficult to extract from plants.

[0004] The use of applied chemical methods to synthesize Danshensu has many synthesis steps, harsh reaction conditions, environmental pollution and other problems. The use of biotransformation methods to prepare catechol structure compounds such as Danshensu by ortho-hydroxylation of substances with phenol structures through biocatalysts has the advantages of mild reaction conditions, high selectivity and environmental friendliness, and has therefore attracted much attention and favor.

[0005] At present, 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) has become the preferred enzyme for the preparation of danshensu due to its advantages such as easy expression and high catalytic efficiency. 4HPA3H is a key enzyme in bacterial degradation of 4-hydroxyphenylacetic acid (4HPA). It can not only ortho-hydroxylate 4HPA, but also has good catalytic activity towards many phenolic substances, showing a broad substrate spectrum ([1] Deng Y, Faivre B, Back O, et al. Structural and Functional Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase from Escherichia coli[J]. Chembiochem, 2020, 21(1-2): 163-170. [2] Y. Lin, Y. Yan. Biotechnological Production of Plant-Specific Hydroxylated Phenylpropanoids[J]. Biotechnology and Bioengineering, 2014, 111(9)1895-1899.).

[0006] Although 4HPA3H can catalyze p-hydroxyphenyllactic acid to produce danshensu, its catalytic efficiency for danshensu is significantly lower than that of its natural substrate 4HPA, which seriously restricts its application in non-natural substrates such as p-hydroxyphenyllactic acid. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention provides two 4-hydroxyphenylacetic acid-3-hydroxylase mutants and applications thereof. The present invention obtains two 4-hydroxyphenylacetic acid-3-hydroxylase mutants, which have higher enzyme activity than the wild-type 4-hydroxyphenylacetic acid-3-hydroxylase and can efficiently catalyze the synthesis of danshensu.

[0008] A 4-hydroxyphenylacetic acid-3-hydroxylase mutant, characterized in that it is obtained by mutating a wild-type 4HPA3H enzyme from Escherichia coli, wherein the amino acid sequence of the wild-type 4HPA3H enzyme is shown in SEQ ID No. 1, and the specific mutation is any one of the following:

[0009] (1)M205F;

[0010] (2)T398S.

[0011] Among them, M205F means that the amino acid at position 205 mutates from methionine to phenylalanine; T398S means that the amino acid at position 398 mutates from threonine to serine.

[0012] The present invention also provides a gene encoding the 4-hydroxyphenylacetic acid-3-hydroxylase mutant, wherein the gene sequence of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant with the mutation site M205F is shown in SEQ ID No. 3; the gene sequence of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant with the mutation site T398S is shown in SEQ ID No. 4.

[0013] The invention also provides a recombinant plasmid comprising the gene.

[0014] The present invention also provides a genetically engineered cell comprising the gene. Specifically, the gene encoding the wild-type 4HPA3H enzyme in Escherichia coli may be mutated into the gene.

[0015] Alternatively, the gene is exogenously introduced into Escherichia coli.

[0016] The present invention also provides the use of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant, the gene, or the genetically engineered cell in catalyzing the production of danshensu from p-hydroxyphenyllactic acid. Compared with the wild-type enzyme, the mutant enzyme has better catalytic activity and is more suitable for industrial application.

[0017] The present invention also provides a method for catalyzing p-hydroxyphenyllactic acid to produce danshensu, comprising the following steps: using p-hydroxyphenyllactic acid as a substrate, adding the 4-hydroxyphenylacetic acid-3-hydroxylase mutant or the genetically engineered cell to carry out a catalytic reaction to produce danshensu.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The 4-hydroxyphenylacetic acid-3-hydroxylase mutant of the present invention is obtained by mutating the wild-type 4HPA3H enzyme from Escherichia coli, wherein the whole-cell catalytic tanshinone activity of the two mutants M205F and T398S is increased by 1.70 and 1.24 times relative to the WT. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the reaction of preparing danshensu from p-hydroxyphenyllactic acid catalyzed by 4HPA3H.

[0021] Figure 2 The figure shows the purification of WT and mutant proteins.

[0022] Figure 3 The amount of tanshinone produced by WT and mutants. DETAILED DESCRIPTION

[0023] The invention introduces mutations at sites 205 and 398 on the substrate pocket domain through site-directed mutagenesis PCR on the basis of 4HPA3H (EC 1.14.13.3) of Escherichia coli, thereby obtaining mutants M205F and T398S whose catalytic efficiency of tanshinone is better than that of WT.

[0024] The amino acid sequence of the wild-type 4-hydroxyphenylacetic acid-3-hydroxylase (4HPA3H) is shown in SEQ ID No. 1. The nucleotide sequence encoding the wild-type 4-hydroxyphenylacetic acid-3-hydroxylase (4HPA3H) is shown in SEQ ID No. 2, the nucleotide sequence encoding the M205F mutant is shown in SEQ ID No. 3, and the nucleotide sequence encoding the T398S mutant is shown in SEQ ID No. 4.

[0025] Example 1: Construction of mutants

[0026] 1. Obtaining site-directed mutagenesis PCR products

[0027] According to the E. coli 4HPA3H gene sequence (GenBank accession number: NC 011751.1), PCR primers were designed to amplify the 4HPA3H gene in the E.coil BL21 (DE3) genome, and BamH I and Hind III were added to the 5′ end of the upstream and downstream primers, respectively. The primer sequences are as follows:

[0028] HpaBCF: 5′-ACGCGGATCCGATGAAACCAGAAGATTTCCGCG-3′(BamH I),

[0029] HpaBCR: 5'-ACCCAAGCTTAAATCGCAGCTTCCATTTCC-3' (Hind III).

[0030] The genome of Escherichia coli BL21 (DE3) was extracted according to the method of Omega bacterial genome extraction kit. PCR amplification reaction was performed with HpaBCF / R primers. PCR program: 94℃, 5min; (94℃, 1min; 50℃, 30s; 72℃, 1min) × 32 cycles; 72℃, 5min; finally, cool to 4℃ for storage.

[0031] The target gene was digested with two restriction endonucleases, BamH I and Hind III, and then connected with the plasmid pETDuet that had been treated with the same double enzymes to obtain the recombinant plasmid pETDuet-4HPA3H.

[0032] Using pETDuet-4HPA3H plasmid as template, fixed-point PCR amplification was performed. The PCR reaction system was: 10 μL 5× PCR buffer, 4 μL dNTPs (2.5 mmol / L), 1 μL upstream primer (10 mmol / L), 1 μL downstream primer (10 mmol / L), 1 μL plasmid template (50 ng / μL), Fast Pfu DNA polymerase 5U, and high-temperature sterilized ultrapure water to a total volume of 50 μL. Fixed-point PCR amplification program: 94°C denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 6 min, 30 cycles; 72°C extension for 4 min.

[0033] M205F:

[0034] Upstream primer: 5′-GCGCTGACTCACTACAACttcATTGGCTTCGG-3′,

[0035] Downstream primer: 5′-CAATgaaGTTGTAGTGAGTCAGCGCCGAGTTG-3′.

[0036] T398S:

[0037] Upstream primer: 5′-CATTATCGAACGCAACGTTagcAGTGGC-3′,

[0038] Downstream primer: 5′-GGAGATAGATCAGGCCACTgctAACGTTGCG-3′.

[0039] The lowercase letters represent the bases that were replaced.

[0040] 2. Transformation and verification of site-directed mutagenesis PCR products

[0041] The site-directed mutagenesis PCR product obtained above was digested with Dpn I enzyme to degrade the plasmid template and eliminate the possibility of wild-type individuals in the transformants, thereby effectively screening mutant clones. The digestion system is: 40-50 μL of PCR product, 1 μL of Dpn I, gently pipette and mix, briefly centrifuge to collect into the bottom tube, and place at 37°C for 1-2 hours.

[0042] The digested product was treated with CaCl 2 The method was used to transform Escherichia coli BL21 (DE3) competent cells. The specific operation is as follows:

[0043] (1) Take 100 μL of competent cell suspension from the -70°C refrigerator and thaw on ice;

[0044] (2) Add 5 μL of site-directed mutagenesis PCR digestion product, mix gently, and place on ice for 30 min;

[0045] (3) Heat shock in a 42°C water bath for 60 seconds and then immediately place on ice for 2 minutes;

[0046] (4) Add 400 μL of SOC medium to the tube, mix well, and culture at 37°C with shaking for 1 h to allow the cells to recover;

[0047] (5) Shake the bacterial solution and spread an appropriate amount on an LB plate containing 100 μg / mL ampicillin. Place the plate face up for half an hour. After the bacterial solution is absorbed by the culture medium, invert the plate and culture it at 37°C overnight.

[0048] (6) After the transformants grew, several clones were randomly selected, plasmids were extracted, and fully automated DNA sequencing was performed to confirm the expected mutation sites ATG→TTC and ACC→AGC at positions 205 and 398.

[0049] Example 2: Expression and purification of mutant enzymes

[0050] A single colony was picked and inoculated into 5 mL LB liquid medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 200 r / min. Subsequently, the overnight culture was inoculated into 100 mL LB medium containing 100 μg / mL ampicillin at a ratio of 2% (V / V) and cultured at 37°C until OD 600 The value reached 0.6-0.8. Then, IPTG was added to a final concentration of 0.5 mmol / L, and the bacteria were collected by centrifugation after induction culture at 28°C and 150 r / min for 6 hours. The bacteria were washed twice with PBS buffer (pH 7.4). After removing the culture medium, the cells were resuspended in PBS buffer with a volume of 1 / 5 of the original fermentation broth. The cells were ultrasonically broken in an ice bath, and the supernatant was collected by centrifugation for 10 minutes to obtain a crude enzyme solution containing 4HPA3H. The obtained crude enzyme solution was centrifuged at 12,000 × g at 4°C for 30 minutes, and the His-tagged WT and mutant enzymes were purified from the clarified lysate using Ni affinity chromatography. It was concentrated using a 10 kDa cutoff membrane (Millipore, Billerica, MA, USA). The purified product was detected by electrophoresis, and the results are as shown Figure 2 As shown, its size is as expected.

[0051] Example 3: Determination of WT and mutant whole-cell catalytic activity

[0052] After the cells induced in Example 2 were washed twice with phosphate buffer, the collected cells were resuspended in PBS buffer and the OD 600 10. Take 1mL of bacterial suspension and centrifuge at 12000rpm for 10min to collect the bacteria, then suspend them with 1mL of reaction solution (20mM p-hydroxyphenyllactic acid, 100mM KPi, pH 7.8). Put the suspension into a metal bath, 800rpm, 37℃, react for 1h, then add 50μL of 20% hydrochloric acid to terminate the reaction. The amount of danshensu produced was analyzed by high performance liquid chromatography. Schematic diagram of the reaction of preparing danshensu with p-hydroxyphenyllactic acid catalyzed by 4HPA3H is shown in Figure 1 shown.

[0053] HPLC operating conditions are as follows: the chromatographic separation column is Shimadzu Shim-pack GIS C18 (250 mm×4.6 mm) (Shimadzu Corporation), the UV detection wavelength is 210 nm, the injection volume is 10 μL, the column temperature is controlled at 30°C, the mobile phase A is 100% methanol, the mobile phase B is water containing 0.05% acetic acid, the flow rate is 1 mL / min, and the gradient elution program is shown in Table 1.

[0054] Table 1 HPLC gradient elution program

[0055] T / min 0 16 A％ 20 20 B％ 80 80

[0056] The result is as follows Figure 3 .Depend on Figure 3 It can be seen that under the same treatment conditions, the specific activities of the mutant enzymes M205F and T398S were higher than that of the wild-type 4HPA3H, increasing by 1.70 and 1.24 times, respectively.

[0057] Example 4: Determination of mutant enzyme kinetic parameters

[0058] The initial reaction velocity was measured at different substrate concentrations (10-1000 μM).

[0059] Using p-hydroxyphenyllactic acid as substrate, the Michaelis-Menten kinetic constants of the wild-type enzyme, mutant enzyme M205F and mutant enzyme T398S were determined at pH 7.8 and 37°C. The catalytic rate was determined at a substrate concentration of 10-1000 μM. OriginPro8 TM The nonlinear regression of the Michaelis-Menten equation was performed to determine the catalytic kinetic parameters. The calculation results are shown in Table 2.

[0060] Table 24 Kinetic parameters of HPA3H and mutants M205F and T398S for substrate p-hydroxyphenyllactic acid

[0061] mutation <![CDATA[K m (μM)]]> <![CDATA[K cat (min -1 )]]> <![CDATA[K cat / K m (s -1 μM -1 )]]> WT 874.46±168.03 18.16±2.19 346.12 M205F 363.82±46.80 18.99±1.88 869.94 T398S 225.84±31.00 9.74±0.44 718.80

[0062] K m Represents the affinity of the enzyme to its substrate.

[0063] K cat Refers to the turnover number or catalytic constant, which indicates the number of substrate molecules converted per second per enzyme molecule or per enzyme active center when the enzyme is saturated with substrate. cat The larger the value, the higher the catalytic efficiency of the enzyme.

[0064] K cat / K m It is the secondary rate constant for the reaction of enzyme and substrate to generate product, and its size can be used to compare the catalytic efficiency of enzyme.

[0065] As shown in Table 2, the K cat / K m They are 2.51 and 2.08 times of WT, respectively. This indicates that the catalytic efficiency of the mutant enzymes M205F and T398S is greater than that of the wild-type 4HPA3H. This is very beneficial for the biopreparation of danshensu using 4HPA3H.

Claims

1. A 4-hydroxyphenylacetic acid-3-hydroxylase mutant with enhanced activity, characterized in that: It is obtained by mutating the wild-type 4HPA3H enzyme from Escherichia coli, the amino acid sequence of the wild-type 4HPA3H enzyme is shown in SEQ ID No. 1, and the specific mutation is any one of the following: (1)M205F; (2)T398S.

2. A gene encoding the 4-hydroxyphenylacetic acid-3-hydroxylase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The gene sequence of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant with the mutation site of M205F or T398S is shown in SEQ ID No. 3 or SEQ ID No. 4, respectively.

4. A recombinant plasmid comprising the gene according to claim 2.

5. A genetically engineered cell comprising the gene of claim 2.

6. The genetically engineered cell according to claim 5, characterized in that The gene encoding the wild-type 4HPA3H enzyme in Escherichia coli is mutated into the gene according to claim 2, Alternatively, the gene according to claim 2 is exogenously introduced into Escherichia coli.

7. Use of the 4-hydroxyphenylacetic acid-3-hydroxylase mutant according to claim 1, the gene according to claim 2 or 3, or the genetically engineered cell according to claim 5 or 6 in catalyzing the production of danshensu from p-hydroxyphenyllactic acid.

8. A method for catalyzing p-hydroxyphenyllactic acid to produce danshensu, characterized in that: The following steps are involved: Taking p-hydroxyphenyllactic acid as a substrate, adding the 4-hydroxyphenylacetic acid-3-hydroxylase mutant described in claim 1 or the genetically engineered cell described in claim 5 or 6 to carry out a catalytic reaction to produce danshensu.

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