4-hydroxyphenylacetic acid 3-hydroxylase mutant and application thereof

Abstract

The invention discloses a 4-hydroxyphenylacetic acid 3-hydroxylase mutant and application thereof, recombinant expression plasmids and corresponding genetically engineered bacteria M214V, R290N and E294I are constructed by using genes for coding the mutants (EcHpaBM214V, EcHpaBR290N and EcHpaBE294I) and nucleotide sequences for coding EcHpaC genes respectively, and after the genetically engineered bacteria disclosed by the invention are subjected to shake flask fermentation in a 30mL M9Y culture medium containing 50mu g / mL chloramphenicol for 24 hours, the yield of the mutants is increased, and the yield of the mutants is increased. The yield of the tanshinol is 748.04 mg / L, 895.55 mg / L and 793.36 mg / L. The method breaks through the limitation that the substrate generated by the 4-hydroxyphenylacetic acid 3-hydroxylase through catalysis is poor in specificity and low in catalysis efficiency, and the yield of the tanshinol is high. The method has the advantages of being high in catalytic efficiency, simple in process and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the fields of enzyme engineering, biology and technology, and specifically relates to a 4-hydroxyphenylacetic acid 3-hydroxylase mutant and application thereof. Background Art

[0002] Salvianic acid A (SAA) is a water-soluble phenylpropanoid compound found in the Chinese herbal medicine Danshen. It is mostly found in the rhizomes of Danshen and Salvia officinalis in nature. Its chemical name is β-(3,4-dihydroxyphenyl) lactic acid. It has multiple pharmacological activities due to its special chemical structure. In clinical studies, danshen has shown the ability to protect the myocardium, anti-thrombosis, antioxidant, and anti-tumor, and has broad market prospects. At present, the main methods for obtaining danshen are plant extraction and chemical synthesis. However, the danshen extract obtained by traditional plant extraction contains polysaccharides, proteins and other substances, which requires higher requirements for downstream separation and purification processes. In addition, the yield of danshen obtained by plant extraction is limited, which cannot meet clinical needs, and it is difficult to meet the requirements of green chemistry and sustainable development. Chemical synthesis requires expensive catalysts, which is costly and has strict requirements on some reaction conditions, which limits the large-scale production of danshen. Using modern molecular biology and bioengineering technology to construct engineered bacteria for producing danshen, using synthetic biology methods to replace traditional plant extraction methods to produce danshen is more in line with the requirements of environmental protection and sustainable development. In recent years, a variety of complex natural products have been able to achieve large-scale production through synthetic biology. The artificial synthesis pathway of tanshinone has been analyzed and preliminarily constructed, but the yield is still some distance away from achieving industrialization.

[0003] 4-Hydroxyphenylacetic acid 3-hydroxylase (EcHpaB) from E. coli BL21 (DE3) is a key enzyme in the biosynthesis of danshensu. It can catalyze the ortho-hydroxylation of the phenolic hydroxyl group of 4-hydroxyphenyllactate, which is similar to the natural substrate structure, to synthesize danshensu by cooperating with the reductase component (EcHpaC) of 4-hydroxyphenylacetic acid 3-hydroxylase from E. coli BL21 (DE3). However, this technology still has problems such as poor substrate specificity and low catalytic efficiency, which limits the yield of danshensu synthesized by microorganisms. Therefore, how to improve the biosynthesis efficiency of danshensu is an urgent problem to be solved in order to promote the green and efficient synthesis of danshensu as soon as possible. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a 4-hydroxyphenylacetic acid 3-hydroxylase mutant.

[0005] The second object of the present invention is to provide a nucleotide sequence containing a gene encoding an EcHpaB mutant and a gene encoding EcHpaC.

[0006] The third object of the present invention is to provide a recombinant expression plasmid containing the above nucleotide sequence.

[0007] The fourth object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0008] The fifth objective of the present invention is to provide the application of the above-mentioned genetically engineered bacteria in fermentation to produce danshensu.

[0009] The technical solution of the present invention is summarized as follows:

[0010] A 4-hydroxyphenylacetate 3-hydroxylase mutant, wherein the amino acid sequence of the 4-hydroxyphenylacetate 3-hydroxylase is shown in SEQ ID NO.1; the 4-hydroxyphenylacetate 3-hydroxylase mutant is one of the following:

[0011] The methionine at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, as shown in SEQ ID NO.3;

[0012] The arginine at position 290 of the amino acid sequence shown in SEQ ID NO.1 is mutated to asparagine, as shown in SEQ ID NO.4;

[0013] The glutamic acid at position 294 of the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine, as shown in SEQ ID NO.5;

[0014] The 4-hydroxyphenylacetic acid 3-hydroxylase is referred to as EcHpaB.

[0015] A nucleotide sequence containing a gene encoding the above-mentioned EcHpaB mutant and a gene encoding EcHpaC;

[0016] The EcHpaC is the abbreviation of the reductase component of 4-hydroxyphenylacetic acid 3-hydroxylase, and the nucleotide sequence encoding the EcHpaC gene is shown in SEQ ID NO.2.

[0017] A recombinant expression plasmid containing the above encoding gene.

[0018] A genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0019] The application of the above genetically engineered bacteria in fermentation to prepare danshensu.

[0020] Advantages of the present invention:

[0021] After the genetically engineered bacteria (M214V, R290N and E294I) of the present invention were shaken and fermented in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol for 24 hours, the yields of danshensu were 748.04 mg / L, 895.55 mg / L and 793.36 mg / L respectively. The present invention breaks through the limitations of poor substrate specificity and low catalytic efficiency of 4-hydroxyphenylacetic acid 3-hydroxylase catalyzing the generation of danshensu, and significantly improves the yield of danshensu. The present invention has the advantages of high catalytic efficiency, simple process and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Candidate mutation sites for the import and export positions of the substrate binding pocket of the HpaB crystal structure derived from E. coli BL21 (DE3);

[0023] Figure 2 Schematic diagram of the expression plasmid pACYC-EchpaB-EchpaC of EcHpaB and EcHpaC;

[0024] Figure 3 This is a graph showing the production of tanshinone by the genetically engineered bacterial strain after fermentation for 24 hours in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol. DETAILED DESCRIPTION

[0025] The invention analyzes and designs the key amino acids at the import and export positions of the substrate binding pocket of HpaB from E. coli BL21 (DE3), uses the expression plasmid paCYCDuet-1 (commercially available) as a carrier to construct a saturation mutation library, and screens out highly active 4-hydroxyphenylacetic acid 3-hydroxylase mutants therefrom by a high-throughput screening method. The expression plasmid paCYCDuet-1 (commercially available) is used to construct a recombinant expression plasmid containing a gene of an EcHpaB mutant and a gene encoding EcHpaC. The recombinant expression plasmid is transformed into Escherichia coli TS6 to obtain a genetically engineered bacterium.

[0026] The above-mentioned E. coli TS6 is based on E. coli W3110 (commercially available) as the starting strain, and has undergone the following modifications: E. coli W3110, ΔlacIZ, ΔtyrR, ΔtehB::aroG S180F , ΔmbhA:tyrA M53I / A354V , ΔpheA, ΔyjgX::BlfldA.

[0027] Table 1 Gene accession numbers

[0028] Gene name accession number (GenBank) lacI BAE76127.1 lacZ BAE76126.1 tyrBAA14905 tehB BAA15059 aroG BAA35416 mbhA WP_167580554.1 tyrA WP_240776599.1 pheA WP_179306759.1 yjgX BDT18761.1 BlfldA WP_060620770.1

[0029] The culture medium formula involved in the following specific embodiments is as follows:

[0030] LB medium: 5 g / L yeast extract, 10 g / L casein peptone, 10 g / L sodium chloride.

[0031] M9Y medium: 2 g / L yeast extract, 20 g / L glucose, 6 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 2 g / L 3-morpholinepropanesulfonic acid (MOPS), 2 mmol / L magnesium sulfate, and 0.1 mmol / L calcium chloride.

[0032] The experimental methods in the following examples without specific conditions are usually carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Primer synthesis and sequencing were performed by Anshengda Biotechnology Co., Ltd.

[0033] The present invention is further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0034] Example 1

[0035] The crystal structure of 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB, amino acid sequence as shown in SEQ ID NO.1) from E. coli BL21 (DE3) (PDB ID: 6QYI) was visualized using Pymol software, and the key positions of substrate import and export of 4-hydroxyphenylacetate 3-hydroxylase were analyzed, and candidate sites M214, R290, E294 were screened out, see Figure 1 The catechol structure of Danshensu will generate quinone substances under the action of strong oxidants, which will appear red. 400 There is a maximum absorption peak at the bottom, and within a certain concentration range, the concentration and OD 400 The values ​​were linearly related. Saturated mutant libraries of M214, R290, and E294 were constructed and then high-throughput screening was performed. After fermentation at 37°C and 180 rpm for 24 h in a 96-well deep-well plate containing M9Y medium, 180 μL of the supernatant was added to a 96-well microplate and then 0.1 M sodium periodate solution was added. After mixing by pipetting, the mixture was allowed to stand for 10 min. The absorbance at 400 nm was detected and the OD 400 The mutant strains with high values ​​were sent to a sequencing company for sequencing to determine that three 4-hydroxyphenylacetic acid 3-hydroxylase mutants were beneficial mutants, and the 4-hydroxyphenylacetic acid 3-hydroxylase mutants were one of the following:

[0036] The methionine at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, as shown in SEQ ID NO.3;

[0037] The arginine at position 290 of the amino acid sequence shown in SEQ ID NO.1 is mutated to asparagine, as shown in SEQ ID NO.4;

[0038] The glutamic acid at position 294 of the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine, as shown in SEQ ID NO.5;

[0039] The 4-hydroxyphenylacetic acid 3-hydroxylase is referred to as EcHpaB.

[0040] Example 2

[0041] Construction of a nucleotide sequence containing a gene encoding an EcHpaB mutant and a gene encoding an EcHpaC gene:

[0042] The EcHpaC is the abbreviation of the reductase component of 4-hydroxyphenylacetic acid 3-hydroxylase, and the nucleotide sequence of the gene encoding EcHpaC is shown in SEQ ID NO.2.

[0043] The primers involved in this invention were synthesized by Anshengda Company.

[0044] The upstream and downstream primers and the intermediate mutation primers required for constructing the fragments containing the EcHpaB and EcHpaC genes were synthesized respectively. The primer names and nucleotide sequences are shown in Table 2.

[0045] The genome of E. coli BL21 (DE3) (commercially available) strain was used as template and P trc -hpaBC-F (SEQ ID NO.12), P trc -hpaBC-R (SEQ ID NO.13) as primers to obtain a fragment P containing the genes encoding EcHpaB and EcHpaC trc -EchpaB-EchpaC (SEQ ID NO.6), after verification by agarose gel electrophoresis, the purified DNA fragment was recovered by gel excision.

[0046] P trc -EchpaB-EchpaC as template, with P trc -hpaBC-F (SEQ ID NO.12), M214V-R (SEQ ID NO.15) were used as primers to amplify P trc -EchpaB M214V -The upstream gene fragment of EchpaC, with P trc -EchpaB-EchpaC as template, using M214V-F (SEQ ID NO.14), P trc -hpaBC-R (SEQ ID NO.13) was used as primer to amplify Ptrc -EchpaB M214V -EchpaC downstream gene fragment; primer P trc -hpaBC-F,P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB M214V -The upstream gene fragment of EchpaC and P trc -EchpaB M214V -EchpaC downstream gene fragment overlap extension to obtain the complete mutant gene P trc -EchpaB M214V -EchpaC, whose sequence is shown in SEQ ID NO.7, was verified by agarose gel electrophoresis and then the gel was cut and recovered to obtain a purified DNA fragment.

[0047] P trc -EchpaB-EchpaC as template, with P trc -hpaBC-F, R290N-R (SEQ ID NO.17) were used as primers to amplify P trc -EchpaB R290N -The upstream gene fragment of EchpaC, with P trc -EchpaB-EchpaC as template, using R290N-F (SEQ ID NO.16), P trc -hpaBC-R was used as primer to amplify P trc -EchpaB R290N -EchpaC downstream gene fragment; primer P trc -hpaBC-F,P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB R290N -The upstream gene fragment of EchpaC and P trc -EchpaB R290N -EchpaC downstream gene fragment overlap extension to obtain the complete mutant gene P trc -EchpaB R290N -EchpaC, whose sequence is shown in SEQ ID NO.8, was verified by agarose gel electrophoresis and then the gel was cut and recovered to obtain a purified DNA fragment.

[0048] P trc -EchpaB-EchpaC as template, with P trc -hpaBC-F, E294I-R (SEQ ID NO.19) were used as primers to amplify P trc -EchpaB E294I-EchpaC upstream gene fragment, newly added: P trc -EchpaB-EchpaC as template, using E294I-F (SEQ ID NO.18), P trc -hpaBC-R was used as primer to amplify P trc -EchpaB E294I -EchpaC downstream gene fragment; primer P trc -hpaBC-F,P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB E294I -The upstream gene fragment of EchpaC and P trc -EchpaB E294I -EchpaC downstream gene fragment overlap extension to obtain the complete mutant gene P trc -EchpaB E294I -EchpaC, whose sequence is shown in SEQ ID NO.9, was verified by agarose gel electrophoresis and then the gel was cut and recovered to obtain a purified DNA fragment.

[0049] Table 2 Primer sequence list

[0050]

[0051] Example 3

[0052] The recombinant expression plasmid containing the gene encoding the EcHpaB mutant and the nucleotide sequence encoding the EcHpaC gene was constructed as follows:

[0053] The paCYCDuet-1 empty plasmid (commercially available) was selected, and PCR amplification was performed using primers PACYC-hpaBC-R (SEQ ID NO.10) and PACYC-hpaBC-F (SEQ ID NO.11) in Table 2. The template was digested and purified using DpnI enzyme to obtain a linearized fragment of the pACYCDuet-1 empty plasmid.

[0054] The gene fragment P amplified in Example 2 was cloned using the seamless cloning enzyme 2X MultiF Seamless Assembly Mix from Ibotek. trc -EchpaB-EchpaC, P trc -EchpaB M214V -EchpaC,P trc -EchpaB R290N -EchpaC and P trc -EchpaB E294I-EchpaC were connected with the linearized fragment of pACYCDuet-1 empty plasmid, and the mixture of the fragment to be connected and the linearized fragment of pACYCDuet-1 empty plasmid was reacted at 50°C for 30 minutes. The reaction system was transduced into Escherichia coli DH5α competent cells, and then spread on LB solid plates containing 50 μg / mL chloramphenicol and cultured at 37°C overnight to obtain single clones. Use P in Table 2 trc -hpaBC-F and hpaBC-JD-R (SEQ ID NO.20) primers were used for preliminary colony PCR verification and further sequencing verification, and plasmids were extracted from the verified correct strains to finally obtain the recombinant expression plasmids pACYC-EchpaB-EchpaC and pACYC-EchpaB M214V -EchpaC, pACYC-EchpaB R290N -EchpaC and pACYC-EchpaB E294I -EchpaC.

[0055] Example 4

[0056] Construct pACYC-EchpaB-EchpaC, pACYC-EchpaB M214V -EchpaC, pACYC-EchpaB R290N -EchpaC and pACYC-EchpaB E294I -EchpaC genetic engineering bacteria, the specific steps are as follows:

[0057] The constructed recombinant expression plasmids pACYC-EchpaB-EchpaC and pACYC-EchpaB M214V -EchpaC, pACYC-EchpaB R290N -EchpaC and pACYC-EchpaB E294I The E. coli cells into which the expression plasmids were transferred were spread on LB solid medium plates containing 50 μg / mL chloramphenicol and cultured in a 37°C incubator overnight to obtain the control strain TS6 / pACYC-EchpaB-EchpaC (abbreviated as the control strain), three E. coli strains expressing 4-hydroxyphenylacetic acid 3-hydroxylase mutants TS6 / pACYC-EchpaB M214V -EchpaC (abbreviated as M214V), TS6 / pACYC-EchpaB R290N -EchpaC (abbreviated as R290N) and TS6 / pACYC-pACYC-EchpaB E294I -EchpaC (abbreviated as E294I).

[0058] Single colonies of the control strain, M214V, R290N and E294I were picked up in turn and transferred to 5 mL of LB liquid culture medium containing 50 μg / mL chloramphenicol for overnight culture. 1 mL of the bacterial solution was transferred to a bacterial preservation tube, 1 mL of 30% (v / v) glycerol was added and mixed, and the tubes were marked in turn and stored in a -80°C refrigerator.

[0059] Example 5

[0060] The method of producing danshensu by using genetically engineered bacterial strains for shake flask fermentation comprises the following steps:

[0061] The control strain, M214V, R290N and E294I were used for shake flask fermentation, and three parallel experiments were set for each strain. 5 μL of bacterial solution was transferred from the bacterial preservation tube to 5 mL of LB medium containing 50 μg / mL chloramphenicol, and cultured at 220 rpm in a 37°C shaker for 12 hours, and then transferred to 30 mL of M9Y medium containing 50 μg / mL chloramphenicol, and fermented at 180 rpm in a 37°C shaker. At 4 hours, IPTG with a final concentration of 0.1 mM was added for protein induction expression. After 24 hours of fermentation, the culture was stopped to determine the product yield in the fermentation broth.

[0062] Example 6

[0063] The concentration of danshensu in the fermentation broth was detected by HPLC, as follows:

[0064] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 10 min, and then take the supernatant and pass it through a membrane for HPLC detection.

[0065] The detection conditions are as follows: Bona C18 (250 mm × 4.6 mm) chromatographic column; PDA detector; detection wavelength 281 nm; flow rate 1 mL / min. The mobile phase is: 20% methanol, 79.9% water and 0.1% formic acid.

[0066] like Figure 3 As shown, after 24 h of shake flask fermentation in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol, the tanshinone production of M214V, R290N and E294I reached 748.04 mg / L, 895.55 mg / L and 793.36 mg / L, respectively, which were increased by 51.12%, 80.09% and 60.27% compared with 497.28 mg / L of the control strain.

[0067] The results show that the 4-hydroxyphenylacetic acid 3-hydroxylase mutant EcHpaB M214V 、EcHpaB R290N and EcHpaB E294IThe efficiency of producing danshensu in Escherichia coli was significantly improved, overcoming the limitations of poor substrate specificity and low catalytic efficiency of natural hydroxylases, and has broad application prospects in industry.

[0068] The above-mentioned embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention. All simple changes and modifications made according to the claims and the contents of the specification of the present invention are within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A 4-hydroxyphenylacetate 3-hydroxylase mutant, wherein the amino acid sequence of the 4-hydroxyphenylacetate 3-hydroxylase is shown in SEQ ID NO.1; characterized in that The 4-hydroxyphenylacetic acid 3-hydroxylase mutant is one of the following: The methionine at position 214 of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, as shown in SEQ ID NO.3; the arginine at position 290 of the amino acid sequence shown in SEQ ID NO.1 is mutated to asparagine, as shown in SEQ ID NO.4; the glutamic acid at position 294 of the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine, as shown in SEQ ID NO.5; The 4-hydroxyphenylacetic acid 3-hydroxylase is referred to as EcHpaB.

2. A nucleotide sequence comprising a gene encoding the EcHpaB mutant of claim 1 and a gene encoding EcHpaC; The EcHpaC is the abbreviation of the reductase component of 4-hydroxyphenylacetic acid 3-hydroxylase, and the nucleotide sequence encoding the EcHpaC gene is shown in SEQ ID NO.

2.

3. A recombinant expression plasmid containing the coding gene according to claim 2.

4. A genetically engineered bacterium containing the recombinant expression plasmid according to claim 3.

5. Use of the genetically engineered bacteria according to claim 4 in fermenting and preparing danshensu.

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