A 4-hydroxyphenylacetic acid 3-hydroxylase mutant and its application

Abstract

This invention discloses a 4-hydroxyphenylacetic acid 3-hydroxylase mutant and its applications, using the encoding of the above mutant (EcHpaB) M214V EcHpaB R290N and EcHpaB E294I The gene and the nucleotide sequence encoding the EcHpaC gene were used to construct recombinant expression plasmids and corresponding genetically engineered bacteria M214V, R290N, and E294I. After 24 hours of shake-flask fermentation in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol, the above-mentioned genetically engineered bacteria produced tanshinone yields of 748.04 mg / L, 895.55 mg / L, and 793.36 mg / L, respectively. This invention overcomes the limitations of poor substrate specificity and low catalytic efficiency in the production of tanshinone catalyzed by 4-hydroxyphenylacetic acid 3-hydroxylase, achieving high tanshinone yield. This invention has the advantages of high catalytic efficiency, simple process, and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering, biology and technology, and specifically relates to a 4-hydroxyphenylacetic acid 3-hydroxylase mutant and its applications. Background Technology

[0002] Salvianic acid A (SAA) is a water-soluble phenylpropanoid compound found in the Chinese herbal medicine *Salvia miltiorrhiza*. It is naturally found in the rhizomes of *Salvia miltiorrhiza* and *Salvia miltiorrhiza*, and its chemical name is β-(3,4-dihydroxyphenyl)lactic acid. Due to its unique chemical structure, it possesses various pharmacological activities. Clinical studies have shown that tanshinone has protective effects on the myocardium, antithrombotic effects, antioxidant effects, and antitumor effects, indicating a broad market prospect. Currently, the main methods for obtaining tanshinone are plant extraction and chemical synthesis. However, traditional plant extraction methods for *Salvia miltiorrhiza* extract contain polysaccharides and proteins, requiring more sophisticated downstream separation and purification processes. Furthermore, the yield of tanshinone obtained through plant extraction is limited, failing to meet clinical needs and the requirements of green chemistry and sustainable development. Chemical synthesis requires expensive catalysts, resulting in high costs and stringent requirements for certain reaction conditions, thus limiting the large-scale production of tanshinone. Constructing engineered bacteria to produce tanshinone using modern molecular biology and bioengineering techniques, and employing synthetic biology methods to replace traditional plant extraction methods, is more in line with environmental protection and sustainable development requirements. In recent years, many complex natural products have been able to be produced on a large scale through synthetic biology. The artificial synthesis pathway of tanshinone has been elucidated and preliminarily constructed, but the yield is still some distance away from industrialization.

[0003] The 4-hydroxyphenylacetic acid 3-hydroxylase (EcHpaB) derived from *E. coli* BL21(DE3) is a key enzyme in the biosynthesis of tanshinone. It synergistically works with the reductase component (EcHpaC) of the 4-hydroxyphenylacetic acid 3-hydroxylase from *E. coli* BL21(DE3) to catalyze the ortho-hydroxylation of the phenolic hydroxyl group of 4-hydroxyphenyllactic acid, which has a structure similar to that of the natural substrate, to synthesize tanshinone. However, this technology still suffers from poor substrate specificity and low catalytic efficiency, limiting the yield of tanshinone synthesized by microorganisms. Therefore, improving the biosynthetic efficiency of tanshinone is an urgent problem to be solved to promote the green and efficient synthesis of tanshinone. Summary of the Invention

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

[0005] A second objective of this invention is to provide nucleotide sequences containing genes encoding the EcHpaB mutant and genes encoding the EcHpaC.

[0006] A third objective of this invention is to provide a recombinant expression plasmid containing the above-described nucleotide sequence.

[0007] A fourth objective of this invention is to provide genetically engineered bacteria containing the above-described recombinant expression plasmid.

[0008] The fifth objective of this invention is to provide an application for the fermentation production of tanshinone by the above-mentioned genetically engineered bacteria.

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

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

[0011] The amino acid sequence shown in SEQ ID NO.1 has a methionine mutation at position 214, which is replaced by valine, as shown in SEQ ID NO.3;

[0012] The amino acid sequence shown in SEQ ID NO.1 has an arginine mutation at position 290, which is replaced by asparagine, as shown in SEQ ID NO.4;

[0013] The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 294 where glutamic acid is replaced with isoleucine, as shown in SEQ ID NO.5;

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

[0015] It contains nucleotide sequences encoding the gene for the above-mentioned EcHpaB mutant and the gene encoding the EcHpaC gene;

[0016] EcHpaC is an abbreviation for 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] Recombinant expression plasmids containing the above-mentioned encoding genes.

[0018] Genetically engineered bacteria containing the above recombinant expression plasmid.

[0019] Applications of the above-mentioned genetically engineered bacteria fermentation preparation of tanshinone.

[0020] Advantages of this invention:

[0021] The genetically engineered bacteria (M214V, R290N, and E294I) of this invention, after fermentation in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol for 24 h in shake flasks, produced tanshinone yields of 748.04 mg / L, 895.55 mg / L, and 793.36 mg / L, respectively. This invention overcomes the limitations of poor substrate specificity and low catalytic efficiency in the production of tanshinone catalyzed by 4-hydroxyphenylacetic acid 3-hydroxylase, significantly improving the yield of tanshinone. This invention has the advantages of high catalytic efficiency, simple process, and environmental friendliness. Attached Figure Description

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

[0023] Figure 2 A schematic diagram of the expression plasmids pACYC-EchpaB-EchpaC for EcHpaB and EcHpaC;

[0024] Figure 3 The figure shows the tanshinone yield of the genetically engineered bacterial strain after fermentation in 30 mL of M9Y medium containing 50 μg / mL chloramphenicol for 24 h. Detailed Implementation

[0025] This invention analyzes and designs key amino acids at the substrate-binding pocket inlet and outlet positions of HpaB derived from E. coli BL21(DE3). A saturated mutant library is constructed using the commercially available expression plasmid paCYCDuet-1 as a vector. High-activity 4-hydroxyphenylacetic acid 3-hydroxylase mutants are screened from this library using a high-throughput screening method. Recombinant expression plasmids containing the EcHpaB mutant gene and the gene encoding EcHpaC are constructed using the commercially available expression plasmid paCYCDuet-1. These recombinant expression plasmids are transformed into E. coli TS6 to obtain genetically engineered bacteria.

[0026] The aforementioned Escherichia coli TS6 was derived from commercially available Escherichia coli W3110, modified as follows: E. coli W3110, ΔlacIZ, ΔtyrR, ΔtehB::aroG S180F ,ΔmbhA:tyrA M53I / A354V ,ΔpheA,ΔyjgX::BlfldA.

[0027] Table 1 Gene Accession Numbers

[0028] Gene Name Registry Number (GenBank) lacI BAE76127.1 lacZ BAE76126.1 tyrR BAA14905 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 formulations involved in the following specific embodiments are 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-morpholinopropanesulfonic acid (MOPS), 2 mmol / L magnesium sulfate, 0.1 mmol / L calcium chloride.

[0032] Unless otherwise specified, experimental methods in the following examples were performed under standard 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 will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Example 1

[0035] The crystal configuration (PDB ID: 6QYI) of 4-hydroxyphenylacetic acid 3-hydroxylase (EcHpaB, amino acid sequence as shown in SEQ ID NO. 1) derived from E. coli BL21(DE3) was visualized using Pymol software. Key substrate entry and exit points of the 4-hydroxyphenylacetic acid 3-hydroxylase were analyzed, and candidate sites M214, R290, and E294 were selected. Figure 1 The catechol structure of tanshinone reacts with strong oxidizing agents to form quinone compounds, which appear red. (OD) 400 It has a maximum absorption peak below, and within a certain concentration range, the concentration is related to the OD. 400 Based on the linear relationship between the values, saturated mutant libraries of M214, R290, and E294 were constructed and subjected to high-throughput screening. After fermentation at 37℃ and 180 rpm for 24 h in 96-well deep-well plates containing M9Y medium, 180 μL of the supernatant was added to a 96-well microplate, followed by the addition of 0.1 M sodium periodate solution. After mixing by pipetting and aspiration, the absorbance was measured at 400 nm after standing for 10 min. The OD values ​​were then selected. 400 The high-value mutant strains were sent to a sequencing company for sequencing, which identified three 4-hydroxyphenylacetic acid 3-hydroxylase mutants as beneficial mutants. The 4-hydroxyphenylacetic acid 3-hydroxylase mutant was one of the following:

[0036] The amino acid sequence shown in SEQ ID NO.1 has a methionine mutation at position 214, which is replaced by valine, as shown in SEQ ID NO.3;

[0037] The amino acid sequence shown in SEQ ID NO.1 has an arginine mutation at position 290, which is replaced by asparagine, as shown in SEQ ID NO.4;

[0038] The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 294 where glutamic acid is replaced with isoleucine, as shown in SEQ ID NO.5;

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

[0040] Example 2

[0041] Construction of nucleotide sequences containing the gene encoding the EcHpaB mutant and the gene encoding the EcHpaC:

[0042] EcHpaC is an abbreviation for 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] All primers involved in this invention were synthesized by Ansonda Corporation.

[0044] Upstream and downstream primers and intermediate mutation primers required to construct fragments encoding the EcHpaB and EcHpaC genes were synthesized, respectively. The primer names and nucleotide sequences are shown in Table 2.

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

[0046] With P trc Using -EchpaB-EchpaC as a template, with P trc Using -hpaBC-F (SEQ ID NO.12) and M214V-R (SEQ ID NO.15) as primers, P was amplified. trc -EchpaB M214V -EchpaC upstream gene fragment, with P trc Using -EchpaB-EchpaC as a template, and M214V-F (SEQ ID NO.14), P trc -hpaBC-R (SEQ ID NO.13) was used as a primer to amplify Ptrc -EchpaB M214V -Downstream gene fragment of EchpaC; using primer P trc -hpaBC-F、P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB M214V -EchpaC upstream gene fragment and P trc -EchpaB M214V -EchpaC downstream gene fragment overlaps and extends to obtain the complete mutant gene P trc -EchpaB M214V -EchpaC, whose sequence is shown in SEQ ID NO.7, was purified by gel extraction after verification by agarose gel electrophoresis.

[0047] With P trc Using -EchpaB-EchpaC as a template, with P trc Using -hpaBC-F and R290N-R (SEQ ID NO.17) as primers, P was amplified. trc -EchpaB R290N -EchpaC upstream gene fragment, with P trc Using -EchpaB-EchpaC as a template, and with R290N-F (SEQ ID NO.16) and P trc -hpaBC-R was used as a primer to amplify P trc -EchpaB R290N -Downstream gene fragment of EchpaC; using primer P trc -hpaBC-F、P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB R290N -EchpaC upstream gene fragment and P trc -EchpaB R290N -EchpaC downstream gene fragment overlaps and extends 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 purified DNA fragment was obtained by gel extraction and recovery.

[0048] With P trc Using -EchpaB-EchpaC as a template, with P trc Using -hpaBC-F and E294I-R (SEQ ID NO.19) as primers, P was amplified. trc -EchpaB E294I-Upstream gene fragment of EchpaC, newly added: with P trc Using EchpaB-EchpaC as a template, and with E294I-F (SEQ ID NO.18) and P trc -hpaBC-R was used as a primer to amplify P trc -EchpaB E294I -Downstream gene fragment of EchpaC; using primer P trc -hpaBC-F、P trc -hpaBC-R was used for fusion PCR, and P trc -EchpaB E294I -EchpaC upstream gene fragment and P trc -EchpaB E294I -EchpaC downstream gene fragment overlaps and extends to obtain the complete mutant gene P trc -EchpaB E294I -EchpaC, whose sequence is shown in SEQ ID NO.9, was purified by gel extraction after verification by agarose gel electrophoresis.

[0049] Table 2 Primer sequence list

[0050]

[0051] Example 3

[0052] Recombinant expression plasmids containing nucleotide sequences encoding the EcHpaB mutant and the EcHpaC gene were constructed, as follows:

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

[0054] The gene fragment P amplified in Example 2 was processed using Ibotek's seamless cloning enzyme 2X MultiF Seamless Assembly Mix. trc -EchpaB-EchpaC、P trc -EchpaB M214V -EchpaC、P trc -EchpaB R290N -EchpaC and P trc -EchpaB E294I-EchpaC was ligated to the linearized fragment of the pACYCDuet-1 empty plasmid. The mixture of the fragment to be ligated and the linearized fragment of the pACYCDuet-1 empty plasmid was reacted at 50°C for 30 min. The resulting system was then transformed into E. coli DH5α competent cells. After plating on LB agar plates containing 50 μg / mL chloramphenicol, the cells were incubated overnight at 37°C to obtain single colonies. Using P in Table 2... trc Preliminary colony PCR verification and further sequencing verification were performed using primers -hpaBC-F and hpaBC-JD-R (SEQ ID NO.20). Plasmids were extracted from the correctly verified strains to 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 and pACYC-EchpaB respectively M214V -EchpaC、pACYC-EchpaB R290N -EchpaC and pACYC-EchpaB E294I The genetically engineered bacteria of -EchpaC are produced through the following steps:

[0057] The constructed recombinant expression plasmids pACYC-EchpaB-EchpaC and pACYC-EchpaB were used. M214V -EchpaC、pACYC-EchpaB R290N -EchpaC and pACYC-EchpaB E294I -EchpaC was electroporated into E. coli TS6 competent cells. The E. coli cells transformed with the expression plasmid were plated on LB agar plates containing 50 μg / mL chloramphenicol and incubated overnight at 37°C to obtain the control strain TS6 / pACYC-EchpaB-EchpaC (abbreviated as control strain), and three E. coli strains expressing the 4-hydroxyphenylacetic acid 3-hydroxylase mutant 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 sequentially transferred to 5 mL of LB liquid medium containing 50 μg / mL chloramphenicol and cultured overnight. 1 mL of the bacterial culture was transferred to a preservation tube, 1 mL of 30% (v / v) glycerol was added and mixed well. The tubes were then labeled and stored at -80°C.

[0059] Example 5

[0060] The production of tanshinone using a genetically engineered bacterial strain through shake-flask fermentation includes the following steps:

[0061] Shake-flask fermentation was performed using the control strain, M214V, R290N, and E294I, with three replicates for each strain. 5 μL of bacterial culture was transferred from the culture tube to 5 mL of LB medium containing 50 μg / mL chloramphenicol and cultured at 37°C and 220 rpm for 12 h in a shaker. The culture was then transferred to 30 mL of M9Y medium containing 50 μg / mL chloramphenicol and fermented at 37°C and 180 rpm in a shaker. At 4 h, IPTG was added to a final concentration of 0.1 mM to induce protein expression. After 24 h of fermentation, the culture was stopped, and the product yield in the fermentation broth was determined.

[0062] Example 6

[0063] The concentration of tanshinone in the fermentation broth was detected by HPLC, as detailed below:

[0064] Take 1 mL of each fermentation broth, centrifuge at 12000 rpm for 10 min, and then take the supernatant for HPLC detection via membrane filtration.

[0065] The detection conditions were as follows: Bona C18 (250 mm × 4.6 mm) column; PDA detector; detection wavelength 281 nm; flow rate 1 mL / min. The mobile phase was: 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 yields of M214V, R290N, and E294I reached 748.04 mg / L, 895.55 mg / L, and 793.36 mg / L, respectively, which were 51.12%, 80.09%, and 60.27% higher than that of the control strain (497.28 mg / L).

[0067] The results showed that the 4-hydroxyphenylacetic acid 3-hydroxylase mutant EcHpaB of the present invention... M214V EcHpaB R290N and EcHpaB E294IIt significantly improves the efficiency of tanshinone production in Escherichia coli, overcomes the limitations of poor substrate specificity and low catalytic efficiency of natural hydroxylases, and has broad application prospects in industry.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any simple variations and modifications made in accordance with the claims and description of this application shall be within the scope of protection of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A mutant of 4-hydroxyphenylacetic acid 3-hydroxylase, wherein the amino acid sequence of the 4-hydroxyphenylacetic acid 3-hydroxylase is shown in SEQ ID NO. 1; characterized in that... The 4-hydroxyphenylacetic acid 3-hydroxylase mutant is one of the following: Only 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 amino acid sequence shown in SEQ ID NO.1 has an arginine mutation at position 290, which is replaced by asparagine, as shown in SEQ ID NO.4; Alternatively, the amino acid sequence shown in SEQ ID NO.1 may have a mutation at position 294, where glutamic acid is replaced with isoleucine, as shown in SEQ ID NO.

5.

2. A nucleic acid molecule containing the gene encoding the 4-hydroxyphenylacetic acid 3-hydroxylase mutant of claim 1 and the EcHpaC gene, wherein the nucleotide sequence encoding the EcHpaC gene is shown in SEQ ID NO.

2.

3. A recombinant expression plasmid containing the nucleic acid molecule of claim 2.

4. Genetically engineered bacteria containing the recombinant expression plasmid as described in claim 3.

5. Application of the genetically engineered bacteria of claim 4 for the fermentation preparation of tanshinone.

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