4-Hydroxyphenylacetic acid 3-hydroxylase mutants, their encoding genes, and their applications

Abstract

This invention discloses a 4-hydroxyphenylacetic acid 3-hydroxylase mutant, its encoding gene, and its applications. The 4-hydroxyphenylacetic acid 3-hydroxylase mutant is one of the following: glutamine at position 212 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, as shown in SEQ ID NO.3; or glutamic acid at position 216 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine, as shown in SEQ ID NO.4. The genetically engineered bacteria (Q212A and E216A) of this invention, after shake-flask fermentation in M9Y medium containing 100 μg / mL ampicillin for 24 h, achieved tanshinone yields of 757.18 mg / L and 744.36 mg / L, respectively. This invention demonstrates good substrate specificity, high catalytic efficiency, simple process, and environmental friendliness in tanshinone biosynthesis.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and synthetic biology, specifically relating to 4-hydroxyphenylacetic acid 3-hydroxylase mutants, their encoding genes, and their applications. Background Technology

[0002] Salvianic acid A (SAA) is one of the main active components of Salvia miltiorrhiza. It is a phenylpropanoid compound with various biological activities, found in the rhizomes of Salvia miltiorrhiza and Salvia miltiorrhiza in nature. It is named after its primary extraction source from the traditional Chinese plant Salvia miltiorrhiza, and its chemical name is β-(3,4-dihydroxyphenyl)lactic acid. Clinical studies have shown that tanshinone possesses various pharmacological activities, including myocardial protection, antioxidant activity, and antitumor activity, making it a highly valuable medicinal component with broad market prospects. Currently, the main methods for obtaining tanshinone are plant extraction and chemical synthesis. The former involves cumbersome extraction steps and low yields, while the latter's complex chemical synthesis and difficulties in subsequent purification and separation limit the large-scale production of tanshinone.

[0003] In recent years, many complex natural products have been able to be produced on a large scale using synthetic biology techniques. Constructing engineered bacteria to produce tanshinone using modern molecular biology and bioengineering technologies, and replacing traditional methods with synthetic biology approaches, better aligns with environmental protection and sustainable development requirements. Although artificial biosynthetic pathways for tanshinone have been constructed through metabolic pathway analysis and enzyme discovery, producing tanshinone in *E. coli*, the yield remains low, hindering the industrialization of tanshinone.

[0004] 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

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a 4-hydroxyphenylacetic acid 3-hydroxylase mutant.

[0006] A second objective of this invention is to provide nucleotide sequences containing genes encoding the EcHpaB mutant and genes encoding the EcHpaC.

[0007] A third objective of this invention is to provide a recombinant expression plasmid containing the above-described nucleotide sequence.

[0008] A fourth objective of this invention is to provide genetically engineered bacteria containing the above-described recombinant expression plasmid.

[0009] The fifth objective of this invention is to provide an application for the fermentation production of tanshinone by the above-mentioned genetically engineered bacteria.

[0010] The technical solution of this invention is summarized as follows:

[0011] 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:

[0012] The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 212 where glutamine is replaced by alanine, as shown in SEQ ID NO.3; the amino acid sequence shown in SEQ ID NO.1 has a mutation at position 216 where glutamic acid is replaced by alanine, as shown in SEQ ID NO.4;

[0013] The 4-hydroxyphenylacetic acid 3-hydroxylase is abbreviated as EcHpaB.

[0014] Nucleotide sequences containing the gene encoding the EcHpaB mutant and the gene encoding the EcHpaC gene;

[0015] 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.

[0016] Recombinant expression plasmids containing the above nucleotide sequences.

[0017] Genetically engineered bacteria containing the above recombinant expression plasmid.

[0018] Application of the above-mentioned genetically engineered bacteria in the fermentation production of tanshinone.

[0019] The beneficial effects of this invention are:

[0020] The genetically engineered bacteria (Q212A and E216A) of the present invention achieved tanshinone yields of 757.18 mg / L and 744.36 mg / L, respectively, after shake-flask fermentation in M9Y medium containing 100 μg / mL ampicillin for 24 h. The present invention exhibits good substrate specificity, high catalytic efficiency, simple process, and is environmentally friendly in tanshinone biosynthesis. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the expression plasmids pTrc99a-EchpaB-EchpaC for EcHpaB and EcHpaC.

[0022] Figure 2 The results of shake-flask fermentation of tanshinone by genetically engineered bacterial strains. Detailed Implementation

[0023] Compared to 4-hydroxyphenylacetic acid, the natural substrate of 4-hydroxyphenylacetic acid 3-hydroxylase (EcHpaB), the 4-hydroxyphenyllactic acid molecule, a precursor of tanshinone, occupies a larger space. Correspondingly, the binding pocket of EcHpaB needs to be enlarged to better accommodate the substrate molecule for catalysis. Based on this assumption, this invention performs site-directed mutagenesis on EcHpaB (amino acid sequence shown in SEQ ID NO.1), sequentially mutating the key substrate recognition amino acids I206-P218 to the smaller, nonpolar amino acid alanine, thereby enlarging the substrate binding pocket. Two EcHpaB mutants, EcHpaB, were then screened from these mutants. Q212A (SEQ ID NO.3) and EcHpaB E216A (SEQ ID NO.4). A recombinant expression plasmid containing the gene encoding the EcHpaB mutant and the gene encoding EcHpaC (SEQ ID NO.2) was constructed using the expression plasmid pTrc99a (commercially available).

[0024] The recombinant expression plasmid was transformed into Escherichia coli TS6 to obtain the genetically engineered bacteria.

[0025] Escherichia coli TS6 is 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.

[0026] Table 1 Gene Accession Numbers

[0027] 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

[0028] The culture medium formulations used in the following specific implementation examples are as follows:

[0029] LB medium: 5 g / L yeast extract, 10 g / L casein peptone, 10 g / L sodium chloride.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Example 1

[0034] 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:

[0035] The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 212 where glutamine is replaced by alanine, as shown in SEQ ID NO.3;

[0036] The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 216 where glutamic acid is replaced with alanine, as shown in SEQ ID NO.4;

[0037] The 4-hydroxyphenylacetic acid 3-hydroxylase is abbreviated as EcHpaB.

[0038] Example 2

[0039] Construction of nucleotide sequences containing the gene encoding the EcHpaB mutant and the gene encoding the EcHpaC:

[0040] 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.

[0041] All primers involved in this invention were synthesized by Ansonda Corporation.

[0042] Upstream and downstream primers and intermediate mutation primers required to construct fragments encoding the EcHpaB and EcHpaC genes were synthesized, and the primer names and nucleotide sequences are shown in Table 2.

[0043] Using the genome of E. coli BL21(DE3) (commercially available) strain as a template, and P trc -hpaBC-F (SEQ ID NO.10), P trc Using -hpaBC-R (SEQ ID NO.11) as primers, fragment P containing the genes encoding EcHpaB and EcHpaC was amplified. trc -EchpaB-EchpaC (SEQ ID NO.5) was validated by agarose gel electrophoresis, and the purified DNA fragment was recovered by gel excision.

[0044] With P trc Using -EchpaB-EchpaC as a template, with P trc Using hpaBC-F (SEQ ID NO. 10) and hpaB-Q212A-R (SEQ ID NO. 13) as primers, P was amplified. trc -EchpaB Q212A -Upstream gene fragments of EchpaC;

[0045] With P trc Using -EchpaB-EchpaC as a template, and with hpaB-Q212A-F (SEQ ID NO.12) and P trc Using -hpaBC-R (SEQ ID NO.11) as primers, P was amplified. trc -EchpaB Q212A Downstream gene fragments of -EchpaC;

[0046] With primer P trc -hpaBC-F (SEQ ID NO.10), P trc -hpaBC-R (SEQ ID NO.11) was used for fusion PCR, and P was added. trc -EchpaB Q212A -EchpaC upstream gene fragment and P trc -EchpaB Q212A -EchpaC downstream gene fragment overlaps and extends to obtain the complete mutant gene P trc -EchpaB Q212A -EchpaC (SEQ ID NO.6) was validated by agarose gel electrophoresis, and the purified DNA fragment was recovered by gel excision.

[0047] With P trc Using -EchpaB-EchpaC as a template, with P trc Using hpaBC-F (SEQ ID NO.10) and hpaB-E216A-R (SEQ ID NO.15) as primers, P was amplified.trc -EchpaBC E216A -EchpaC upstream gene fragment,

[0048] With P trc Using -EchpaB-EchpaC as a template, and with hpaB-E216A-F (SEQ ID NO.14) and P trc Using -hpaBC-R (SEQ ID NO.11) as primers, P was amplified. trc -EchpaB E216A Downstream gene fragments of -EchpaC;

[0049] With primer P trc -hpaBC-F (SEQ ID NO.10), P trc -hpaBC-R (SEQ ID NO.11) was used for fusion PCR, and P was added. trc -EchpaB E216A -EchpaC upstream gene fragment and P trc -EchpaB E216A -EchpaC downstream gene fragment overlaps and extends to obtain the complete mutant gene P trc -EchpaB E216A -EchpaC (SEQ ID NO.7) was validated by agarose gel electrophoresis, and the purified DNA fragment was recovered by gel cutting.

[0050] Table 2 Primer sequence list

[0051]

[0052] Example 3

[0053] Recombinant expression plasmids containing nucleotide sequences encoding the EcHpaB mutant and the EcHpaC gene were constructed, as follows:

[0054] The pTrc99a empty vector plasmid (commercially available) was selected as a template. The plasmid was amplified by PCR using primers 99a-xianxing-R (SEQ ID NO. 8) and 99a-xianxing-F (SEQ ID NO. 9) in Table 2. The template was digested and purified using DpnI enzyme to obtain the linearized fragment of the pTrc99a empty plasmid.

[0055] The gene fragment P amplified in Example 1 was processed using Ibotek's 2X MultiF Seamless Assembly Mix seamless cloning enzyme. trc -EchpaB-EchpaC、P trc -EchpaBQ212A -EchpaC and P trc -EchpaB E216A -EchpaC was ligated to the linearized fragment of the pTrc99a empty plasmid. The mixture of the fragment to be ligated and the linearized fragment of the pTrc99a empty plasmid was reacted at 50°C for 30 min, then transformed into E. coli DH5α competent cells. After being plated on LB agar plates containing 100 μg / ml ampicillin, single colonies were obtained by incubation at 37°C overnight. Using P in Table 2 trc Using hpaBC-F (SEQ ID NO. 10) and hpaBC-JD-R (SEQ ID NO. 16) as primers, preliminary colony PCR verification and further sequencing verification were performed, ultimately yielding the recombinant expression plasmids pTrc99a-EchpaB-EchpaC and pTrc99a-EchpaB. Q212A -EchpaC、pTrc99a-EchpaB E216A -EchpaC.

[0056] Example 4

[0057] Construct pTrc99a-EchpaB-EchpaC and pTrc99a-EchpaB respectively Q212A -EchpaC、pTrc99a-EchpaB E216A The genetically engineered bacteria of EchpaC are produced through the following steps:

[0058] The constructed recombinant expression plasmids pTrc99a-EchpaB-EchpaC and pTrc99a-EchpaB were used. Q212A -EchpaC、pTrc99a-EchpaB E216A -EchpaC was electroporated into E. coli TS6 competent cells. The cells were plated on LB agar plates containing 100 μg / ml ampicillin and incubated overnight at 37°C to obtain the control strain TS6 / pTrc99a-EchpaB-EchpaC (abbreviated as control strain). Figure 2 Two *E. coli* strains, TS6 / pTrc99a-EchpaB, expressing the 4-hydroxyphenylacetic acid 3-hydroxylase mutant (changed to control strain), were used. Q212A -EchpaC (abbreviated as Q212A), and TS6 / pTrc99a-EchpaB E216A -EchpaC (abbreviated as E216A).

[0059] Preservation procedure: Pick single colonies sequentially and transfer them to 5 mL of LB liquid medium containing 100 μg / mL ampicillin and incubate overnight. Transfer 1 mL of the bacterial culture to a preservation tube, add 1 mL of 30% (v / v) glycerol, mix well, label, and store in a -80°C refrigerator.

[0060] Example 5

[0061] The application of genetically engineered bacterial strains in the production of tanshinone via shake-flask fermentation includes the following steps:

[0062] The control strain constructed in Example 3, as well as Q212A and E216A, were selected for shake-flask fermentation, with three parallel experiments set up for each strain. 5 μL of bacterial culture was transferred from the preservation tube to 5 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37°C and 220 rpm for 12 h in a shaker. Subsequently, it was transferred to 30 mL of M9Y medium containing 100 μg / mL ampicillin 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.

[0063] Example 6

[0064] The concentration of tanshinone in the fermentation broth was detected by HPLC, as detailed below:

[0065] 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.

[0066] 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.

[0067] Yield measurement results as follows Figure 2 As shown, the tanshinone yields of the control strain, Q212A, and E216A were 516.64 mg / L, 757.18 mg / L, and 744.36 mg / L, respectively. The yields of Q212A and E216A were 46.70% and 44.22% higher than those of the control strain, respectively.

[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 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; characterized in that... The 4-hydroxyphenylacetic acid 3-hydroxylase mutant is one of the following: The amino acid sequence shown in SEQ ID NO.1 has a mutation at position 212 where glutamine is replaced by alanine, as shown in SEQ ID NO.3; the amino acid sequence shown in SEQ ID NO.1 has a mutation at position 216 where glutamic acid is replaced by alanine, as shown in SEQ ID NO.4; The 4-hydroxyphenylacetic acid 3-hydroxylase is abbreviated as EcHpaB.

2. A nucleotide sequence containing the gene encoding the EcHpaB mutant of claim 1 and the gene encoding EcHpaC; 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.

3. A recombinant expression plasmid containing the nucleotide sequence of claim 2.

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

5. The application of the genetically engineered bacteria of claim 4 for the fermentation production of tanshinone.

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