4-hydroxyphenylacetic acid 3-hydroxylase mutant, coding gene and application

Abstract

The invention discloses a 4-hydroxyphenylacetic acid 3-hydroxylase mutant, a coding gene of the 4-hydroxyphenylacetic acid 3-hydroxylase mutant and application of the 4-hydroxyphenylacetic acid 3-hydroxylase mutant and the coding gene of the 4-hydroxyphenylacetic acid 3-hydroxylase mutant. The 4-hydroxyphenylacetic acid 3-hydroxylase mutant is one of the following: glutamine at the 212th site of an amino acid sequence as shown in SEQ ID NO.1 is mutated into alanine, as shown in SEQ ID NO.3; the glutamic acid at the 216th site of the amino acid sequence as shown in SEQ ID NO.1 is mutated into alanine, as shown in SEQ ID NO.4; after the genetically engineered bacteria (Q212A and E216A) are subjected to shake flask fermentation in an M9Y culture medium containing 100 [mu] g / mL of ampicillin for 24 h, the tanshinol yield reaches 757.18 mg / L, and the tanshinol yield reaches 744.36 mg / L. The genetically engineered bacteria are good in substrate specificity in tanshinol biosynthesis, high in catalytic efficiency, simple in process and environmentally friendly.

Description

Technical Field

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

[0002] Salvianic acid A (SAA) is one of the main active ingredients of Danshen, a phenylpropanoid compound with multiple biological activities. In nature, it is mostly found in the rhizomes of Danshen and Salvia officinalis. It is named because it is mainly extracted from the traditional Chinese plant Danshen. Its chemical name is β-(3,4-dihydroxyphenyl) lactic acid. Clinical studies have shown that danshen has multiple pharmacological activities such as myocardial protection, anti-oxidation, and anti-tumor. It is a chemical component with great medicinal value and has broad market prospects. At present, the main methods for obtaining danshen are mainly plant extraction and chemical synthesis. The former has cumbersome extraction steps and low yield; the latter has complex chemical synthesis and difficulty in subsequent purification and separation, which limits the large-scale production of danshen.

[0003] In recent years, a variety of complex natural products have been able to achieve large-scale production through synthetic biology. Modern molecular biology and bioengineering technologies have been used to construct engineered bacteria that produce danshensu. Using synthetic biology methods to replace traditional methods to produce danshensu is more in line with the requirements of environmental protection and sustainable development. Although people have constructed an artificial biosynthetic pathway for danshensu through metabolic pathway analysis and enzyme mining, and produced danshensu in Escherichia coli, the yield is still low, making it difficult to achieve industrialization of danshensu.

[0004] 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

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a 4-hydroxyphenylacetic acid 3-hydroxylase mutant.

[0006] 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.

[0007] The third object of the present invention is to provide a recombinant expression plasmid containing the above nucleotide sequence.

[0008] The fourth object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0009] The fifth objective of the present invention is to provide the application of the above-mentioned genetically engineered bacteria in the fermentation production of danshensu.

[0010] The technical solution of the present 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 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; the 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;

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

[0014] A nucleotide sequence containing a gene encoding an EcHpaB mutant and a gene encoding an EcHpaC;

[0015] 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.

[0016] A recombinant expression plasmid containing the above nucleotide sequence.

[0017] A genetically engineered bacterium containing the above-mentioned recombinant expression plasmid.

[0018] The application of the above genetically engineered bacteria in the fermentation production of danshensu.

[0019] Beneficial effects of the present invention:

[0020] The genetically engineered bacteria (Q212A and E216A) of the present invention can produce 757.18 mg / L and 744.36 mg / L of danshensu respectively after shaking flask fermentation for 24 hours in an M9Y culture medium containing 100 μg / mL ampicillin. The present invention has good substrate specificity and high catalytic efficiency in the biosynthesis of danshensu, simple process, and is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Schematic diagram of the expression plasmid pTrc99a-EchpaB-EchpaC of EcHpaB and EcHpaC.

[0022] Figure 2 These are the results of shake flask fermentation of danshensu using genetically engineered bacterial strains. DETAILED DESCRIPTION

[0023] Compared with 4-hydroxyphenylacetic acid, the natural substrate of 4-hydroxyphenylacetic acid 3-hydroxylase (abbreviated as EcHpaB), the space occupied by the 4-hydroxyphenyllactic acid molecule, the precursor of danshensu, is larger. Accordingly, the binding pocket of EcHpaB also needs to be enlarged to better accommodate the substrate molecule for catalysis. Based on this assumption, the present invention performs site-directed mutagenesis on EcHpaB (amino acid sequence as shown in SEQ ID NO.1), and mutates the key amino acids I206-P218 of the enzyme substrate recognition into alanine, a non-polar amino acid with a smaller residue, one by one, so that the substrate binding pocket is enlarged, and two EcHpaB mutants EcHpaB are screened out. Q212A (SEQ ID NO. 3) and EcHpaB E216A (SEQ ID NO. 4) A recombinant expression plasmid containing a gene encoding an EcHpaB mutant and a gene encoding EcHpaC (SEQ ID NO. 2) was constructed using expression plasmid pTrc99a (commercially available).

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

[0025] E. coli TS6 was based on E. coli W3110 (commercially available) and was 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 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

[0028] The culture medium formula involved in the following specific implementation examples is 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-morpholinepropanesulfonic acid (MOPS), 2 mmol / L magnesium sulfate, and 0.1 mmol / L calcium chloride.

[0031] 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.

[0032] 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.

[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 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;

[0036] The 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;

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

[0038] Example 2

[0039] Construction of a nucleotide sequence containing a gene encoding an EcHpaB mutant and a gene encoding an EcHpaC gene:

[0040] 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.

[0041] The primers involved in the present invention were all commissioned to be synthesized by Anshengda Company.

[0042] 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.

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

[0044] P trc -EchpaB-EchpaC as template, with P trc -hpaBC-F (SEQ ID NO.10), hpaB-Q212A-R (SEQ ID NO.13) were used as primers to amplify P trc -EchpaB Q212A -Upstream gene fragment of EchpaC;

[0045] P trc -EchpaB-EchpaC as template, using hpaB-Q212A-F (SEQ ID NO.12), P trc -hpaBC-R (SEQ ID NO.11) was used as primer to amplify P trc -EchpaB Q212A - Downstream gene fragment of EchpaC;

[0046] Primer P trc -hpaBC-F (SEQ ID NO.10), P trc -hpaBC-R (SEQ ID NO.11) was subjected to fusion PCR, and P trc -EchpaB Q212A -EchpaC upstream gene fragment and P trc -EchpaB Q212A -EchpaC downstream gene fragment overlap extension to obtain the complete mutant gene P trc -EchpaB Q212A -EchpaC (SEQ ID NO.6) 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 (SEQ ID NO.10), hpaB-E216A-R (SEQ ID NO.15) were used as primers to amplify Ptrc -Echpa BC E216A -The upstream gene fragment of EchpaC,

[0048] P trc -EchpaB-EchpaC as template, using hpaB-E216A-F (SEQ ID NO.14), P trc -hpaBC-R (SEQ ID NO.11) was used as primer to amplify P trc -EchpaB E216A - Downstream gene fragment of EchpaC;

[0049] Primer P trc -hpaBC-F (SEQ ID NO.10), P trc -hpaBC-R (SEQ ID NO.11) was subjected to fusion PCR, and P trc -EchpaB E216A -EchpaC upstream gene fragment and P trc -EchpaB E216A -EchpaC downstream gene fragment overlap extension to obtain the complete mutant gene P trc -EchpaB E216A -EchpaC (SEQ ID NO.7) was verified by agarose gel electrophoresis and the purified DNA fragment was obtained by gel excision and recovery.

[0050] Table 2 Primer sequence list

[0051]

[0052] Example 3

[0053] The recombinant expression plasmid containing the gene encoding the EcHpaB mutant and the nucleotide sequence encoding the EcHpaC gene was constructed as follows:

[0054] The pTrc99a empty plasmid (commercially available) was selected as a template, and 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 a linearized fragment of the pTrc99a empty plasmid.

[0055] The gene fragment P amplified in Example 1 was cloned using the seamless cloning enzyme 2X MultiF Seamless Assembly Mix from Ibotek. trc -EchpaB-EchpaC, P trc -EchpaBQ212A -EchpaC and P trc -EchpaB E216A -EchpaC were connected with the linearized fragment of pTrc99a empty plasmid, and the mixture of the fragment to be connected and the linearized fragment of pTrc99a empty plasmid was reacted at 50°C for 30 minutes, and then transformed into E. coli DH5α competent cells, and then spread on LB solid plates containing 100 μg / ml ampicillin and cultured at 37°C overnight to obtain single clones. trc -hpaBC-F (SEQ ID NO.10) and hpaBC-JD-R (SEQ ID NO.16) were used as primers for preliminary colony PCR verification and further sequencing verification, and finally the recombinant expression plasmids pTrc99a-EchpaB-EchpaC and pTrc99a-EchpaB were obtained. Q212A -EchpaC, pTrc99a-EchpaB E216A -EchpaC.

[0056] Example 4

[0057] Construct pTrc99a-EchpaB-EchpaC, pTrc99a-EchpaB Q212A -EchpaC, pTrc99a-EchpaB E216A -EchpaC genetic engineering bacteria, the steps are as follows:

[0058] The constructed recombinant expression plasmids pTrc99a-EchpaB-EchpaC, pTrc99a-EchpaB Q212A -EchpaC, pTrc99a-EchpaB E216A -EchpaC were electroporated into E. coli TS6 competent cells. They were spread on LB solid medium plates containing 100 μg / ml ampicillin and cultured in a 37°C incubator overnight to obtain the control strain TS6 / pTrc99a-EchpaB-EchpaC (abbreviated as control strain, Figure 2 coli strains TS6 / pTrc99a-EchpaB expressing 4-hydroxyphenylacetic acid 3-hydroxylase mutants Q212A -EchpaC (abbreviated as Q212A), and TS6 / pTrc99a-EchpaB E216A -EchpaC (abbreviated as E216A).

[0059] Preservation operation: Pick single colonies in turn and transfer them to 5 mL of LB liquid culture medium containing 100 μg / ml ampicillin for overnight culture. Pipette 1 mL of bacterial solution and transfer it to a bacterial preservation tube. Add 1 mL of 30% (v / v) glycerol and mix well. After marking, store in a -80°C refrigerator.

[0060] Example 5

[0061] The application of genetically engineered bacterial strains in the production of danshensu by shake flask fermentation comprises the following steps:

[0062] The control strain, Q212A and E216A constructed in Example 3 were selected 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 100 μg / ml ampicillin, and cultured at 220 rpm in a 37°C shaker for 12 h, then transferred to 30 mL of M9Y medium containing 100 μg / ml ampicillin, and fermented at 180 rpm in a 37°C shaker. IPTG with a final concentration of 0.1 mM was added at 4 h for protein induction expression, and the culture was stopped after 24 h of fermentation to determine the product yield in the fermentation broth.

[0063] Example 6

[0064] The concentration of danshensu in the fermentation broth was detected by HPLC, as follows:

[0065] 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.

[0066] 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.

[0067] The yield determination results are as follows Figure 2 As shown, the tanshinone production of the control strain, Q212A and E216A were 516.64 mg / L, 757.18 mg / L and 744.36 mg / L, respectively. The production of Q212A and E216A increased by 46.70% and 44.22% respectively compared with the control strain.

[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 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; the 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 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 nucleotide sequence of 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 fermentation to produce danshensu.

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