CN109266625B

CN109266625B - Coding gene of S-type 1-phenyl ethanol synthetase and application thereof

Info

Publication number: CN109266625B
Application number: CN201811139441.4A
Authority: CN
Inventors: 东方; 周瀛; 杨子银; 彭琪媛
Original assignee: Guangdong Food and Drugs Vocational College
Current assignee: Guangdong Food and Drugs Vocational College
Priority date: 2018-09-28
Filing date: 2018-09-28
Publication date: 2021-08-17
Anticipated expiration: 2038-09-28
Also published as: CN109266625A

Abstract

The invention discloses a protein, the amino acid sequence of which is shown as SEQ ID No.1, or the protein which has the same or higher activity after the amino acid sequence shown as SEQ ID No.1 is substituted, deleted and/or added with one or more amino acids and/or the tail end is modified, and can be used as S-type 1-phenyl ethanol synthetase. The invention also discloses a method for synthesizing the S-type 1-phenyl ethanol, the ee percent of the S-type 1-phenyl ethanol synthesized by the method is more than 99 percent, the conversion efficiency of the acetophenone is higher, the method is low in cost and simple in operation steps, and the environmental pollution is avoided.

Description

Coding gene of S-type 1-phenyl ethanol synthetase and application thereof

Technical Field

The invention relates to an S-type 1-phenyl ethanol synthetase, a gene for coding the synthetase, a vector containing the gene and application thereof.

Background

The demand of chiral compounds in the pharmaceutical and fragrance markets is increasing, S-type 1-phenylethanol is an intermediate of many compounds used in pharmacy, and S-type 1-phenylethanol has a mixed fragrance similar to hyacinth, gardenia and strawberry, and is widely used in the cosmetic industry as a fragrance substance. In the plant kingdom, 1-phenylethanol accumulates in large amounts in plants of the camellia family, but there are technical barriers to obtaining 1 phenylethanol in a single configuration by direct plant isolation, and the feasibility is very low.

The S-type 1-phenyl ethanol is obtained by a chemical synthesis method mostly. The chemical synthesis route requires expensive catalysts and cumbersome protection/deprotection steps and harsh reaction conditions. Therefore, the demand for designing a more economical and more environment-friendly S-type 1-phenyl ethanol synthesis method is urgent. Biosynthesis is a method of compound synthesis that has emerged following chemical synthesis. In the aspect of chiral compound synthesis application, biosynthesis has the characteristics of low cost, low energy consumption, no environmental pollution, single chiral structure of a product and the like, thereby receiving wide attention.

There are currently documents (Dong F, Zhou Y, Zeng L, et al. optimization of the Production of 1-phenyl ethanol Using Enzymes from Flowers of Tea (Camellia sinensis) Plants [ J ]. Molecules,2017,22(1):131.) reporting synthetases that specifically produce 1-phenyl ethanol (mixture of R-type 1-phenyl ethanol and S-type 1-phenyl ethanol), but there has been no report on the specific Production of S-type 1-phenyl ethanol synthetases in Plants. Therefore, it is very important to provide a plant-derived synthase capable of specifically synthesizing S-type 1-phenylethanol.

Disclosure of Invention

The invention aims to provide an S-type 1-phenyl ethanol synthetase, a gene for coding the synthetase, a vector containing the gene and application thereof.

The technical scheme adopted by the invention is as follows:

a protein, the amino acid sequence of which is shown as SEQ ID No.1, or the protein which has the same or higher activity after the amino acid sequence shown as SEQ ID No.1 is substituted, deleted and/or added with one or more amino acids and/or the end is modified;

SEQ ID No.1：

MAKKVESSERGLRWSLAGMTALVTGGTRGIGHDVVEELADKGAIVHTCSRNESELSRCLQEWSAKGFTVTGSVCDVSSRPQREKMLDEVSSLFNGKLNILINNVGTTLWKPTMEYSAEEYSMLMATNLESAFHLCQLAYPLLKASGVGSIVFISSVAGLVHTGSGSVYGASKGAMNQLTKNLACEWAKDNIRSNCVTPFCIRTPLLQHLMDKQEFLDKVRSRTPLGRPGEPNEISSLVAFLCMPTSSYITGQIISVDGGMTVYGFSPN。

sequences encoding the above proteins.

Further, the sequence is shown as SEQ ID No. 2;

SEQ ID No.2：

ATGGCGAAGAAGGTGGAGAGCAGTGAGAGAGGTTTGAGATGGTCTCTCGCCGGAATGACCGCTCTCGTCACCGGCGGCACTCGCGGCATCGGGCACGATGTTGTGGAGGAACTAGCCGATAAAGGGGCGATCGTGCACACCTGTTCTCGCAACGAATCCGAGCTCAGTCGTTGCTTGCAGGAATGGTCCGCCAAGGGTTTCACGGTCACCGGTTCGGTCTGCGATGTATCGTCTCGGCCTCAAAGAGAGAAGATGTTGGATGAAGTCTCTTCTCTCTTCAATGGCAAGCTCAATATCCTTATAAACAATGTTGGCACAACCCTCTGGAAGCCTACTATGGAGTATAGTGCTGAAGAATACTCAATGCTCATGGCTACCAATCTTGAGTCCGCTTTCCATCTCTGCCAACTTGCATATCCTCTTCTAAAAGCTTCTGGAGTTGGAAGCATTGTGTTTATTTCCTCTGTTGCTGGTTTGGTGCACACCGGTTCTGGATCCGTTTATGGAGCTAGCAAAGGTGCAATGAATCAACTTACAAAAAATTTGGCTTGTGAGTGGGCAAAAGATAACATCCGAAGTAATTGTGTTACACCCTTTTGTATCAGAACCCCACTCTTACAACATTTGATGGACAAGCAAGAGTTCCTGGATAAGGTAAGATCTAGAACTCCTCTTGGACGCCCTGGAGAGCCAAATGAAATATCATCCTTGGTGGCATTCCTTTGTATGCCTACTTCATCTTACATCACTGGTCAGATCATTTCTGTTGACGGAGGAATGACTGTGTATGGTTTCTCACCTAATTAG。

a cloning vector comprising the above sequence.

An expression vector containing the above sequence.

A preparation method of S-type 1-phenylethanol synthetase comprises introducing the expression vector into host cells, and expressing to obtain S-type 1-phenylethanol synthetase.

A biosynthesis method of S-type 1-phenyl ethanol, which uses the protein as a synthetase catalytic reaction.

Further, acetophenone is used as a substrate.

Further, coenzyme NADPH is required to be added into the system.

Further, the pH value is 7-8, and the reaction temperature is 45-55 ℃.

The invention has the beneficial effects that:

1. the synthetic method of the S-type 1-phenyl ethanol has low cost and simple operation steps, and simultaneously avoids environmental pollution.

2. The ee percent of the S-type 1-phenyl ethanol generated by catalyzing acetophenone by the S-type 1-phenyl ethanol synthetase is more than 99 percent, and the conversion efficiency of the acetophenone is high.

3. The invention discloses an amino acid sequence of S-type 1-phenyl ethanol synthetase and a coding gene sequence thereof, which can be used as a skeleton for future gene modification and provide a foundation for designing S-type 1-phenyl ethanol synthetase with higher catalytic efficiency through means such as amino acid substitution in the future and artificial intelligence.

Drawings

FIG. 1 shows the result of SDS-PAGE protein electrophoresis of S-type 1-phenylethanol synthase;

FIG. 2 shows the efficiency of enzymatic reactions under different reaction conditions;

FIG. 3 is a graph showing GC-MS identification of acetophenone products catalyzed by S-type 1-phenylethanol synthase.

Detailed Description

The present invention will be further illustrated with reference to the following examples, which are intended to illustrate the invention and are not intended to limit the scope of the invention.

The technical scheme adopted by the invention is as follows:

1. construction of S-type 1-phenylethanol synthase S1PES expression vector

Cloning the S-type 1-phenyl ethanol synthetase S1PES gene shown in SEQ ID No.2 into pET32a prokaryotic expression vector.

SEQ ID No.2：

2. Prokaryotic expression of S-type 1-phenyl ethanol synthetase S1PES

The constructed vector S1PES-pET32a is transferred into an expression strain Rosetta, and then monoclonal shake bacteria are picked. OD of bacterial liquid₆₀₀Adding at about 0.6The final concentration of 0.1mM isopropyl thiogalactoside for protein induction expression. The temperature for inducing and expressing the protein is 18-25 ℃, and the induction time is 12-16 hours.

3. Purification of the recombinant protein R1PES-pET32a

Coli Rosetta 12,000g induced for expression was centrifuged for 10 min. With lysis buffer (50mM NaH)₂PO₄300mM NaCl, 10mM imidazole, pH 8.0), and centrifuged at 12,000g for 30min after sonication. The supernatant was incubated with Ni-NTA agarose gel at 4 ℃ for 1 hour and then applied to the column. Rinsing solution (50mM NaH)₂PO₄300mM NaCl, 20mM imidazole, pH 8.0) was washed 4 times with an eluent (50mM NaH)₂PO₄300mM NaCl, 250mM imidazole, pH 8.0) eluting the protein from the column to give a purified S1PES recombinant protein. The SDS-PAGE result of the purified S1PES recombinant protein is shown in FIG. 1.

4. Synthesis of S-type 1-phenyl ethanol

Carrying out in-vitro enzymatic reaction by using the obtained S1PES recombinant protein, and synthesizing S-type 1-phenyl ethanol by using acetophenone as a substrate. The specific method comprises the following steps: the concentration of acetophenone added in the reaction system is 0.25mM, the concentration of coenzyme NADPH is 1.5mM, the reaction pH is 7-8, the reaction temperature is 45-55 ℃, and the reaction time is 1-1.5 hours. The efficiency of the enzymatic reaction under different reaction conditions is shown in FIG. 2.

GC-MS identifies the product of S1PES catalysis acetophenone, the identification result is shown in figure 3, and the identification result shows that S1PES can catalyze acetophenone to specifically form S type 1-phenyl ethanol.

Example 1

The S-type 1-phenyl ethanol synthetase gene shown in SEQ ID No.2 is cloned into pET32a prokaryotic expression vector.

The S1PES-pET32a vector was transformed into E.coli Rosetta. Selecting a single clone, inoculating the single clone into 100mL LB culture medium, rotating at 37 ℃ and 230 ℃ to OD₆₀₀Approximately equal to 0.6, isopropyl thiogalactoside was added to a final concentration of 0.1 mM. Thereafter, the cultivation was continued at 20 ℃ for 16 hours under 180 rpm.

The cells were collected by centrifugation at 12,000g for 10 minutes, and 10mL of a lysate (50mM NaH) was added₂PO₄300mM NaCl, 10mM imidazole, pH 8.0) and then sonicating the cells. The parameters of the ultrasonographer were: 40% power, sonicate for 3 seconds, pause for 5 seconds. After 30 minutes of sonication, the supernatant was collected by centrifugation at 12,000g for 30 minutes. mu.L of Ni-NTA agarose gel was added to the supernatant, and the mixture was incubated at 4 ℃ for 1 hour at 200 rpm. The mixture of protein and Ni-NTA agarose gel was packed into a column and the flow-through was discarded. Rinsing solution (50mM NaH)₂PO₄300mM NaCl, 20mM imidazole, pH 8.0) and 3mL of eluent (50mM NaH) were added after 4 washes₂PO₄300mM NaCl, 250mM imidazole, pH 8.0) the recombinant protein was eluted from the sepharose column. The amount of recombinant protein obtained was 3.6 mg.

5 mu g of recombinant protein is added into a reaction system, the concentration of acetophenone in the reaction system is 0.25mM, the concentration of coenzyme NADPH is 1.5mM, the reaction pH is 7.5, the reaction temperature is 45 ℃, and the reaction time is 1 hour. After the reaction, the amount of S-type 1-phenylethanol formed was determined to be about 25nmol, and the conversion efficiency was determined to be about 25%.

Example 2

The S1PES-pET32a vector was transformed into E.coli Rosetta. Selecting a single clone, inoculating the single clone into 400mL LB culture medium, rotating at 37 ℃ and 230 ℃ to OD₆₀₀Approximately equal to 0.6, isopropyl thiogalactoside was added to a final concentration of 0.1 mM. Thereafter, the cultivation was continued at 20 ℃ for 16 hours under 180 rpm.

The cells were collected by centrifugation at 12,000g for 10 minutes, and 10mL of a lysate (50mM NaH) was added₂PO₄300mM NaCl, 10mM imidazole, pH 8.0) and then sonicating the cells. The parameters of the ultrasonographer were: 40% power, sonicate for 3 seconds, pause for 5 seconds. After 30 minutes of sonication, the supernatant was collected by centrifugation at 12,000g for 30 minutes. mu.L of Ni-NTA agarose gel was added to the supernatant, and the mixture was incubated at 4 ℃ for 1 hour at 200 rpm. The mixture of protein and Ni-NTA agarose gel was packed into a column and the flow-through was discarded. Rinsing solution (50mM NaH)₂PO₄300mM NaCl, 20mM imidazole, pH 8.0) and 3mL of eluent (50mM NaH) were added after 4 washes₂PO₄，300mM NaCl, 250mM imidazole, pH 8.0) the recombinant protein was eluted from the sepharose column. The amount of recombinant protein obtained was 7.6 mg.

5 mu g of recombinant protein is added into a reaction system, the concentration of acetophenone in the reaction system is 0.25mM, the concentration of coenzyme NADPH is 1.5mM, the reaction pH is 7.5, the reaction temperature is 55 ℃, and the reaction time is 1 hour. The amount of S-type 1-phenylethanol formed after the reaction was determined to be about 21nmol with a conversion efficiency of about 21%.

SEQUENCE LISTING

<110> Guangdong food and drug profession school

<120> coding gene of S-type 1-phenyl ethanol synthetase and application thereof

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 268

<212> PRT

<213> Artificial Synthesis

<400> 1

Met Ala Lys Lys Val Glu Ser Ser Glu Arg Gly Leu Arg Trp Ser Leu

1 5 10 15

Ala Gly Met Thr Ala Leu Val Thr Gly Gly Thr Arg Gly Ile Gly His

20 25 30

Asp Val Val Glu Glu Leu Ala Asp Lys Gly Ala Ile Val His Thr Cys

35 40 45

Ser Arg Asn Glu Ser Glu Leu Ser Arg Cys Leu Gln Glu Trp Ser Ala

50 55 60

Lys Gly Phe Thr Val Thr Gly Ser Val Cys Asp Val Ser Ser Arg Pro

65 70 75 80

Gln Arg Glu Lys Met Leu Asp Glu Val Ser Ser Leu Phe Asn Gly Lys

85 90 95

Leu Asn Ile Leu Ile Asn Asn Val Gly Thr Thr Leu Trp Lys Pro Thr

100 105 110

Met Glu Tyr Ser Ala Glu Glu Tyr Ser Met Leu Met Ala Thr Asn Leu

115 120 125

Glu Ser Ala Phe His Leu Cys Gln Leu Ala Tyr Pro Leu Leu Lys Ala

130 135 140

Ser Gly Val Gly Ser Ile Val Phe Ile Ser Ser Val Ala Gly Leu Val

145 150 155 160

His Thr Gly Ser Gly Ser Val Tyr Gly Ala Ser Lys Gly Ala Met Asn

165 170 175

Gln Leu Thr Lys Asn Leu Ala Cys Glu Trp Ala Lys Asp Asn Ile Arg

180 185 190

Ser Asn Cys Val Thr Pro Phe Cys Ile Arg Thr Pro Leu Leu Gln His

195 200 205

Leu Met Asp Lys Gln Glu Phe Leu Asp Lys Val Arg Ser Arg Thr Pro

210 215 220

Leu Gly Arg Pro Gly Glu Pro Asn Glu Ile Ser Ser Leu Val Ala Phe

225 230 235 240

Leu Cys Met Pro Thr Ser Ser Tyr Ile Thr Gly Gln Ile Ile Ser Val

245 250 255

Asp Gly Gly Met Thr Val Tyr Gly Phe Ser Pro Asn

260 265

<210> 2

<211> 807

<212> DNA

<213> Artificial Synthesis

<400> 2

atggcgaaga aggtggagag cagtgagaga ggtttgagat ggtctctcgc cggaatgacc 60

gctctcgtca ccggcggcac tcgcggcatc gggcacgatg ttgtggagga actagccgat 120

aaaggggcga tcgtgcacac ctgttctcgc aacgaatccg agctcagtcg ttgcttgcag 180

gaatggtccg ccaagggttt cacggtcacc ggttcggtct gcgatgtatc gtctcggcct 240

caaagagaga agatgttgga tgaagtctct tctctcttca atggcaagct caatatcctt 300

ataaacaatg ttggcacaac cctctggaag cctactatgg agtatagtgc tgaagaatac 360

tcaatgctca tggctaccaa tcttgagtcc gctttccatc tctgccaact tgcatatcct 420

cttctaaaag cttctggagt tggaagcatt gtgtttattt cctctgttgc tggtttggtg 480

cacaccggtt ctggatccgt ttatggagct agcaaaggtg caatgaatca acttacaaaa 540

aatttggctt gtgagtgggc aaaagataac atccgaagta attgtgttac acccttttgt 600

atcagaaccc cactcttaca acatttgatg gacaagcaag agttcctgga taaggtaaga 660

tctagaactc ctcttggacg ccctggagag ccaaatgaaa tatcatcctt ggtggcattc 720

ctttgtatgc ctacttcatc ttacatcact ggtcagatca tttctgttga cggaggaatg 780

actgtgtatg gtttctcacc taattag 807

Claims

1. The amino acid sequence of the protein is shown as SEQ ID No. 1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the sequence of the nucleic acid molecule is shown as SEQ ID No. 2.

4. A cloning vector comprising the nucleic acid molecule of claim 2 or 3.

5. An expression vector comprising the nucleic acid molecule of claim 2 or 3.

6. A method for producing S-type 1-phenylethanol synthase, comprising introducing the expression vector of claim 5 into a host cell, and expressing to obtain S-type 1-phenylethanol synthase.

7. A biosynthesis method of S-type 1-phenyl ethanol is characterized by comprising the following steps: use of the protein of claim 1 as a synthetase catalytic reaction.

8. The biosynthesis method according to claim 7, wherein: acetophenone is used as a substrate.

9. The biosynthesis method according to claim 8, wherein: the coenzyme NADPH is also required to be added into the reaction system.

10. The biosynthetic method of claim 9, wherein: the reaction pH is 7-8, and the reaction temperature is 45-55 ℃.