Arbutin synthetase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly provides an arbutin synthetase mutant and application thereof. The arbutin synthetase mutant is obtained through rational design and enzyme evolution screening on the basis of an amino acid sequence as shown in SEQ ID NO: 1, and compared with wild arbutin synthetase, the arbutin synthetase mutant is changed in protein structure and function. The catalytic activity of the arbutin synthetase mutant provided by the invention is obviously improved, and higher conversion rate and conversion rate from arbutin synthetase to beta-arbutin can be obtained. A shikimic acid pathway is enhanced in escherichia coli, and an arbutin synthetase mutant gene is introduced to construct a high-yield beta-arbutin producing strain. The method for producing beta-arbutin has the characteristics of simplicity in operation, low cost, high product synthesis efficiency and reduction of environmental pollution, and has a good industrialization prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a mutant of arbutin synthase (AS) and its application, and more specifically to the application in the production of β-arbutin. Background Art

[0002] β-Arbutin, also known as hydroquinone glucoside and 4-hydroxyphenyl-β-D-glucopyranoside, exists in the leaves of plants such as arbutus trees, blueberry trees, pear trees, and holly trees. It has the ability to inhibit tyrosinase and prevent the formation of melanin, and has the effect of whitening and skin care, and is widely used in the medical and cosmetic industries. There are mainly four methods to obtain arbutin: plant extraction method, chemical synthesis method, enzyme conversion method, and biosynthesis method. The production process of the plant extraction method is complex, and it is affected by the environment and season, resulting in low yield; the method of chemically synthesizing arbutin is not the first choice due to low catalytic efficiency and low selectivity; the enzyme conversion method is affected by the toxicity of hydroquinone, which inhibits the activity of the enzyme, and the separation of the substrate and the product is difficult, resulting in waste of energy; the biosynthesis method can use glucose as a carbon source to de novo synthesize arbutin in the host bacterium construction pathway, but the current pathway has low production yield and high cost.

[0003] Based on the defects of various current methods, it is very necessary to study a method for efficiently industrializing the production of β-arbutin. Summary of the Invention

[0004] The purpose of the present invention is to provide a mutant of arbutin synthase enzyme and its application to solve the problems of low yield and low conversion rate in the production of β-arbutin.

[0005] To achieve the above purpose, the present invention first provides a mutant of arbutin synthase. The amino acid sequence of the mutant of arbutin synthase has the following amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 1: L239G, L239G + V155Y, L239G + W193G, L239G + W193K.

[0006] The above mutants screened in this application have been verified through a large number of experiments, and finally it is proved that the catalytic activity of the enzyme-catalyzed reaction is significantly improved.

[0007] The present invention also provides a nucleic acid encoding the above arbutin synthase.

[0008] The present invention also provides a genetically engineered cell expressing the above arbutin synthase, which contains a nucleic acid encoding the arbutin synthase.

[0009] According to the embodiment of the present invention, the genetically engineered cell is a recombinant strain obtained by constructing an expression cassette of the nucleic acid and then introducing it into a host cell.

[0010] The present invention also provides a method for constructing an Escherichia coli engineering bacterium expressing arbutin synthase.

[0011] The present invention provides a method for producing β - arbutin, which uses the above - mentioned recombinant Escherichia coli bacterium with glucose as a carbon source to produce β - arbutin; the recombinant Escherichia coli bacterium expresses the arbutin synthase gene.

[0012] The temperature of the culture is 30 - 40 °C; the culture time is 24 - 96 h; preferably, the culture is carried out under stirring or shaking conditions, and more preferably, the shaking speed is 250 rpm.

[0013] In one embodiment, in the system for whole - cell transformation production, it includes a wet cell weight of 0.1 - 200 g / L and 3 - dehydroshikimic acid of 1 - 200 g / L.

[0014] The arbutin synthase mutant obtained by rational design and enzyme evolution screening based on the amino acid sequence shown in SEQ ID NO: 1 in the present invention has changes in protein structure and function compared with the wild - type arbutin synthase. In practical applications, the catalytic efficiency of the arbutin synthase mutant is significantly improved, a higher synthesis rate of arbutin synthase can be obtained, and thus a higher yield of β - arbutin can be obtained. At the same time, further strengthening of the shikimic acid pathway (as shown in Figure 1 ) in Escherichia coli and introducing the arbutin synthase gene to construct an Escherichia coli with a higher yield of β - arbutin; providing a powerful enzyme and strain for the biosynthesis of Escherichia coli, the present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 β - arbutin synthesis and metabolism pathway. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following are listed examples for better understanding of the present invention.

[0017] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0018] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0019] It should be noted that, without conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0020] Among them, the amino acid sequence of SEQ ID NO: 1 (derived from the arbutin synthase of Coffeaeugenioides ) is as follows: MDQTPRVALLPSPGMGHLIPLVEFAKRLILQHNFSVTIILPTDGPLSKAQTTFLAALPAAIDYILLPPVNFDDLADDVRIETRISLTVTRSLPSLRVALKSLVDTTKLAALVVDLFGTDAFDVANEFKLPPYIFFPSTATALAFFSYLPKLHEMVACEFRDLPGPIRVPGCVPIHGRDLLDPAQDRKNDAYKWLLHHARRYSLAEGILVNSFKDLEPGPLKALQEQEPGKPPVYPVGPLIQMGSGDKRGEESECLKWLDDQPSGSVLYISFGSGGTLSHNQLIELAFGLEMSEQRFLWVVRSPNDGVANATYFTVHSQNDPLAFMPEGFLDRIKGRGFLLPSWAPQAKILGHSSTGGFLTHCGWNSTLESVVEGIPLIAWPLYAEQKMNAVMLAEDLKVALRPKADENGFVGRVEIANMVKRLMEGEEGKRLRSRMKELKDAAAKVLSQDGSSAEALAQVAGKWQAKICT。

[0021] The corresponding specific nucleic acid sequence is SEQ ID NO: 2:

[0022] Example 1: Specific methods or steps for rational design screening The specific methods or steps of the present invention using rational design and evolutionary screening are as follows: Using SEQ ID NO: 2 as a template, designing site-directed mutagenesis primers, and obtaining mutant genes by means of site-directed mutagenesis; Among them, site-directed mutagenesis: refers to introducing the required changes (usually changes representing the favorable direction) into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including base addition, deletion, point mutation, etc.

[0023] Through fermentation culture, functional verification of the obtained mutants was carried out to obtain the L239G mutant with greatly improved activity.

[0024] The L239G mutant with improved activity obtained by screening was subjected to combinatorial mutagenesis to obtain mutants L239G + V155Y, L239G + W193G, and L239G + W193K with better enzyme activity.

[0025] Furthermore, the mutant expression cassette was ligated to the pET30a vector, transformed into Escherichia coli cells, and subjected to fermentation culture. The fermentation broth was taken and the β-arbutin yield was detected by HPLC, and finally the activity of the relevant mutants was detected.

[0026] Example 2: Functional verification of arbutin synthase mutants Escherichia coli engineering strains were respectively activated in LB liquid medium, and seed liquid was prepared in LB liquid medium (37 °C, 250 rpm, 16 h). It was inoculated into a 100 mL Erlenmeyer flask containing 15 mL of fermentation liquid medium (macronutrients: glucose, yeast extract, citric acid, KH2PO4, (NH4)2SO4, MgSO4·7H2O; micronutrients: FeSO4·7H2O, MnSO4·H2O, Na2SO4, ZnSO4, CoCl2·6H2O, CuSO4·5H2O) at an inoculation amount of 10%, cultured at 37 °C and 250 rpm for 72 hours. Finally, the liquid in the Erlenmeyer flask was transferred to a 50 ml centrifuge tube, centrifuged at 8000 rpm for 5 min, and the supernatant was collected and the β-arbutin yield was detected by HPLC. Quantification was carried out using β-arbutin standard products with different concentrations. The results are shown in Table 1.

[0027] Table 1. Determination of β-arbutin yield of different mutants under shake flask conditions

[0028] Example 3. Functional verification of arbutin synthase mutants The engineered Escherichia coli strains were activated separately in LB liquid medium, and seed solutions were prepared in LB liquid medium (37 °C, 250 rpm, 16 h). They were inoculated into a 5-L fermenter containing 2 L of fermentation liquid medium (macronutrients: glucose, yeast extract, citric acid, KH2PO4, (NH4)2SO4, MgSO4·7H2O; micronutrients: FeSO4·7H2O, MnSO4·H2O, Na2SO4, ZnSO4, CoCl2·6H2O, CuSO4·5H2O) at an inoculation amount of 10%, and cultured at 37 °C for 80 h. Glucose was added as a carbon source during the process, and samples were taken to detect the growth of the engineered bacteria cells and the yield of β-arbutin. The yield of β-arbutin was detected by HPLC, and the results are shown in Table 2.

[0029] Table 2 Determination of β-arbutin yield under fermentation conditions of different mutants in a 2-L fermenter

[0030] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An arbutin synthase mutant, characterized in that: The amino acid sequence of the arbutin synthase mutant has the following amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 1: L239G, L239G and V155Y, L239G and W193G, L239G and W193K.

2. A nucleic acid, characterized in that Encoding the arbutin synthase mutant according to claim 1.

3. A recombinant expression cassette or expression vector, characterized in that: The recombinant expression cassette or expression vector contains the nucleic acid according to claim 2.

4. A recombinant genetically engineered cell, characterized in that: It contains the nucleic acid according to claim 2 and the recombinant expression cassette or expression vector according to claim 3.

5. The recombinant genetically engineered cell according to claim 4, characterized in that: It is a prokaryotic cell or a eukaryotic cell, preferably, it is Escherichia coli.

6. Use of the arbutin synthase mutant according to claim 1, the nucleic acid according to claim 2, the recombinant expression cassette or the expression vector according to claim 3 in the preparation of β-arbutin.

7. A method for producing β-arbutin, characterized in that: The arbutin synthase mutant according to claim 1 is used as a catalyst, and 3-dehydroshikimic acid is used as a substrate to catalyze the reaction to produce β-arbutin.

8. The method according to claim 7, characterized in that The method comprises culturing the recombinant genetically engineered cell as claimed in claim 4 or 5 to produce β-arbutin; Specifically, the culture temperature is 30-40°C and the culture time is 24-96h.

9. The method according to claim 8, characterized in that The culture is carried out under stirring or shaking conditions, more preferably at a shaking speed of 200-300 rpm.

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