Tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid

Abstract

The embodiment of the invention provides a tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, and L248M + F497I + M4T + V482L. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO. 2 is taken as a template, a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of bioenzymes, and particularly to a tyrosine ammonia lyase mutant and its use in the synthesis of p - coumaric acid. Background Art

[0002] p - Coumaric acid (trans - 4 - hydroxycinnamic acid, also known as p - hydroxycinnamic acid) is a natural compound widely present in plants, especially in leguminous plants with relatively high content. It has a wide range of application fields, covering multiple industries such as medicine, food, daily necessities, feed, and chemical industry. p - Coumaric acid has various biological activities, including antioxidant, anti - inflammatory, immunomodulatory, anti - tumor, cardiovascular protection, prevention and improvement of diabetes, and neuroprotective effects. In addition, p - Coumaric acid can inhibit the monophenolase and diphenolase activities of tyrosinase, thereby reducing melanin production and delaying skin aging, so it has important application value in the cosmetic field. p - Coumaric acid can also form polyhydroxystyrene derivatives through decarboxylation and polymerization reactions as the main component of photoresist; at the same time, it can also be used to synthesize photosensitive p - hydroxy polyimide for application in liquid crystal display devices.

[0003] Currently, the preparation methods of p - coumaric acid mainly include chemical synthesis method and plant extraction method. The traditional chemical synthesis method usually uses p - hydroxybenzaldehyde and malonic acid to react in pyridine, but this method has problems such as serious environmental pollution and difficulty in removing impurities, and it needs to be purified multiple times to meet the quality standards of photoresist raw materials. The plant extraction method is to use the alkali hydrolysis of lignocellulose and ion exchange resin adsorption extraction technology, but this method has disadvantages such as low content, complex process, and high cost.

[0004] In recent years, with the development of protein engineering and metabolic engineering, the preparation of p - coumaric acid by bioenzyme method has gradually become an important research direction. p - Coumaric acid can be generated from phenylalanine through the catalytic action of phenylalanine ammonia lyase and cinnamic acid hydroxylase (C4H), or directly generated from L - tyrosine under the catalysis of tyrosine ammonia lyase. However, due to the low biological activity of natural enzymes, it has become the key bottleneck restricting the improvement of p - coumaric acid production. Therefore, exploring suitable tyrosine ammonia lyase and modifying its structure to optimize the activity and / or conversion efficiency of tyrosine ammonia lyase is of great significance for promoting the industrial production of p - coumaric acid. Summary of the Invention

[0005] The purpose of the embodiments of the present application is to provide a tyrosine ammonia lyase mutant and its use in the synthesis of p - coumaric acid.

[0006] To achieve the above purpose, the embodiments of the present application propose the following technical solutions: In a first aspect, an embodiment of the present application provides a tyrosine ammonia-lyase mutant, which has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, L248M + F497I + M4T + V482L.

[0007] In a second aspect, an embodiment of the present application provides a DNA molecule, which is a nucleotide sequence encoding the tyrosine ammonia-lyase mutant described in the first aspect or its complementary sequence.

[0008] In a third aspect, an embodiment of the present application provides a recombinant plasmid, which contains the DNA molecule described in the second aspect.

[0009] In a fourth aspect, an embodiment of the present application provides a recombinant strain, which contains the recombinant plasmid described in the third aspect.

[0010] As an implementation manner, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

[0011] As an implementation manner, the host cell of the recombinant strain is a competent cell.

[0012] As an implementation manner, the competent cell is Escherichia coli BL21(DE3).

[0013] In a fifth aspect, an embodiment of the present application provides the use of the tyrosine ammonia-lyase mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant plasmid described in the third aspect, or the recombinant strain described in the fourth aspect in the catalytic synthesis of p-coumaric acid from L-tyrosine.

[0014] As an implementation manner, the preparation method of p-coumaric acid includes: Preparing a recombinant plasmid containing a coding sequence for the tyrosine ammonia-lyase mutant; Transforming the recombinant plasmid into a host cell to obtain a recombinant strain; Using the recombinant strain to catalyze the production of p-coumaric acid from L-tyrosine under the conditions of a temperature of 15 - 50 °C and a pH value of 9.5.

[0015] As an implementation manner, the preparation of the recombinant plasmid containing a coding sequence for the tyrosine ammonia-lyase mutant includes: Using an expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, performing mutations at corresponding sites to obtain a recombinant plasmid containing a coding sequence for the tyrosine ammonia-lyase mutant.

[0016] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects: Taking the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), the key active sites of this template (basis) were mutated and modified to construct a mutant library, and a series of mutants with significantly improved catalytic activity and / or conversion rate were screened; the tyrosine ammonia-lyase mutants provided by the embodiments of the present application can selectively catalyze L-tyrosine to generate p-coumaric acid, and have excellent yield and / or higher productivity, indicating that the mutants have more significant application prospects in catalyzing L-tyrosine to generate p-coumaric acid.

[0017] Additional aspects and advantages of the present application will be given in part in the following description, and these will become apparent from the following description or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the standard curve of p-coumaric acid; Figure 2 HPLC chromatogram showing the production of p-coumaric acid from L-tyrosine substrate by mutant M4 (F497I / L248M / M4T / V482L); Figure 3 HPLC chromatogram showing the production of p-coumaric acid from 20 g / L L-tyrosine substrate by mutant M4 (F497I / L248M / M4T / V482L) at a content of 100 g / L. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The technical solutions in the embodiments will be clearly and completely described below in conjunction with the embodiments of the present application and the drawings. Obviously, the embodiments to be described below are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0020] Some terms and raw materials involved in this embodiment will be explained below to facilitate the understanding of those skilled in the art.

[0021] L-tyrosine: L-Tyr.

[0022] p-coumaric acid: pCA, or p-CA for short.

[0023] Unless otherwise specified, the specific composition of the culture medium used in the following embodiments is shown as follows: LB medium: peptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L.

[0024] TB medium: 24 g / L of yeast extract, 12 g / L of peptone, 12.54 g / L of dipotassium hydrogen phosphate, 2.31 g / L of potassium dihydrogen phosphate, 4 mL / L of glycerol.

[0025] The tyrosine ammonia-lyase mutant of this example and its use in the synthesis of p-coumaric acid will be described in detail below.

[0026] First, the tyrosine ammonia-lyase mutant of the first aspect of this example will be described.

[0027] Tyrosine ammonia-lyase mutant In the prior art, p-coumaric acid can be generated from phenylalanine through the catalytic action of phenylalanine ammonia-lyase and cinnamic acid hydroxylase (C4H), or directly generated from L-tyrosine under the catalysis of tyrosine ammonia-lyase. However, due to the low biological activity of natural enzymes, it has become a key bottleneck restricting the improvement of p-coumaric acid production.

[0028] In view of this, this example provides a tyrosine ammonia-lyase mutant, which has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, L248M + F497I + M4T + V482L.

[0029] Among them, the amino acid sequence of tyrosine ammonia-lyase (SEQ ID NO. 2) is: MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAALSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQFPPLETDRPLGQEIAALATHLLQQSPV。

[0030] It is understood that in this embodiment, the amino acid sequence shown in SEQ ID NO. 2 is used as a template (basis), and on the basis of this amino acid sequence, the key active sites of the tyrosine ammonia-lyase are mutated and transformed to construct a mutant library; the tyrosine ammonia-lyase mutants provided in this embodiment can selectively catalyze L-tyrosine to produce p-coumaric acid, and have excellent yield and / or higher productivity, and have excellent yield and / or higher productivity.

[0031] In this embodiment, the nucleotide sequence of the tyrosine ammonia-lyase is as shown in SEQ ID NO. 1, and the amino acid sequence is as shown in the above SEQ ID NO. 2.

[0032] Specifically, the nucleotide sequence (SEQ ID NO. 1) of the above tyrosine ammonia-lyase is:

[0033] As a preferred embodiment of this example, any one of the following amino acid mutations occurs in the amino acid sequence shown in SEQ ID NO. 2 in this example: F497I + L248M + M4T, L248M + F497I + M4T + V482L.

[0034] Specifically, the amino acid sequence of mutant F497I is shown in SEQ ID NO. 3, the amino acid sequence of mutant F497I + L248M is shown in SEQ ID NO. 4, the amino acid sequence of mutant F497I + L248M + M4T is shown in SEQ ID NO. 5, and the amino acid sequence of mutant L248M + F497I + M4T + V482L is shown in SEQ ID NO. 6.

[0035] Among them, the amino acid sequence of mutant F497I (SEQ ID NO. 3) is: MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAALSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV.

[0036] The amino acid sequence of mutant F497I + L248M (SEQ ID NO. 4) is: MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAAMSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV。

[0037] The amino acid sequence of mutant F497I + L248M + M4T (SEQ ID NO. 5) is as follows: MLATSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAAMSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV。

[0038] The amino acid sequence of mutant L248M+F497I+M4T+V482L (SEQ ID NO. 6) is as follows: MLATSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAAMSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGLSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV。

[0039] It is understandable that the tyrosine ammonia-lyase mutant of this embodiment can be mutated at corresponding sites through existing site-directed mutagenesis techniques to obtain the target mutant. Among them, mutants with multiple mutation sites can be subjected to superimposed mutation on the basis of a single mutant, that is, on the basis of a single mutant, mutation is carried out at another amino acid mutation site, so as to obtain a mutant with multiple mutation sites.

[0040] In summary, the tyrosine ammonia-lyase mutant provided in this embodiment can catalyze L-tyrosine to produce p-coumaric acid, and has excellent yield and / or higher productivity, indicating that this mutant has a more significant application prospect in catalyzing L-tyrosine to produce p-coumaric acid.

[0041] Among them, the productivity is defined as: actual yield (mol) of the target product / theoretical yield (mol) of the target product × 100%.

[0042] Next, the DNA molecule of the second aspect of this embodiment will be described.

[0043] DNA molecule It is understandable that the DNA molecule provided in this embodiment is a DNA molecule encoding the nucleotide sequence of any of the above tyrosine ammonia-lyase mutants or its complementary sequence.

[0044] Next, the recombinant plasmid of the third aspect of this embodiment will be described.

[0045] Recombinant plasmid The recombinant plasmid provided in this embodiment is a DNA molecule encoding the nucleotide sequence of any of the above tyrosine ammonia-lyase mutants or its complementary sequence.

[0046] Exemplarily, the recombinant plasmid of this embodiment can be selected from any of the following: pET-21b(+), pET-22b(+), pET-3a (+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), p ET-23a(+), p ET-23b(+), pET-24a(+), p ET-25b (+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a (+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a (+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, pUC-19, pPICZ A, pPICZα B, pPICZα C, pPIC3K, pPIC3.5K.

[0047] It is understandable that the recombinant plasmid of this embodiment can be prepared by using existing known preparation methods. For example: Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations are made at corresponding sites to obtain a recombinant plasmid encoding a tyrosine ammonia-lyase mutant.

[0048] For example, the above expression vector can be selected from pET-28a(+) (purchased from Sangon Biotech (Shanghai) Co., Ltd.); the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 can be obtained by a gene synthesis company (such as Tianyi Huiyuan Gene Technology Co., Ltd.) synthesizing the corresponding gene sequence onto the pET-28a(+) vector to obtain the corresponding template.

[0049] Next, the recombinant strain of the fourth aspect of this embodiment will be described.

[0050] Recombinant strain It can be understood that the recombinant strain provided in this embodiment contains the recombinant plasmid described in the third aspect.

[0051] In this embodiment, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and preferably the eukaryotic cell is a yeast cell.

[0052] Among them, the host cell can be a competent cell, and preferably the competent cell is Escherichia coli BL21(DE3).

[0053] The recombinant strain of this embodiment can be prepared by existing preparation methods. For example: The prepared recombinant plasmid is transferred into Escherichia coli BL21(DE3) for culture to obtain the recombinant strain.

[0054] Among them, the prepared recombinant strain usually needs to be stored in a refrigerator (such as -80°C).

[0055] Secondly, the use of the tyrosine ammonia lyase mutant of the fifth aspect of this embodiment will be described.

[0056] Use of tyrosine ammonia-lyase mutant As described above, the tyrosine ammonia lyase mutant of this embodiment can be used to catalyze the synthesis (generation) of p-coumaric acid from L-tyrosine.

[0057] The preparation method of p-coumaric acid will be introduced in detail below.

[0058] Exemplarily, the preparation method of p-coumaric acid in this embodiment includes: (1) Prepare a recombinant plasmid containing the coding sequence of the tyrosine ammonia lyase mutant; (2) Transform the recombinant plasmid into a host cell to obtain a recombinant strain; (3) Using L-tyrosine as a substrate, under the conditions of a temperature of 15 - 50°C and a pH value of 9.5, use the recombinant strain to catalyze the generation of p-coumaric acid from L-tyrosine.

[0059] The steps of the above preparation method will be further described below.

[0060] In step (1), a recombinant plasmid containing the coding sequence of the tyrosine ammonia-lyase mutant is prepared, including: Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, site-directed mutagenesis is performed at corresponding sites to obtain a recombinant plasmid containing the coding sequence of the tyrosine ammonia-lyase mutant.

[0061] Among them, site-directed mutagenesis can adopt existing mutagenesis techniques, such as the polymerase chain reaction (PCR) method.

[0062] In step (2), the recombinant plasmid is transformed into a host cell to obtain a recombinant strain, including: The recombinant plasmid obtained in step (1) is transferred into Escherichia coli BL21(DE3) for cultivation to obtain a recombinant strain.

[0063] In step (3), using L-tyrosine as a substrate, under the conditions of a temperature of 15-50 °C and a pH value of 9.5, the recombinant strain is used to catalyze the production of p-coumaric acid from L-tyrosine, including: (3.1) The obtained recombinant bacteria are inoculated into LB medium and TB medium, and after shaking culture, the cells are collected by centrifugation; (3.2) The cells are resuspended in a buffer (0.1 M glycine-sodium hydroxide buffer, pH = 9.5), and under the conditions of a temperature of 15-50 °C and a pH value of 9.5, L-tyrosine is added for reaction to obtain p-coumaric acid.

[0064] Generally, the reaction conditions in step (3) are: the rotation speed is 250 ± 20 rpm, and the reaction time is 1-48 h.

[0065] Preferably, the reaction temperature is 50 °C, the reaction time is 10-36 h, and more preferably 20-24 h.

[0066] Exemplarily, in a 10 mL centrifuge tube, 600 μL or 800 μL of wet cells are added, and then 400 μL or 200 μL of L-tyrosine is added to make the final concentration reach 10-20 g / L. The mixture is placed on a shaker at 50 °C and stirred at 250 rpm for 24 hours. After the culture is completed, the conversion solution is collected. 500 μL of 3 mol / L HCl is added to the collected conversion solution to terminate the reaction, and then 2.5 mL of methanol is added. Then, it is centrifuged at 12000 rpm for 5 minutes, and the supernatant is collected. The supernatant is diluted with methanol to 20-30 times the original concentration and then filtered through a 0.22 μm nylon 66 filter membrane. Finally, the content of p-coumaric acid is detected by high performance liquid chromatography.

[0067] It can be understood that the L-tyrosine described in this example is the substrate of the reaction.

[0068] In summary, the tyrosine ammonia lyase mutant provided in this embodiment can catalyze L-tyrosine to produce p-coumaric acid, and has excellent yield and / or higher productivity, indicating that this mutant has a more significant application prospect in catalyzing L-tyrosine to produce p-coumaric acid.

[0069] The following will further elaborate on this application in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain this application and not to limit the scope of this application.

[0070] In the following embodiments, the materials, reagents, and instruments used, unless otherwise specified, can be obtained from commercial channels.

[0071] In the following embodiments, the quantitative analysis conditions for p-coumaric acid are as follows: Chromatographic conditions: The conversion solution is detected using a waters high-performance liquid chromatograph (equipped with a 2489 UV / Vis detector). The chromatographic column is Welch Ultimate ® XS-C18 4.6x250mm, column temperature 30 °C. The mobile phase is 0.1% acetic acid: methanol = 55:45, flow rate 1.0 mL / min. Injection volume 10 μL, detection wavelength 280nm.

[0072] Under the above chromatographic conditions, a curve fitting the peak area and concentration of p-coumaric acid standard products with different concentrations (concentration range is 5 - 500 μg / mL, specifically 5, 20, 50, 100, 188, 250, 500 μg / mL) is obtained for the quantitative analysis of p-coumaric acid; Figure 1 A schematic diagram of the standard curve of p-coumaric acid is shown, where the vertical coordinate Area is the peak area of the p-coumaric acid standard product measured by liquid chromatography.

[0073] In the following embodiments, the method for measuring the activity of tyrosine ammonia lyase: Use a tyrosine ammonia lyase (TAL) activity detection kit (Solarbio ® , product number: BC4065), and refer to the instruction manual for activity measurement.

[0074] Example 1 Synthesis and recombinant expression of the parental plasmid pET-28a(+)-RsTAL.

[0075] In this embodiment, the wild-type tyrosine ammonia lyase is used as a template (the amino acid sequence is WP_011339422.1, hereinafter referred to as WT), and the selected tyrosine ammonia lyase gene (the nucleotide sequence is as shown in SEQ ID NO. 1, and the amino acid sequence is as shown in SEQ ID NO. 2) is synthesized by Sangon Biotech Co., Ltd. and codon-optimized to improve its expression efficiency in Escherichia coli.

[0076] Specifically: The optimized gene fragment was cloned into the pET-28a(+) plasmid vector, with the restriction enzyme sites at both ends being BamH I and EcoRI, respectively, to construct the recombinant plasmid pET-28a(+)-RsTAL.

[0077] The recombinant plasmid was transformed into Escherichia coli competent cells BL21(DE3), and positive clones were screened by kanamycin (Kana). Single colonies were selected for sequencing verification, and the amino acid sequence of the cloned tyrosine ammonia-lyase was confirmed to be SEQ ID NO: 2.

[0078] The recombinant Escherichia coli was diluted by volume ratio to an initial OD 600 of 0.2 and inoculated into TB medium to induce protein expression. The culture was carried out at 37 °C and 200 rpm. When the OD 600 in the logarithmic growth phase of the cells reached 0.6 - 0.8, an inducer IPTG with a final concentration of 0.2 mM was added. Induced expression was carried out at 16 °C and 200 rpm for 12 hours. After the induced expression was completed, the culture was centrifuged at 4 °C and 6500 rpm for 10 minutes to collect the cell precipitate for subsequent enzyme activity assays and application experiments.

[0079] Example 2 Construction of tyrosine ammonia-lyase mutants.

[0080] In this example, the plasmid pET-28a(+)-RsTAL was used as a template to design mutant primers, and the bases to be mutated were placed in the middle of the primers.

[0081] Among them, the PCR amplification system and PCR reaction program refer to the PCR instruction manual of Vazyme 2 × Phanta Flash Master Mix (P510-02).

[0082] Among them, the sequences of the upstream and downstream mutant primers used in this example are shown in Table 1 below: Table 1: Sequences of upstream and downstream mutant primers

[0083] Among them, after the PCR program is completed, DNA gel electrophoresis is carried out to verify whether the band size is correct. After using DNPI to digest the template for transformation, the Snap Gene software is used to check whether the sequencing result is successfully mutated. In this example, the amino acid sequence of the mutated tyrosine ammonia-lyase M1 (F497I) mutant is shown in SEQ ID NO: 3, the amino acid sequence of the mutated tyrosine ammonia-lyase M2 (F497I / L248M) mutant is shown in SEQ ID NO: 4, the amino acid sequence of the mutated tyrosine ammonia-lyase M3 (F497I / L248M / M4T) mutant is shown in SEQ ID NO: 5, and the amino acid sequence of the mutated tyrosine ammonia-lyase M4 (F497I / L248M / M4T / V482L) mutant is shown in SEQ ID NO: 6.

[0084] Among them, for mutants with multiple mutation sites, the above-mentioned same method is used to perform superimposed mutations on the basis of single mutants to obtain mutants with multiple mutation sites; the induced expression of the mutants is the same as that of the above-mentioned template.

[0085] Example 3 In this example, L-tyrosine was used as the substrate to measure the catalytic activities of the above-mentioned mutants (M1-M4).

[0086] Among them, the induced expression of the mutants is the same as that of the above-mentioned template.

[0087] The single colonies grown after transformation were inoculated into LB medium and cultured at 37 °C and 200 rpm for 12 hours. Then, according to the inoculation amount with a final OD 600 of approximately 0.2, it was inoculated into TB medium and continued to be cultured at 37 °C and 200 rpm. When OD 600 reached 0.6-0.8, IPTG with a final concentration of 0.2 mM was added, and then it was cultured at 16 °C and 200 rpm for 12 hours. After the culture was completed, the recombinant engineering bacteria pET28a-RsTAL (after mutation)- E. coli BL21(DE3) were collected by centrifugation. According to the wet weight ratio of 1:8, it was resuspended with 0.1 M glycine-sodium hydroxide buffer at pH = 9.5 to obtain wet bacterial cells.

[0088] Specifically: In a 10 mL centrifuge tube, add the substrate L-tyrosine, the above-mentioned wet bacterial cells (M1 or M2 or M3 or M4), and glycine-sodium hydroxide buffer (pH 9.5) with a final volume of 1 mL, such that the concentration of L-tyrosine is 10 g / L and the concentration of wet bacterial cells is 60 g / L; then, react at 50 °C, pH 9.5, and a rotation speed of 250 rpm for 24 h to obtain a conversion solution. After the reaction, add 500 μL of 3 mol / L HCl to terminate the reaction, and then add 2.5 mL of methanol. Next, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant. Dilute the supernatant to 20 times the original concentration with methanol, filter it through a 0.22 μm nylon 66 filter membrane, and detect the content by HPLC. The detection results are shown in Table 2: Table 2: Effects of different strains on the pCA yield and productivity

[0089] The productivity is defined as: (actual yield of the target product (mol) / theoretical yield of the target product (mol))×100%.

[0090] Figure 2 The HPLC chromatogram of the mutant M4 (F497I / L248M / M4T / V482L) catalyzing L-Tyr is shown; combined with the HPLC detection results in Table 2 and Figure 2 the results show that the content of p-coumaric acid in the wild-type strain (template, or WT) is 4.67 ± 0.21 g / L, and the productivity is 51.50 ± 2.37%. Among the screened mutants, at the same concentration, the pCA yields and productivities of M1, M2, M3, and M4 are all improved; among them, M4 shows the highest productivity, reaching 80.40 ± 0.60%, and the content of coumaric acid is 7.28 ± 0.05 g / L.

[0091] Therefore, compared with the prior art, in this example, the key active sites of the template WT are mutated and transformed to construct a mutant library, and a series of mutants with significantly improved catalytic activity and / or conversion rate are screened. The tyrosine ammonia-lyase mutants provided in this example can selectively catalyze L-tyrosine to produce p-coumaric acid and have excellent yields and / or higher productivities.

[0092] Example 4 In this example, the M4 mutant is used as an exemplary mutant to further determine the catalytic activity of mutants with different addition amounts on L-Tyr.

[0093] Specifically: In a 10 mL centrifuge tube, add the substrate L-tyrosine, the above-mentioned wet bacterial cells (M4), and glycine-sodium hydroxide buffer (pH 9.5) to a final volume of 1 mL, such that the concentration of L-tyrosine is 20 g / L and the concentration of wet bacterial cells is 60 g / L; then, under the reaction conditions of 50 °C, pH 9.5, rotation speed of 250 rpm, after a reaction time of 24 h, a conversion solution is obtained. After the reaction is completed, add 500 μL of 3 mol / L HCl to terminate the reaction, and then add 2.5 mL of methanol. Then, centrifuge at 12000 rpm for 5 minutes and collect the supernatant. Dilute the supernatant with methanol to 20 times the original concentration, filter through a 0.22 μm nylon 66 membrane, and detect the content by HPLC. The detection results are shown in Table 3 as follows: Table 3: Effects of Different Strains on pCA Yield and Productivity

[0094] According to the HPLC detection results in Table 3, the content of p-coumaric acid in the wild-type strain (WT) is 6.17 ± 0.19 g / L, and the productivity is 34.04 ± 1.02%. The productivity of M4 reaches 54.32 ± 0.92%, and the content of p-coumaric acid is 10.23 ± 0.67 g / L.

[0095] In a 10 mL centrifuge tube, add the substrate L-tyrosine, the above-mentioned wet bacterial cells (M4), and glycine-sodium hydroxide buffer (pH 9.5) to a final volume of 1 mL, such that the concentration of L-tyrosine is 20 g / L and the concentration of wet bacterial cells is 100 g / L; then, under the reaction conditions of 50 °C, pH 9.5, rotation speed of 250 rpm, after a reaction time of 24 h, a conversion solution is obtained. After the reaction is completed, add 500 μL of 3 mol / L HCl to terminate the reaction, and then add 2.5 mL of methanol. Then, centrifuge at 12000 rpm for 5 minutes and collect the supernatant. Dilute the supernatant with methanol to 20 times the original concentration, filter through a 0.22 μm nylon 66 membrane, and detect the content by HPLC. The detection results are shown in Table 4 as follows: Table 4: Effects of Different Strains on pCA Yield and Productivity

[0096] Among them, Figure 3 shows the HPLC chromatogram of the mutant M4 (F497I / L248M / M4T / V482L) catalyzing the generation of p-coumaric acid from 20 g / L L-tyrosine substrate at a content of 100 g / L.

[0097] According to Figure 3The HPLC detection results in Table 4 show that the content of p-coumaric acid in the wild-type strain (WT) is 11.98 ± 0.24 g / L, and the yield is 66.14 ± 1.32%. The yield of M4 reaches 85.64 ± 0.30%, and the content of p-coumaric acid is 15.52 ± 0.05 g / L. That is, M4 can better catalyze L-Tyr to produce pCA, and pCA has excellent yield and / or higher productivity.

[0098] Example 5 In this example, the M4 mutant was used as an exemplary mutant to further determine the catalytic activity of the above mutant on different concentrations of L-Tyr.

[0099] Specifically, in a 10 mL centrifuge tube, add the substrate L-tyrosine, the above wet cells (M4), and glycine-sodium hydroxide buffer (pH 9.5) with a final volume of 1 mL, such that the concentration of L-tyrosine is 20 - 100 g / L and the concentration of wet cells is 200 g / L; then, under the reaction conditions of 50 °C, pH 9.5, rotation speed of 250 rpm, and reaction time of 24 h, the conversion solution is obtained. After the reaction is completed, add 500 μL of 3 mol / L HCl to terminate the reaction, and then add 2.5 mL of methanol. Then, centrifuge at 12,000 rpm for 5 minutes to collect the supernatant. The supernatant is diluted to 20 times the original concentration with methanol, filtered through a 0.22 μm nylon 66 filter membrane, and the content is detected by HPLC. The detection results are shown in Table 5: Table 5: pCA production and yield of M4 at different concentrations of L-tyrosine L-Tyr concentration (g / L) pCA production (g / L) Yield rate (%) 20 15.94±0.14 87.95±0.75 40 21.55±0.43 59.48±1.20 60 30.34±1.03 55.81±1.89 80 44.83±2.57 61.85±3.54 100 37.76±1.46 41.68±1.62 According to the HPLC detection results in Table 5, M4 can better catalyze L-Tyr to produce pCA at different concentrations of L-Tyr, and pCA has excellent yield and / or higher productivity. Among them, when the concentration of L-tyrosine is 80 g / L, the yield reaches 61.85 ± 3.54%, and the content of p-coumaric acid is 44.83 ± 2.57 g / L.

[0100] Example 6 Based on the catalytic yields of the above mutant strains, the enzyme activities of the mutant strains were further tested in this example; among them, the determination of tyrosine ammonia-lyase activity is described in detail in the tyrosine ammonia-lyase (TAL) activity detection kit (Solarbio ® , product number: BC4065). In this example, according to the tyrosine ammonia-lyase activity detection kit, the enzyme activities of the mutant strain M4 and the original enzyme were compared, and the test results are shown in Table 6 below: Table 6: Comparison of enzyme activities of tyrosine ammonia-lyase mutant strains (U / g) Strain Enzyme activity (U / g) WT 68.50 M4 128.75 Combined with the test results in Table 6, the enzyme activity of strain M4 was increased to 187.95% compared with the original RsTAL. That is, the mutant provided in this example has higher catalytic activity and can selectively catalyze L-tyrosine to produce p-coumaric acid.

[0101] In summary, in this example, using the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), the key active sites of this template were mutated and transformed to construct a mutant library, and a series of mutants with significantly improved catalytic activity and / or conversion rate were screened; the tyrosine ammonia-lyase mutant provided in this example can selectively catalyze L-tyrosine to produce p-coumaric acid, and has excellent yield and / or higher productivity, indicating that this mutant has a more significant application prospect in catalyzing L-tyrosine to produce p-coumaric acid.

[0102] The technical solutions provided in the embodiments of the present application have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present application. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present application; at the same time, for those of ordinary skill in the art, according to the embodiments of the present application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. A tyrosine ammonia-lyase mutant, characterized in that, The mutant has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, L248M + F497I + M4T + V482L.

2. A DNA molecule, characterized in that, The DNA molecule is a nucleotide sequence encoding the tyrosine ammonia-lyase mutant as described in claim 1 or its complementary sequence.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the DNA molecule as described in claim 2.

4. A recombinant strain, characterized in that, The recombinant strain contains the recombinant plasmid as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, The host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

6. The recombinant strain according to claim 4, characterized in that, The host cell of the recombinant strain is a competent cell.

7. The recombinant strain according to claim 6, wherein The competent cell is Escherichia coli BL21(DE3).

8. Use of the tyrosine ammonia-lyase mutant as described in claim 1, the DNA molecule as described in claim 2, the recombinant plasmid as described in claim 3, or the recombinant strain as described in any one of claims 4-7 in the catalytic synthesis of p-coumaric acid from L-tyrosine.

9. The use according to claim 8, characterized in that, The method for preparing p-coumaric acid includes: Preparing a recombinant plasmid containing a coding sequence for a tyrosine ammonia-lyase mutant; Transforming the recombinant plasmid into a host cell to obtain a recombinant strain; Using the recombinant strain to catalyze the production of p-coumaric acid from L-tyrosine under the conditions of a temperature of 15-50 °C and a pH value of 9.

5.

10. The use according to claim 9, characterized in that, The preparation of the recombinant plasmid containing a coding sequence for a tyrosine ammonia-lyase mutant includes: Using an expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, performing mutations at corresponding sites to obtain a recombinant plasmid containing a coding sequence for a tyrosine ammonia-lyase mutant.

Images