Tyrosine ammonia-lyase mutant and its use in synthesizing p-coumaric acid

Abstract

The present application embodiment proposes a tyrosine ammonia lyase mutant and its use in the synthesis of p-coumaric acid. The mutant undergoes 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. The present application embodiment uses the amino acid sequence shown in SEQ ID NO.2 as a template, performs mutation modification on the key active sites of the template, constructs a mutant library, and screens a series of mutants with significantly improved catalytic activity and / or conversion rate; the tyrosine ammonia lyase mutant provided in the present application embodiment can selectively catalyze L-tyrosine to produce p-coumaric acid, and has excellent yield and / or higher yield.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of bioenzyme technology, and specifically to a tyrosine ammonia lyase mutant and its use in synthesizing p-coumaric acid. Background Art

[0002] p-Coumaric acid (trans-4-hydroxycinnamic acid, also known as p-hydroxycinnamic acid) is a natural compound widely found in plants, particularly in leguminous plants. Its applications span a wide range of industries, including medicine, food, daily necessities, feed, and chemicals. p-Coumaric acid exhibits multiple biological activities, including antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, cardiovascular protection, diabetes prevention and improvement, and neuroprotection. Furthermore, p-Coumaric acid can inhibit the monophenolase and diphenolase activities of tyrosinase, thereby reducing melanin production and delaying skin aging, thus possessing important application value in the cosmetics field. p-Coumaric acid can also form polyhydroxystyrene derivatives through decarboxylation and polymerization reactions, which serve as the main component of photoresists. It can also be used to synthesize photosensitivity p-hydroxypolyimides for use in liquid crystal display devices.

[0003] Currently, the main methods for preparing p-coumaric acid include chemical synthesis and plant extraction. Traditional chemical synthesis typically involves reacting p-hydroxybenzaldehyde with malonic acid in pyridine. However, this method poses significant environmental risks and is difficult to remove impurities, requiring multiple purification steps to meet photoresist raw material quality standards. Plant extraction, on the other hand, utilizes alkaline hydrolysis of lignocellulose and ion exchange resin adsorption extraction techniques. However, this method suffers from low yields, complex processes, and high costs.

[0004] In recent years, with the development of protein engineering and metabolic engineering, the preparation of p-coumaric acid by bioenzymatic methods has gradually become an important research direction. P-coumaric acid can be generated from phenylalanine by the catalytic action of phenylalanine ammonia lyase and cinnamate hydroxylase (C4H), or it can be 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 that limits the improvement of p-coumaric acid production. Therefore, digging suitable tyrosine ammonia lyases and modifying their structures, optimizing the activity and / or conversion efficiency of tyrosine ammonia lyases, is of great significance to 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 objectives, the present application proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a tyrosine ammonia lyase mutant, wherein the mutant has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2:

[0008] F497I, F497I +L248M, F497I +L248M+M4T, L248M+F497I+M4T+V482L.

[0009] In a second aspect, an embodiment of the present application provides a DNA molecule, wherein the DNA molecule is a nucleotide sequence encoding the tyrosine ammonia lyase mutant as described in the first aspect or its complementary sequence.

[0010] In a third aspect, an embodiment of the present application provides a recombinant plasmid, wherein the recombinant plasmid contains the DNA molecule as described in the second aspect.

[0011] In a fourth aspect, an embodiment of the present application provides a recombinant strain, wherein the recombinant strain contains the recombinant plasmid as described in the third aspect.

[0012] As an embodiment, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

[0013] As an embodiment, the host cell of the recombinant strain is a competent cell.

[0014] In one embodiment, the competent cells are Escherichia coli BL21 (DE3).

[0015] In a fifth aspect, the embodiments of the present application propose 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 catalyzing the synthesis of p-coumaric acid from L-tyrosine.

[0016] As an embodiment, the preparation method of p-coumaric acid comprises:

[0017] preparing a recombinant plasmid containing a tyrosine ammonia lyase mutant;

[0018] Transforming the recombinant plasmid into host cells to obtain a recombinant strain;

[0019] With L-tyrosine as a substrate, the recombinant strain is used to catalyze L-tyrosine to produce p-coumaric acid under the conditions of a temperature of 15-50°C and a pH value of 9.5.

[0020] As an embodiment, the preparation of a recombinant plasmid encoding a tyrosine ammonia lyase mutant comprises:

[0021] Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations at the corresponding sites were performed to obtain a recombinant plasmid containing the encoding tyrosine ammonia lyase mutant.

[0022] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects:

[0023] In the examples of the present application, the amino acid sequence shown in SEQ ID NO. 2 was used as a template (basis), and key active sites of the template (basis) were mutated to construct a mutant library. A series of mutants with significantly improved catalytic activity and / or conversion rate were screened and obtained. The tyrosine ammonia lyase mutants provided in the examples of the present application can selectively catalyze L-tyrosine to produce p-coumaric acid with excellent yield and / or higher yield, suggesting that the mutants have more significant application prospects in catalyzing L-tyrosine to produce p-coumaric acid.

[0024] Additional aspects and advantages of the present application will be given in part in the following description, which will become apparent from the following description, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of the standard curve of p-coumaric acid is shown;

[0026] Figure 2 HPLC chart showing the production of p-coumaric acid from L-tyrosine substrate by mutant M4 (F497I / L248M / M4T / V482L);

[0027] Figure 3 The HPLC chart of mutant M4 (F497I / L248M / M4T / V482L) catalyzing 20 g / L L-tyrosine substrate to produce p-coumaric acid at a content of 100 g / L is shown. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only some of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0029] Some of the terms and raw materials involved in this embodiment are explained below to facilitate understanding by those skilled in the art.

[0030] L-Tyrosine: L-Tyr.

[0031] p-Coumaric acid: pCA, or p-CA.

[0032] Unless otherwise specified, the specific composition of the culture medium used in the following examples is as follows:

[0033] LB medium: peptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L.

[0034] TB medium: yeast extract 24 g / L, peptone 12 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L.

[0035] The tyrosine ammonia lyase mutant of this example and its use in synthesizing p-coumaric acid will be described in detail below.

[0036] First, the tyrosine ammonia lyase mutant according to the first aspect of this embodiment will be described.

[0037] Tyrosine ammonia lyase mutant

[0038] In the prior art, p-coumaric acid can be produced from phenylalanine via the catalytic action of phenylalanine ammonia lyase and cinnamate hydroxylase (C4H), or directly from L-tyrosine via the catalysis of tyrosine ammonia lyase. However, the low biological activity of natural enzymes has become a key bottleneck limiting the increase in p-coumaric acid production.

[0039] In view of this, this embodiment provides a tyrosine ammonia lyase mutant, wherein the mutant has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2:

[0040] F497I, F497I +L248M, F497I +L248M+M4T, L248M+F497I+M4T+V482L.

[0041] Among them, the amino acid sequence of tyrosine ammonia lyase (SEQ ID NO. 2) is:

[0042] .

[0043] It will be understood that this example uses the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), and on the basis of this amino acid sequence, the key active sites of the tyrosine ammonia lyase are mutated to construct a mutant library; the tyrosine ammonia lyase mutants provided in this example can selectively catalyze L-tyrosine to produce p-coumaric acid, and have excellent yield and / or higher yield, and have excellent yield and / or higher yield.

[0044] The nucleotide sequence of tyrosine ammonia lyase in this example is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2 above.

[0045] Specifically, the nucleotide sequence of the above-mentioned tyrosine ammonia lyase (SEQ ID NO. 1) is:

[0046]

[0047] As a preferred embodiment of this example, the mutant of this example undergoes any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: F497I + L248M + M4T, L248M + F497I + M4T + V482L.

[0048] 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.

[0049] Among them, the amino acid sequence of mutant F497I (SEQ ID NO. 3) is:

[0050] .

[0051] The amino acid sequence of the mutant F497I + L248M (SEQ ID NO. 4) is:

[0052] MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAAMSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV。

[0053] The amino acid sequence of mutant F497I + L248M + M4T (SEQ ID NO. 5) is:

[0054] MLATSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAAMSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQIPPLETDRPLGQEIAALATHLLQQSPV。

[0055] The amino acid sequence of mutant L248M + F497I + M4T + V482L (SEQ ID NO. 6) is as follows:

[0056] .

[0057] It is understood that the tyrosine ammonia lyase mutants of this embodiment can be mutated at the corresponding sites using existing site-directed mutagenesis techniques to obtain target mutants. Mutants with multiple mutation points can be subjected to additive mutations based on a single mutant, i.e., mutations are performed on a single mutant at another amino acid mutation site to obtain mutants with multiple mutation points.

[0058] In summary, the tyrosine ammonia lyase mutant provided in this example can catalyze L-tyrosine to produce p-coumaric acid with excellent yield and / or higher yield, suggesting that the mutant has more significant application prospects in catalyzing L-tyrosine to produce p-coumaric acid.

[0059] The yield is defined as: actual yield of target product (mole) / theoretical yield of target product (mole) × 100%.

[0060] Next, the DNA molecule according to the second aspect of this embodiment will be described.

[0061] DNA molecules

[0062] It is understood that the DNA molecule provided in this embodiment is a DNA molecule encoding a nucleotide sequence of any of the above-mentioned tyrosine ammonia lyase mutants or a complementary sequence thereof.

[0063] Hereinafter, the recombinant plasmid according to the third aspect of this embodiment will be described.

[0064] Recombinant plasmid

[0065] The recombinant plasmid provided in this embodiment is a DNA molecule encoding the nucleotide sequence of any of the above-mentioned tyrosine ammonia lyase mutants or its complementary sequence.

[0066] Illustratively, the recombinant plasmid of this embodiment can be selected from any one of the following:

[0067] 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(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-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.

[0068] It is understood that the recombinant plasmid of this embodiment can be prepared using existing known preparation methods, for example:

[0069] Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations at the corresponding sites were performed to obtain a recombinant plasmid containing the encoding tyrosine ammonia lyase mutant.

[0070] For example, the above-mentioned expression vector can be selected from pET-28a(+) (purchased from Sangon Biotech (Shanghai) Co., Ltd.); the above-mentioned expression vector containing the amino acid sequence shown in SEQ ID NO. 2 can be synthesized into the pET-28a(+) vector by a gene synthesis company (such as Tianyi Huiyuan Gene Technology Co., Ltd.) to obtain the corresponding template.

[0071] Next, the recombinant strain according to the fourth aspect of this embodiment will be described.

[0072] Recombinant strains

[0073] It can be understood that the recombinant strain provided in this embodiment contains the recombinant plasmid described in the third aspect.

[0074] 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.

[0075] The host cell may be a competent cell, and the competent cell is preferably Escherichia coli BL21 (DE3).

[0076] The recombinant strain of this embodiment can be prepared using existing preparation methods, for example:

[0077] The prepared recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for culture to obtain a recombinant strain.

[0078] The prepared recombinant strain usually needs to be stored in a refrigerator (such as -80°C).

[0079] Next, the use of the tyrosine ammonia lyase mutant according to the fifth aspect of this embodiment will be described.

[0080] Uses of tyrosine ammonia lyase mutants

[0081] As mentioned above, the tyrosine ammonia lyase mutant of this example can be used to catalyze the synthesis (production) of p-coumaric acid from L-tyrosine.

[0082] The preparation method of p-coumaric acid will be introduced in detail below.

[0083] Illustratively, the method for preparing p-coumaric acid in this embodiment comprises:

[0084] (1) preparing a recombinant plasmid encoding a tyrosine ammonia lyase mutant;

[0085] (2) transforming the recombinant plasmid into host cells to obtain a recombinant strain;

[0086] (3) Using L-tyrosine as a substrate, the recombinant strain catalyzes L-tyrosine to produce p-coumaric acid at a temperature of 15-50°C and a pH of 9.5.

[0087] The steps of the above preparation method will be further described below.

[0088] In step (1), preparing a recombinant plasmid encoding a tyrosine ammonia lyase mutant comprises:

[0089] Using the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations at the corresponding sites (site-directed mutagenesis) were performed to obtain a recombinant plasmid containing the encoding tyrosine ammonia lyase mutant.

[0090] The site-directed mutagenesis can be carried out using existing mutagenesis techniques, such as the polymerase chain reaction (PCR) method.

[0091] In step (2), the recombinant plasmid is transformed into a host cell to obtain a recombinant strain, comprising:

[0092] The recombinant plasmid obtained in step (1) was transferred into Escherichia coli BL21 (DE3) for cultivation to obtain a recombinant strain.

[0093] In step (3), L-tyrosine is used as a substrate, and the recombinant strain is used to catalyze L-tyrosine to produce p-coumaric acid at a temperature of 15-50° C. and a pH of 9.5, comprising:

[0094] (3.1) The obtained recombinant bacteria were inoculated into LB medium and TB medium, cultured with shaking, and then centrifuged to collect the cells;

[0095] (3.2) The cells are resuspended in a buffer solution (0.1 M glycine-sodium hydroxide buffer, pH 9.5). L-tyrosine is added to the solution at a temperature of 15-50°C and a pH of 9.5 to produce p-coumaric acid.

[0096] Typically, the reaction conditions of step (3) are: a rotation speed of 250±20 rpm, and a reaction time of 1-48 h.

[0097] Preferably, the reaction temperature is 50° C., and the reaction time is 10-36 h, more preferably 20-24 h.

[0098] For example, in a 10 mL centrifuge tube, 600 μL or 800 μL of wet bacteria were added, and then 400 μL or 200 μL of tyrosine was added to a final concentration of 10-20 g / L. The mixture was placed on a shaker at 50 ° C and stirred at 250 rpm for 24 hours. After the culture was completed, the conversion liquid was collected, 500 μL of 3 mol / L HCl was added to the collected conversion liquid to terminate the reaction, and then 2.5 mL of methanol was added. Then, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. After the supernatant was diluted with methanol to 20-30 times the original concentration, it was filtered through a 0.22 μm nylon 66 filter membrane, and finally the content of p-coumaric acid was detected by high performance liquid chromatography.

[0099] It is understood that the L-tyrosine described in this embodiment is the substrate for the reaction.

[0100] In summary, the tyrosine ammonia lyase mutant provided in this example can catalyze L-tyrosine to produce p-coumaric acid with excellent yield and / or higher yield, suggesting that the mutant has more significant application prospects in catalyzing L-tyrosine to produce p-coumaric acid.

[0101] The present application will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate / explain the present application and are not intended to limit the scope of the present application.

[0102] In the following examples, all materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0103] In the following examples, the quantitative analysis conditions for p-coumaric acid are as follows: Chromatographic conditions: The conversion solution was detected using a Waters high performance liquid chromatograph (equipped with a 2489 UV / Vis detector). The chromatographic column was a Welch Ultimate ® XS-C18 column, 4.6x250mm, column temperature 30°C. Mobile phase: 0.1% acetic acid: methanol = 55:45, flow rate 1.0 mL / min. Injection volume 10 μL, detection wavelength 280 nm.

[0104] Under the above chromatographic conditions, a curve fitting of the peak area and concentration of p-coumaric acid standards at different concentrations (concentration range 5-500 μg / mL, specifically 5, 20, 50, 100, 188, 250, 500 μg / mL) was obtained for the quantitative analysis of p-coumaric acid; Figure 1 A schematic diagram of the standard curve of p-coumaric acid is shown, wherein the ordinate Area is the peak area of the p-coumaric acid standard determined by liquid chromatography.

[0105] In the following examples, the tyrosine ammonia lyase activity was determined using a tyrosine ammonia lyase (TAL) activity detection kit (Solarbio ® , Catalog Number: BC4065), enzyme activity was determined according to the instructions.

[0106] Example 1

[0107] Synthesis and recombinant expression of the parental plasmid pET-28a(+)-RsTAL.

[0108] In this example, wild-type tyrosine ammonia lyase was used as a template (amino acid sequence WP_011339422.1, hereinafter referred to as WT). The selected tyrosine ammonia lyase gene (nucleotide sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2) was synthesized by Sangon Biotech Co., Ltd. and codon-optimized to improve its expression efficiency in Escherichia coli.

[0109] Specifically:

[0110] The optimized gene fragment was cloned into the pET-28a(+) plasmid vector with restriction enzyme sites of BamH I and EcoRI at both ends to construct the recombinant plasmid pET-28a(+)-RsTAL.

[0111] The recombinant plasmid was transformed into E. coli competent cells BL21 (DE3), and positive clones were screened by kanamycin. 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.

[0112] The recombinant E. coli was diluted to the initial OD 600 The cells were inoculated into TB medium to induce protein expression. The cells were cultured at 37 °C and 200 rpm. When the cells reached the logarithmic growth phase, the OD 600 When the p-value reaches 0.6-0.8, add the inducer IPTG to a final concentration of 0.2 mM. Induce expression at 16°C and 200 rpm for 12 hours. After induction, centrifuge the culture at 4°C and 6500 rpm for 10 minutes to collect the cell pellet for subsequent enzyme activity determination and application experiments.

[0113] Example 2

[0114] Construction of tyrosine ammonia lyase mutants.

[0115] In this example, plasmid pET-28a(+)-RsTAL was used as a template to design mutation primers, and the base to be mutated was placed in the middle of the primer.

[0116] The PCR amplification system and PCR reaction procedures refer to the Vazyme 2 × Phanta Flash Master Mix (P510-02) PCR instructions.

[0117] The sequences of the upstream and downstream mutation primers used in this example are shown in Table 1 below:

[0118] Table 1: Sequences of upstream and downstream mutation primers

[0119]

[0120] After the PCR program is completed, DNA gel electrophoresis is performed to verify whether the band size is correct, and after transformation using the DNPI digestion template, the sequencing results are checked using Snap Gene software to check whether the mutation is successful. In this embodiment, 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.

[0121] Among them, mutants with multiple mutation points are obtained by superimposing mutations on a single mutant using the same method as above; the induced expression of the mutants is consistent with the induced expression of the above template.

[0122] Example 3

[0123] In this example, L-tyrosine was used as a substrate to measure the catalytic activities of the mutants (M1-M4).

[0124] Among them, the induced expression of the mutant is consistent with the induced expression of the above template.

[0125] The single colony grown after transformation was inoculated into LB medium and cultured at 37°C and 200 rpm for 12 hours. 600 The inoculum size was about 0.2, and it was inoculated into TB medium and cultured at 37°C and 200 rpm. 600When the pH value reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and then cultured at 16 °C and 200 rpm for 12 hours. After the culture was completed, the recombinant engineered bacteria pET28a-RsTAL (after mutation)- E. coli BL21(DE3) cells were resuspended in 0.1 M glycine-sodium hydroxide buffer (pH 9.5) at a wet weight ratio of 1:8 to obtain wet cells.

[0126] Specifically, the substrate L-tyrosine, the wet cells (M1, M2, M3, or M4) and glycine-sodium hydroxide buffer (pH 9.5) were added to a 10 mL centrifuge tube to a final volume of 1 mL, resulting in an L-tyrosine concentration of 10 g / L and a wet cell concentration of 60 g / L. The reaction was then incubated at 50°C, pH 9.5, and 250 rpm for 24 h to obtain a conversion solution. After the reaction was complete, 500 μL of 3 mol / L HCl was added to terminate the reaction, followed by 2.5 mL of methanol. The tube was then centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected. The supernatant was diluted with methanol to 20 times its original concentration, filtered through a 0.22 μm nylon 66 filter membrane, and the content was determined by HPLC. The results are shown in Table 2.

[0127] Table 2: Effects of different strains on pCA production and yield

[0128]

[0129] The yield is defined as: actual yield of target product (molar) / theoretical yield of target product (molar) × 100%.

[0130] Figure 2 The HPLC diagram of mutant M4 (F497I / L248M / M4T / V482L) catalyzing L-Tyr is shown; Figure 2 HPLC analysis revealed that the wild-type strain (template, or WT) had a p-coumaric acid content of 4.67 ± 0.21 g / L and a p-coumaric acid yield of 51.50 ± 2.37%. Among the screened mutants, M1, M2, M3, and M4 all showed improved pCA production and yield at the same concentration; M4 exhibited the highest yield, reaching 80.40 ± 0.60%, and a p-coumaric acid content of 7.28 ± 0.05 g / L.

[0131] Therefore, compared with the prior art, this example mutated the key active sites of the template WT, constructed a mutant library, and screened a series of mutants with significantly improved catalytic activity and / or conversion rate. The tyrosine ammonia lyase mutants provided in this example can selectively catalyze L-tyrosine to produce p-coumaric acid, and have excellent yield and / or higher yield.

[0132] Example 4

[0133] In this example, the M4 mutant was used as an exemplary mutant to further determine the catalytic activity of the mutants with different addition amounts toward L-Tyr.

[0134] Specifically: In a 10mL centrifuge tube, the substrate L-tyrosine, the above-mentioned wet bacteria (M4) and glycine-sodium hydroxide buffer (pH 9.5) were added to a final volume of 1mL, so that the concentration of L-tyrosine was 20g / L and the concentration of wet bacteria was 60g / L; then, the reaction conditions were 50°C, pH 9.5, and a speed of 250rpm. After the reaction time was 24h, the conversion solution was obtained. After the reaction was completed, 500 μL of 3 mol / L HCl was added to terminate the reaction, and then 2.5mL of methanol was added. Then, centrifuged at 12000rpm for 5 minutes, and the supernatant was collected. After the supernatant was diluted with methanol to 20 times the original concentration, it was filtered through a 0.22 μm nylon 66 filter membrane and the content was detected by HPLC. The test results are shown in Table 3:

[0135] Table 3: Effects of different strains on pCA production and yield

[0136]

[0137] The HPLC results in Table 3 show that the wild-type strain (WT) had a p-coumaric acid content of 6.17 ± 0.19 g / L and a yield of 34.04 ± 1.02%. The M4 strain had a yield of 54.32 ± 0.92% and a p-coumaric acid content of 10.23 ± 0.67 g / L.

[0138] In a 10 mL centrifuge tube, the substrate L-tyrosine, the wet cells (M4), and glycine-sodium hydroxide buffer (pH 9.5) were added to a final volume of 1 mL, resulting in an L-tyrosine concentration of 20 g / L and a wet cell concentration of 100 g / L. The reaction was then incubated at 50°C, pH 9.5, and 250 rpm for 24 h to obtain a conversion solution. After the reaction was complete, 500 μL of 3 mol / L HCl was added to terminate the reaction, followed by 2.5 mL of methanol. The tube was then centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected. The supernatant was diluted with methanol to 20 times its original concentration, filtered through a 0.22 μm nylon 66 filter, and the content was determined by HPLC. The results are shown in Table 4.

[0139] Table 4: Effects of different strains on pCA production and yield

[0140]

[0141] in, Figure 3 The HPLC chart of mutant M4 (F497I / L248M / M4T / V482L) catalyzing 20 g / L L-tyrosine substrate to produce p-coumaric acid at a content of 100 g / L is shown.

[0142] according to Figure 3 The HPLC results shown in Table 4 show that the wild-type strain (WT) had a p-coumaric acid content of 11.98 ± 0.24 g / L and a yield of 66.14 ± 1.32%. M4 achieved a yield of 85.64 ± 0.30% and a p-coumaric acid content of 15.52 ± 0.05 g / L. This indicates that M4 is better at catalyzing the conversion of L-Tyr to pCA, with superior pCA yield and / or higher yield.

[0143] Example 5

[0144] In this example, the M4 mutant was used as an exemplary mutant to further determine the catalytic activity of the mutant towards different concentrations of L-Tyr.

[0145] Specifically, in a 10 mL centrifuge tube, the substrate L-tyrosine, the above-mentioned wet bacteria (M4) and glycine-sodium hydroxide buffer (pH 9.5) were added to a final volume of 1 mL, so that the concentration of L-tyrosine was 20-100 g / L and the concentration of wet bacteria was 200 g / L; then, the reaction conditions were 50 ° C, pH 9.5, and a speed of 250 rpm. After the reaction time was 24 hours, the conversion solution was obtained. After the reaction was completed, 500 μL of 3 mol / L HCl was added to terminate the reaction, and then 2.5 mL of methanol was added. Then, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. After the supernatant was diluted with methanol to 20 times the original concentration, it was filtered through a 0.22 μm nylon 66 filter membrane and the content was detected by HPLC. The test results are shown in Table 5:

[0146] Table 5: pCA production and yield of M4 at different L-tyrosine concentrations

[0147] L-Tyr concentration (g / L) pCA yield (g / L) Yield (%) 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

[0148] The HPLC results in Table 5 demonstrate that M4 effectively catalyzes the conversion of L-Tyr to PCA at varying L-Tyr concentrations, achieving excellent PCA yields and / or higher yields. At an L-tyrosine concentration of 80 g / L, the yield reached 61.85 ± 3.54%, and the coumaric acid content was 44.83 ± 2.57 g / L.

[0149] Example 6

[0150] Based on the catalytic yield of the mutant strains, this example further tested the enzyme activity of the mutant strains; wherein, the tyrosine ammonia lyase activity assay is detailed in the tyrosine ammonia lyase (TAL) activity assay kit (Solarbio ® , Catalog No.: BC4065). In this example, the enzymatic activity of the mutant strain M4 was compared with that of the original enzyme using a tyrosine ammonia lyase activity assay kit. The test results are shown in Table 6 below:

[0151] Table 6: Comparison of enzyme activity of tyrosine ammonia lyase mutant strains (U / g)

[0152] strains Enzyme activity (U / g) WT 68.50 M4 128.75

[0153] Combined with the test results in Table 6, the enzyme activity of the M4 strain increased to 187.95% compared to the original RsTAL. This means that the mutant provided in this example has higher catalytic activity and can selectively catalyze L-tyrosine to produce p-coumaric acid.

[0154] In summary, this example uses the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), performs mutation modification on the key active sites of the template, constructs a mutant library, and screens a series of mutants with significantly improved catalytic activity and / or conversion rate; 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 yield, suggesting that the mutant has more significant application prospects in catalyzing L-tyrosine to produce p-coumaric acid.

[0155] The above is a detailed introduction to the technical solutions provided in the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there may be changes in the specific implementation methods and application scope. In summary, the contents of this specification should not be understood as limiting 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 +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 according to claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains the DNA molecule according to claim 2.

4. A recombinant strain, characterized in that The recombinant strain contains the recombinant plasmid according to 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 cells of the recombinant strain are competent cells.

7. The recombinant strain according to claim 6, characterized in that The competent cells are Escherichia coli BL21 (DE3).

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

9. The use according to claim 8, characterized in that The preparation method of p-coumaric acid comprises: preparing a recombinant plasmid containing a tyrosine ammonia lyase mutant; Transforming the recombinant plasmid into host cells to obtain a recombinant strain; With L-tyrosine as a substrate, the recombinant strain is used to catalyze L-tyrosine to produce p-coumaric acid under the conditions of a temperature of 15-50°C and a pH value of 9.5.

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