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. 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 obtained through screening; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, has excellent yield and / or higher yield, and has outstanding industrial application prospects in efficient and green synthesis of p-coumaric acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological enzymes, in particular to a tyrosine ammonia lyase mutant and its use in synthesis of p-coumaric acid. BACKGROUND

[0002] P-coumaric acid (trans-4-hydroxycinnamic acid) is a common natural aromatic acid in the plant kingdom, especially in leguminous plants. Its unique phenylpropenoic acid skeleton makes it have multiple biological activities such as antioxidant and anti-inflammatory, and it can also inhibit the activity of tyrosinase to block the synthesis of melanin, which is the core ingredient of high-end cosmetics "whitening + anti-aging". In the field of advanced materials, it can be used to prepare deep ultraviolet photoresist main body resin and low dielectric constant photosensitive polyimide to meet the needs of high-resolution liquid crystal display.

[0003] In the prior art, both traditional chemical synthesis and plant extraction routes face the bottleneck of green manufacturing. The pyridine solvent in chemical method has high toxicity and large amount of waste, and the by-products need to be recrystallized several times to meet the standards; the plant extraction method is limited by the biomass content of less than 2%, high acid and alkali consumption, and long extraction steps, resulting in high cost. In addition, enzyme-catalyzed synthesis has become the focus of the current industry. Through the one-step deamination of tyrosine ammonia lyase (TAL), L-tyrosine is directly converted to p-coumaric acid with a theoretical molar yield of 100% and optimal atom economy. However, the specific activity of natural TAL is generally lower than 0.5 U·mg -1 , which cannot meet the requirements of industrial grade.

[0004] Therefore, it is of great significance to excavate suitable tyrosine ammonia lyase and modify its structure to realize the efficient synthesis of p-coumaric acid and promote its industrial production. SUMMARY

[0005] The present application provides a tyrosine ammonia lyase mutant and its use in synthesis of p-coumaric acid.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, the present application 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: M4T, D192V, A245D, A247T, L248F, V482L, C42I, C42I+G179A, C42I+I126V, C42I+I126V+G179A, C42I+I126V+G179A+L248F, C42I+I126V+G179A+L248F+A245D, C42I+I126V+G179A+L248F+A245D+V482L, C42I+I126V+G179A+L248F+A245D+V482L+M4T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

[0007] In a second aspect, the embodiments of the present application provide a DNA molecule comprising a nucleotide sequence encoding the tyrosine ammonia-lyase mutant according to the first aspect or a complementary sequence thereof.

[0008] In a third aspect, the embodiments of the present application provide a recombinant plasmid comprising the DNA molecule according to the second aspect.

[0009] In a fourth aspect, the embodiments of the present application provide a recombinant strain comprising the recombinant plasmid according to the third aspect.

[0010] In a fifth aspect, the embodiments of the present application provide the use of the tyrosine ammonia-lyase mutant according to the first aspect, the DNA molecule according to the second aspect, the recombinant plasmid according to the third aspect or the recombinant strain according to the fourth aspect in catalyzing the production of p-coumaric acid from L-tyrosine.

[0011] As an implementation form, the method for preparing p-coumaric acid comprises: preparing a recombinant plasmid comprising a nucleotide sequence encoding a tyrosine ammonia-lyase mutant; transforming the recombinant plasmid into a host cell to obtain a recombinant strain; using the recombinant strain to catalyze L-tyrosine under the conditions of a temperature of 15-55°C and a pH value of 8.5-11.5, and obtaining p-coumaric acid by reaction of L-tyrosine.

[0012] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects: The amino acid sequence shown in SEQ ID NO. 2 is taken as a template, the key active sites of the template are mutated and reformed, a mutant library is constructed, and a series of mutants with significantly improved catalytic activity and / or conversion rate are screened. The tyrosine ammonia lyase mutant provided in the embodiment can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

[0013] Meanwhile, the catalytic reaction condition of the tyrosine ammonia lyase mutant is mild, and does not need to use organic solvents, which can meet the requirements of green manufacturing. Thus, the tyrosine ammonia lyase mutant provided in the embodiment shortens the process chain through one-step deamination route, can reduce the separation cost, is suitable for the large-scale production of p-coumaric acid in the medical, cosmetic and electronic material grades, and has an outstanding industrial application prospect in the efficient and green synthesis of p-coumaric acid.

[0014] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A standard curve diagram of p-coumaric acid is shown; Figure 2 An HPLC diagram of the mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V catalyzing the generation of p-coumaric acid from L-tyrosine substrate with a concentration of 20 g / L is shown; Figure 3 An HPLC diagram of the mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V catalyzing the generation of p-coumaric acid from L-tyrosine substrate with a concentration of 100 g / L is shown. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments will be described clearly and completely in combination 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. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0017] First, some terms and materials involved in the embodiments will be explained below, so as to facilitate the understanding of those skilled in the art.

[0018] L-Tyr: L-tyrosine, chemical name 2-amino-3-p-hydroxyphenylpropionic acid; its molecular formula is C9H 11NO3, and the structural formula is: .

[0019] pCA: p-coumaric acid, the English name of p-coumaric acid; pCA is the reaction product of the above L-Tyr under the catalysis of the tyrosine ammonia-lyase mutant; the structural formula of pCA is: .

[0020] The specific composition of the culture medium used in the following examples is as follows unless otherwise specified: LB liquid medium: weigh 2.5 g of proteose peptone, 2.5 g of sodium chloride and 1.25 g of yeast powder, stir and dissolve with an appropriate amount of distilled water, make up to 25 mL, and sterilize at high pressure 121°C for 20 minutes; TB liquid medium: add 3.0 g of proteose peptone, 6.0 g of yeast powder and 1 mL of glycerol, stir and dissolve with an appropriate amount of distilled water, make up to 225 mL, and sterilize at high pressure. In addition, weigh 12.54 g (0.72 M) of potassium phosphate dibasic and 2.31 g (0.17 M) of potassium phosphate monobasic, stir and dissolve with an appropriate amount of distilled water, make up to 100 mL, and prepare a potassium phosphate buffer. Before use, add 2.5 mL of potassium phosphate buffer to each bottle of TB medium.

[0021] The tyrosine ammonia-lyase mutant of the present embodiment and its use in the synthesis of p-coumaric acid will be described in detail below.

[0022] First, the tyrosine ammonia-lyase mutant of the first aspect of the present embodiment will be described.

[0023] Tyrosine ammonia-lyase mutant.

[0024] Tyrosine ammonia-lyase (TAL) is a key catalytic tool in the field of biosynthesis, which can directly catalyze the conversion of L-tyrosine to target product p-coumaric acid through a one-step deamination reaction, without the need for exogenous coenzyme and under mild culture conditions. From the perspective of reaction thermodynamics and atomic utilization efficiency, the theoretical molar yield of this conversion path can reach 100%. However, the specific activity of natural TAL is generally lower than , the catalytic reaction rate is slow, and it is difficult to achieve rapid and efficient conversion of the substrate; at the same time, the natural TAL has poor tolerance to high concentration of substrate (L-Tyr) and is easily affected by substrate inhibition. These performance defects result in that the catalytic efficiency, reaction persistence and scale adaptation of natural TAL cannot meet the stringent requirements of industrial production, directly limiting the production capacity expansion, cost control and industrialization landing of related biosynthesis routes, and have become the core technical bottleneck restricting the technical transformation and market application in this field.

[0025] Therefore, the present embodiment aims to find a tyrosine ammonia-lyase mutant that can greatly increase the yield and / or yield of p-coumaric acid, using wild-type tyrosine ammonia-lyase as the initial template (amino acid sequence WP_011339422.1, hereinafter or WT).

[0026] Specifically, the present embodiment provides a tyrosine ammonia-lyase mutant; the mutant has any one of the following amino acid mutations on the amino acid sequence shown in SEQ ID NO. 2: M4T, D192V, A245D, A247T, L248F, V482L, C42I, C42I+G179A, C42I+I126V, C42I+I126V+G179A, C42I+I126V+G179A+L248F, C42I+I126V+G179A+L248F+A245D, C42I+I126V+G179A+L248F+A245D+V482L, C42I+I126V+G179A+L248F+A245D+V482L+M4T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

[0027] wherein the amino acid sequence of the wild-type tyrosine ammonia-lyase (SEQ ID NO. 2) is: MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAALSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQFPPLETDRPLGQEIAALATHLLQQSPV.

[0028] It can be understood that the present embodiment is based on the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), and the key active site of the tyrosine deaminase is mutated and reformed based on the amino acid sequence to construct a mutant library. The tyrosine deaminase mutant provided in the present embodiment can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

[0029] Meanwhile, the catalytic reaction condition of the tyrosine deaminase mutant is mild, and does not require the use of organic solvents, which can meet the requirements of green manufacturing. Therefore, the tyrosine deaminase mutant provided in the present embodiment shortens the process chain through one-step deamination route, reduces the separation cost, and is suitable for the large-scale production of p-coumaric acid in the medical, cosmetic and electronic material industries, and has outstanding industrial application prospects in the efficient and green synthesis of p-coumaric acid.

[0030] The nucleotide sequence of the tyrosine deaminase in the present embodiment is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0031] Specifically, the nucleotide sequence of the tyrosine deaminase (SEQ ID NO. 1) is as follows:

[0032] As a preferred embodiment of the present embodiment, the mutant of the present embodiment has any one of the following amino acid mutations in the amino acid sequence shown as SEQ ID NO. 2: C42I+I126V+G179A, C42I+I126V+G179A+L248F, C42I+I126V+G179A+L248F+A245D, C42I+I126V+G179A+L248F+A245D+V482L, C42I+I126V+G179A+L248F+A245D+V482L+M4T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

[0033] As a more preferred embodiment of the present embodiment, the mutant of the present embodiment has the following amino acid mutations in the amino acid sequence shown as SEQ ID NO. 2: C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

[0034] It can be understood that the tyrosine deaminase mutant of the present embodiment is based on SEQ ID NO. 2, and 1-9 single / stacked mutations of C42I, I126V, G179A, L248F, A245D, V482L, M4T, A247T, and D192V are introduced by existing site-directed saturation mutation and combinatorial mutation to construct a gradient mutant library. The subsequent sites can be further extended, and the present embodiment stage focuses on verification up to D192V.

[0035] Meanwhile, the above-mentioned mutant provided by the present embodiment retains the core folding (TIM-barrel) of TAL and the conserved catalytic 149-151 ASG, can significantly improve the subsequent utilization rate of L-Tyr, so as to ensure that the molar conversion rate is still ≥80% under high substrate load (≥100 g L -1 ).

[0036] The molar conversion rate is defined as: the number of moles of the converted reactant / the total number of moles of the initial reactant x 100%.

[0037] In summary, the tyrosine deaminase mutant provided by the present embodiment can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield, and has outstanding industrial application prospects in the efficient and green synthesis of p-coumaric acid.

[0038] Next, the use of the above tyrosine ammonia-lyase mutant will be described.

[0039] DNA molecules.

[0040] The present embodiment provides a DNA molecule encoding any of the above tyrosine ammonia-lyase mutants or its complementary sequence.

[0041] The coding sequences of the mutants of the present embodiment are all reverse translated according to the codon bias of E. coli, and an Nde I site (CATATG) is introduced at the 5' end and an Xho I site (CTCGAG) is introduced at the 3' end to facilitate subsequent direct subcloning into pET-28a(+) or other T7 expression vectors. The obtained nucleotide sequences and their complementary sequences together constitute the DNA molecules protected by the present embodiment.

[0042] Recombinant plasmids.

[0043] The recombinant plasmids provided by the present embodiment contain the above DNA molecules.

[0044] Exemplarily, the recombinant plasmids of the present 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(+), 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, pPICZA, pPICZαB, pPICZαC, pPIC3K, pPIC3.5K.

[0045] Exemplarily, the embodiment selects pET-28a(+) as the expression vector, inserts the above-mentioned DNA fragment into the Nde I / Xho I site of pET-28a(+) after double enzyme digestion, and obtains a system expression plasmid with His6-tag at the C-terminus. Then, the expression vector containing the amino acid sequence shown in SEQ ID NO. 2 is used as a template to mutate the corresponding sites, and after sequencing verification, a recombinant plasmid containing a tyrosine ammonia lyase mutant is obtained. The naming rule is pET-TAL-mutant point.

[0046] For example, when the mutant is C21I, the mutant is recorded as M1, and the corresponding recombinant plasmid is pET-TAL-M1; when the mutant is C42I + I126V, the mutant is recorded as M2, and the corresponding recombinant plasmid is pET-TAL-M2.

[0047] Recombinant strains.

[0048] It can be understood that the recombinant strain provided in the embodiment contains the recompliant plasmid described above.

[0049] In the 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. The selection of the host cell includes but is not limited to E. coli BL21 (DE3), Rosetta, Tuner, Pichia pastoris GS115, X-33.

[0050] The host cell can be a competent cell, and the competent cell is preferably Pichia pastoris, E. coli BL21 (DE3) or Transetta (DE3).

[0051] For example, the E. coli BL21 (DE3) is selected as the competent cell in the embodiment.

[0052] The recombinant strain in the embodiment can be prepared by using the existing preparation method, for example: The recombinant plasmid is transformed into the competent cell of E. coli BL21 (DE3) for culture to obtain a recombinant strain containing the tyrosine ammonia-lyase mutant plasmid. The naming rule is E. coli-TAL-corresponding mutant.

[0053] The prepared recombinant strain usually needs to be placed in a refrigerator (such as -80°C) for storage.

[0054] Use of the tyrosine ammonia-lyase mutant.

[0055] As described above, the tyrosine ammonia-lyase mutant in the embodiment can be used to catalyze L-tyrosine to generate p-coumaric acid.

[0056] It can be understood that based on the use of the tyrosine ammonia-lyase mutant, the DNA molecule, the recombinant plasmid and the recombinant strain in the embodiment can also be used to catalyze L-tyrosine to generate p-coumaric acid.

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

[0058] For example, the preparation method of p-coumaric acid in the embodiment includes: (1) constructing a recombinant plasmid containing a coding tyrosine ammonia-lyase mutant; (2) transforming the recombinant plasmid into a host cell to obtain a recombinant strain; (3) using the recombinant strain to catalyze L-tyrosine under the conditions of a temperature of 15-55°C and a pH value of 8.5-11.5, and using L-tyrosine as a substrate to obtain p-coumaric acid by reaction.

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

[0060] In step (1), the preparation of a recombinant plasmid containing a coding tyrosine ammonia-lyase mutant includes: The expression vector containing the amino acid sequence shown in SEQ ID NO. 2 is used as a template to complete site-directed mutation by using conventional reverse PCR-DpnI strategy; multiple mutants use "ladder" superposition, i.e. introducing new sites on the verified single mutant recombinant plasmid, ensuring that the positive rate of each round of mutation is > 90%, and finally obtaining a high-activity mutant library containing 2-9 combined mutations. After sequencing verification, subclone into T7 promoter vectors such as pET-28a(+) to obtain mutant recombinant plasmids.

[0061] In step (2), the recombinant plasmid is transformed into host cells to obtain a recombinant strain, including: The recombinant plasmid obtained in step (1) is transformed into competent cells of E. coli BL21(DE3), and the target strain is selected by kanamycin plate. A single colony is picked and cultured to extract the recombinant plasmid. After sequencing confirmation (target sequence is correct), a positive recombinant strain is obtained.

[0062] In step (3), the recombinant strain is used to catalyze L-tyrosine to produce p-coumaric acid under the conditions of temperature 15-55℃ and pH value 8.5-11.5, with L-tyrosine as the substrate, including: The transformed single colony is inoculated in LB and TB liquid medium, respectively, and the bacterial cells are collected by centrifugation after shaking culture. The wet bacterial cells are resuspended by buffer (0.1 M glycine-sodium hydroxide, pH=9.5).

[0063] In the centrifuge tube, the substrate, wet bacterial cells and the above buffer are added, and the recombinant strain is used to catalyze L-tyrosine to produce p-coumaric acid under the conditions of temperature 15-55℃ and pH value 8.5-11.5.

[0064] Preferably, the reaction temperature is 50℃ and the pH value is 9.5.

[0065] It can be understood that the L-tyrosine described in the embodiment is the substrate of the reaction.

[0066] Therefore, the tyrosine ammonia-lyase mutant provided by the embodiment of the application can selectively catalyze L-tyrosine to produce p-coumaric acid, and has excellent yield and / or higher yield, which has outstanding industrial application prospects in the efficient and green synthesis of p-coumaric acid.

[0067] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the application and not to limit the scope of the application.

[0068] In the following examples, the materials, reagents and instruments used, unless otherwise specified, can be purchased commercially.

[0069] Example 1: Synthesis and recombinant expression of template pET-28a(+)-RsTAL.

[0070] In this example, the wild-type tyrosine ammonia-lyase was used as a template (amino acid sequence WP_011339422.1, hereinafter referred to as WT), and the selected tyrosine ammonia-lyase gene (nucleotide sequence as shown in SEQ ID NO. 1, amino acid sequence as shown in SEQ ID NO. 2) was synthesized by Sheng Wu Biotechnology Co., Ltd. to optimize the codons and introduce BamH I (5' end) and EcoR I (3' end) restriction enzyme sites to improve its expression efficiency in E. coli.

[0071] In this example, the optimized gene fragment and pET-28a (+) vector were simultaneously double-digested using two endonucleases, and then the digested gene fragment and linearized vector were connected by T4 DNA ligase to obtain the recombinant expression plasmid pET-28a(+)-RsTAL.

[0072] The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, and positive clones were selected by kanamycin (Kana) screening. Single colonies were selected for sequencing verification to confirm that the amino acid sequence of the cloned tyrosine ammonia-lyase was SEQ ID NO: 2.

[0073] The single colony obtained by transformation was inoculated in LB medium and cultured at 37°C, 200 rpm for 12 hours. Then, the culture was transferred to TB medium with an inoculum of about 0.2 OD 600 and cultured at 37°C, 200 rpm. When the OD 600 of the bacterial solution reached 0.6-0.8, the inducer IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.2 mM, and the culture was induced to express at 16°C, 200 rpm for 12 hours.

[0074] After the above induction expression was completed, the culture was centrifuged at 4°C, 5000 rpm for 5 minutes, and the supernatant (containing medium components and a small amount of secreted protein) was discarded. The recombinant engineering bacteria pET28a-RsTAL (mutated)-E. coli BL21(DE3) precipitate at the bottom was collected. According to the wet weight ratio of 1:10, the 0.1 M glycine-sodium hydroxide buffer with pH=9.5 was used for resuspension, and then the physiological saline was used for washing twice to obtain the wet bacteria, which was used for subsequent enzyme activity determination and application experiments.

[0075] Example 2: Construction of tyrosine ammonia lyase mutant.

[0076] In this example, the plasmid pET-28a(+)-RsTAL was used as a template, and a mutation primer was designed with the mutation base in the middle of the primer. The PCR amplification system and PCR reaction program refer to the Vazyme 2xPhanta Flash Master Mix(P510-02) PCR instruction manual.

[0077] In this example, the sequences of the upstream and downstream mutation primers are shown in Table 1 as follows: Table 1: Sequences of upstream and downstream mutation primers

[0078] After the PCR program is completed, the band size of the amplification product is first verified by DNA gel electrophoresis to determine whether it is consistent with the target fragment; then the residual template DNA is digested with DpnI enzyme to reduce the interference of non-target plasmids.

[0079] The DpnI-treated PCR product is transformed into E. coli BL21(DE3) competent cells, and after recovery culture, an appropriate amount of bacterial liquid is spread on LB plates containing kanamycin (Kana), and incubated at 37°C overnight. Single colonies on the plate are picked for verification, and 1-3 suspected positive clones are selected for gene sequencing, and the sequencing results are compared by Snap Gene software to confirm the formation of target gene mutations.

[0080] Among them, the mutant with multiple mutation points is obtained by superimposing mutations (gradually introducing I126V, G179A, L248F, A245D, V482L, M4T, A247T, D192V) on the basis of the single mutant (C42I) using the same method as described above.

[0081] In this embodiment, the single mutant C42I is denoted as M1; the multiple mutant C42I+I126V is denoted as M2; the multiple mutant C42I+I126V+G179A is denoted as M3; the multiple mutant C42I+I126V+G179A+L248F is denoted as M4; the multiple mutant C42I+I126V+G179A+L248F+A245D is denoted as M5; the multiple mutant C42I+I126V+G179A+L248F+A245D+V482L is denoted as M6; the multiple mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T is denoted as M7; the multiple mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T is denoted as M8; and the multiple mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V is denoted as M9.

[0082] The induced expression of the mutants is consistent with the induced expression of the templates described above.

[0083] Example 3: Screening of catalytic activity of mutants

[0084] In this embodiment, the catalytic activity of the mutants in Example 2 is determined by taking L-tyrosine as a substrate.

[0085] The construction of the catalytic reaction system and the detection steps are as follows: In a 10 mL centrifuge tube, the substrate L-tyrosine, the wet bacteria described above (M4T, D192V, A245D, A247T, L248F, V482L, F497I, I126V, G179A, C42I+G179A, M1, M2, M3, M4, M5, M6, M7, M8 or M9) and glycine-sodium hydroxide buffer (pH 9.5) are sequentially added, and the volume is made up to 1 mL, so that the concentration of L-tyrosine is 10 g / L and the concentration of the wet bacteria is 60 g / L. The reaction system is reacted at 50°C, pH 9.5 and a rotation speed of 250 rpm for 24 h to obtain a conversion liquid.

[0086] After the reaction is completed, 500 μL of 3 mol / L HCl is added to terminate the reaction, and then 2.5 mL of methanol is added. Next, centrifugation is performed at 12000 rpm for 5 minutes, the supernatant is diluted to 20 times the original concentration with methanol, and then filtered through a 0.22 μm nylon 66 filter membrane. The content is detected by HPLC, and the detection results are shown in Table 2.

[0087] To ensure the accuracy and quantitative reliability of the HPLC detection results, a concentration-peak area standard curve of p-coumaric acid standard is constructed before the experiment for the quantitative analysis of p-coumaric acid.Figure 1 A standard curve of p-coumaric acid is shown, in which the ordinate is the peak area of p-coumaric acid standard determined by liquid chromatography. Figure 1 In the present embodiment, the standard concentration range is set to 5-500 μg / mL, and the peak area has a linear correlation coefficient R 2 >0.999, which meets the linear requirement of quantitative analysis.

[0088] Table 2: Test results of catalytic activity of mutants

[0089] Note: In the present embodiment, the yield is defined as: actual yield of target product (moles) / theoretical yield of target product (moles) x 100%.

[0090] In the present embodiment, the amino acid sequence of mutant M1 is shown in SEQ ID NO. 3; the amino acid sequence of mutant M2 is shown in SEQ ID NO. 4; the amino acid sequence of mutant M3 is shown in SEQ ID NO. 5; the amino acid sequence of mutant M4 is shown in SEQ ID NO. 6; the amino acid sequence of mutant M5 is shown in SEQ ID NO. 7; the amino acid sequence of mutant M6 is shown in SEQ ID NO. 8; the amino acid sequence of mutant M7 is shown in SEQ ID NO. 9; the amino acid sequence of mutant M8 is shown in SEQ ID NO. 10; and the amino acid sequence of mutant M9 is shown in SEQ ID NO. 11.

[0091] In combination with the test results in Table 2, the wild-type strain (template, or WT) has a defect of low yield in the reaction of catalyzing L-tyrosine to generate p-coumaric acid. The present embodiment mutates the key active sites based on the template (WT), and can obtain mutants with significantly improved yield in catalyzing L-tyrosine under different concentrations.

[0092] Among the single mutants, the yield of mutants M1, M4T, D192V, A245D, A247T, L248F, V482L and F497I is significantly improved compared with the template (WT).

[0093] Further, the present embodiment performs superimposed mutation based on mutant M1 to obtain a mutant with multiple mutation points, and simultaneously verifies the influence of other mutants on the yield and yield of catalyzing L-tyrosine to generate p-coumaric acid.

[0094] In the experiment, 20 g / L L-tyrosine is used as the substrate for whole-cell catalysis, and the reaction solution is diluted 50 times and subjected to chromatographic analysis. Figure 2The mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) catalyzes the L-tyrosine substrate with a concentration of 20 g / L to generate the HPLC chart of p-coumaric acid. According to the spectrum results of Figure 2 the chromatogram, the chromatographic peak with a retention time of 2.932 minutes corresponds to unreacted L-tyrosine, and the peak with a retention time of 6.405 minutes is confirmed as the catalytic product p-coumaric acid.

[0095] In combination with the test results of Table 2 and Figure 2 the test results of Table 3, the multi-mutants obtained on the basis of the mutant M1 in this embodiment have significantly improved yield and yield of p-coumaric acid generated by each multi-mutant catalyzing L-tyrosine under the same concentration (10 g / L) compared with the mutant M1. Among them, the multi-mutants M3-M9 are preferred embodiments of this embodiment.

[0096] Further, the WT in this embodiment has a significantly decreased yield when catalyzing a higher concentration of L-Tyr (20 g / L). Under this concentration, the preferred mutants M3-M9 all effectively improve the yield of pCA relative to M1. Among them, the mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) exhibits the highest yield, reaching 16.48±0.17%, and the content of p-coumaric acid is 90.92±0.91 g / L.

[0097] Therefore, compared with the prior art, this embodiment mutates the key active sites of the template WT, 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 embodiment can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

[0098] Example 4: This embodiment takes the mutant M9 as an exemplary mutant to further determine the catalytic activity of the above mutant on high-concentration L-Tyr.

[0099] The liquid chromatography results show that the optimal induction temperature of the mutant M9 is 20-25°C, so the subsequent protein induction expression of the mutant M9 is performed at 20°C. At the same time, in the construction of the catalytic system, it is determined through time exploration experiments that the yield reaches the highest at 10 h of reaction.

[0100] In this embodiment, after whole-cell catalysis is performed on 100 g / L L-tyrosine as the substrate, the reaction solution is diluted by 200 times and subjected to chromatography analysis. Figure 3The mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) catalyzes the HPLC chart of p-coumaric acid generated from the L-tyrosine substrate with a concentration of 100 g / L. According to the spectrum results of Figure 3 The spectrum results show that the chromatographic peak with a retention time of 2.967 minutes corresponds to unreacted L-tyrosine, and the peak with a retention time of 6.472 minutes is identified as the catalytic product p-coumaric acid. The content detection results are shown in Table 3: Table 3: Catalytic activity of M9 in 100 g / L L-tyrosine

[0101] The detection results of Table 3 and Figure 3 show that the yield of M9 reaches 83.23±0.38% when the concentration of L-tyrosine is 100 g / L, while the yield of WT is only 27.22±1.16%, and the yield of M9 is 3.06 times that of WT, and the standard deviation is smaller, indicating that the stability of M9 catalytic reaction is better.

[0102] From the pCA yield, the pCA yield of M9 is 75.40±0.35 g / L, which is 3.06 times that of WT (24.65±1.05 g / L), directly reflecting the high conversion ability of M9 under high substrate concentration, and solving the problem of low catalytic efficiency of WT for high concentration of L-tyrosine.

[0103] In summary, the mutant M9 can better catalyze L-Tyr to generate pCA, and the pCA has excellent yield and / or higher yield.

[0104] Example 5: In this example, the mutant M9 is used as an exemplary mutant, and the tyrosine ammonia lyase (TAL) activity detection kit (Solarbio®, Catalog No.: BC4065) is used to further determine the enzyme activity content according to the instruction manual.

[0105] The detection results are shown in Table 4: Table 4: Determination of different tyrosine ammonia lyase enzyme activities

[0106] According to the enzyme activity detection results of Table 4, the enzyme activity of the wild type strain (WT) is set to 100%, and the enzyme activity of M9 reaches 550%, i.e. the activity of the tyrosine ammonia lyase mutant of M9 is 5.5 times that of WT.

[0107] It can be understood that higher enzyme activity means that mutant M9 can catalyze more L-Tyr to be converted into pCA in unit time, which explains why mutant M9 can reach the maximum yield in 10 h (faster reaction rate) and also provides a key enzymatic basis for achieving high yield under high concentration of substrate (100 g / L-Tyr).

[0108] In summary, the amino acid sequence shown in SEQ ID NO. 2 is used as a template, the key active sites of the template are mutated and reformed, a mutant library is constructed, and a series of mutants with significantly improved catalytic activity and / or conversion rate are screened. The tyrosine deaminase mutant provided in the embodiments of the present application can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield, and has outstanding industrial application prospects in efficient and green synthesis of p-coumaric acid.

[0109] The technical solutions provided in the embodiments of the present application are described in detail above, and specific examples are applied in this paper to describe the principles and implementation modes of the embodiments of the present application. The above description of the 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, the specific implementation modes and application scope will be changed, and the above description of the embodiments should not be understood as a limitation of the present application.

Claims

1. A mutant tyrosine deaminase enzyme, characterized in that, The mutant has any one of the following amino acid mutations in the amino acid sequence as shown in SEQ ID NO. 2: M4T, D192V, A245D, A247T, L248F, V482L, C42I, C42I+G179A, C42I+I126V, C42I+I126V+G179A, C42I+I126V+G179A+L248F, C42I+I126V+G179A+L248F+A245D, C42I+I126V+G179A+L248F+A245D+V482L, C42I+I126V+G179A+L248F+A245D+V482L+M4T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T, C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

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

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

4. The recombinant plasmid of claim 3, wherein, The recombinant plasmid is selected from any one 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(+), 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, pPICZA, pPICZαB, pPICZαC, pPIC3K, pPIC3.5K.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant plasmid of claim 4.

6. The recombinant bacterial strain of claim 5, wherein, The host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

7. The recombinant bacterial strain of claim 5, wherein, The host cell of the recombinant strain is a competent cell.

8. The recombinant bacterial strain of claim 7, wherein, The competent cell is Escherichia coli BL21 (DE3).

9. Use of the tyrosine ammonia-lyase mutant of claim 1, the DNA molecule of claim 2, the recombinant plasmid of claim 3 or 4, or the recombinant strain of any one of claims 5-8 in catalyzing L-tyrosine to produce p-coumaric acid.

10. Use according to claim 9, characterized in that, The method for preparing p-coumaric acid comprises: preparing a recombinant plasmid containing a gene encoding a tyrosine ammonia-lyase mutant; transforming the recombinant plasmid into a host cell to obtain a recombinant strain; using the recombinant strain to catalyze L-tyrosine at a temperature of 15-55°C and a pH value of 8.5-11.5, and a reaction of L-tyrosine to produce p-coumaric acid.

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