Tyrosine ammonia lyase mutants and their use in the synthesis of p-coumaric acid

Abstract

The application provides a tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid. The application takes an amino acid sequence shown in SEQ ID NO. 2 as a template, mutates and reforms key active sites of the template, constructs a mutant library, and selects a series of mutants with significantly improved catalytic activity and / or conversion rate. The tyrosine ammonia lyase mutant provided by the application can selectively catalyze L-tyrosine to generate p-coumaric acid, has excellent yield and / or higher yield, and has an outstanding industrial application prospect 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 phenyl-propenoic 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 atomic 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:

[0007] 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:

[0008] 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.

[0009] In a second aspect, the embodiments of the present application provide a DNA molecule, which comprises a nucleotide sequence encoding the tyrosine ammonia-lyase mutant according to the first aspect or a complementary sequence thereof.

[0010] In a third aspect, the embodiments of the present application provide a recombinant plasmid, which comprises the DNA molecule according to the second aspect.

[0011] In a fourth aspect, the embodiments of the present application provide a recombinant strain, which comprises the recombinant plasmid according to the third aspect.

[0012] 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.

[0013] As an implementation form, the method for preparing p-coumaric acid comprises:

[0014] preparing a recombinant plasmid containing a nucleotide sequence encoding the tyrosine ammonia-lyase mutant;

[0015] transforming the recombinant plasmid into a host cell to obtain a recombinant strain;

[0016] 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.

[0017] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects:

[0018] 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.

[0019] 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.

[0020] Additional aspects and advantages of the present application will be described in part below, which will become apparent from the following description, or will be understood by those skilled in the art from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A standard curve diagram of p-coumaric acid is shown;

[0022] 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;

[0023] 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

[0024] The technical solutions in the embodiments will be described clearly and completely below with reference to the embodiments of the present application and the accompanying drawings. Obviously, the embodiments 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.

[0025] The following first explains some terms and materials involved in the embodiments, so as to facilitate understanding by those skilled in the art.

[0026] L-Tyr: L-tyrosine, chemical name 2-amino-3-p-hydroxyphenylpropionic acid; its molecular formula is C9H 11 NO3, structural formula is: .

[0027] pCA: p-coumaric acid, English name p-coumaric acid; pCA is the reaction product of the above L-Tyr under the catalysis of tyrosine ammonia-lyase mutant; the structural formula of pCA is: .

[0028] The specific composition of the culture medium used in the following examples is as follows unless otherwise specified:

[0029] 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 appropriate amount of distilled water, make up to 25 mL, sterilize at high pressure 121℃ for 20 minutes;

[0030] 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 appropriate amount of distilled water, make up to 225 mL, divide into 22.5 mL / bottle, 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 appropriate amount of distilled water, make up to 100 mL to prepare potassium phosphate buffer. Before use, add 2.5 mL of potassium phosphate buffer to each bottle of TB medium.

[0031] 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.

[0032] First, the tyrosine ammonia-lyase mutant of the first aspect of the present embodiment will be described.

[0033] Tyrosine ammonia-lyase mutant.

[0034] 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 one-step deamination reaction, without 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 adaptability 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.

[0035] 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).

[0036] 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:

[0037] 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.

[0038] Wherein, the amino acid sequence of the wild-type tyrosine ammonia-lyase (SEQ ID NO. 2) is:

[0039] MLAMSPPKPAVELDRHIDLDQAHAVASGGARIVLAPPARDRCRASEARLGAVIREARHVYGLTTGFGPLANRLISGENVRTLQANLVHHLASGVGPVLDWTTARAMVLARLVSIAQGASGASEGTIARLIDLLNSELAPAVPSRGTVGASGDLTPLAHMVLCLQGRGDFLDRDGTRLDGAEGLRRGRLQPLDLSHRDALALVNGTSAMTGIALVNAHACRHLGNWAVALTALLAECLRGRTEAWAAALSDLRPHPGQKDAAARLRARVDGSARVVRHVIAERRLDAGDIGTEPEAGQDAYSLRCAPQVLGAGFDTLAWHDRVLTIELNAVTDNPVFPPDGSVPALHGGNFMGQHVALTSDALATAVTVLAGLAERQIARLTDERLNRGLPPFLHRGPAGLNSGFMGAQVTATALLAEMRATGPASIHSISTNAANQDVVSLGTIAARLCREKIDRWAEILAILALCLAQAAELRCGSGLDGVSPAGKKLVQALREQFPPLETDRPLGQEIAALATHLLQQSPV.

[0040] 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.

[0041] Meanwhile, the catalytic reaction condition participated by the tyrosine deaminase mutant is mild, and does not need to use organic solvent, which can meet the requirement of green manufacturing; thus, the tyrosine deaminase mutant provided in the present 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 outstanding industrial application prospect in the efficient and green synthesis of p-coumaric acid.

[0042] 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.

[0043] Specifically, the nucleotide sequence (SEQ ID NO. 1) of the tyrosine deaminase is as follows:

[0044]

[0045] 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:

[0046] 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.

[0047] 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:

[0048] C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V.

[0049] It can be understood that the tyrosine ammonia lyase mutant of the present embodiment is based on SEQ ID NO. 2, and through existing site-directed saturation mutation and combination mutation, 1-9 single / overlapping mutations of C42I, I126V, G179A, L248F, A245D, V482L, M4T, A247T, and D192V are introduced into the mutant library in stages. The subsequent sites can be further extended, and the present embodiment stage focuses on verification up to D192V.

[0050] 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, and 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 ).

[0051] 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%.

[0052] In summary, the tyrosine ammonia-lyase mutant provided by 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.

[0053] Next, the use of the above tyrosine ammonia-lyase mutant will be described.

[0054] DNA molecule.

[0055] The present embodiment provides a DNA molecule encoding the nucleotide sequence or its complementary sequence of any of the above tyrosine ammonia-lyase mutants.

[0056] The coding sequences of the mutants of the present embodiment are all reverse translated according to the codon bias of Escherichia 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 sequence and its complementary sequence together constitute the DNA molecule protected by the present embodiment.

[0057] Recombinant plasmid

[0058] The present embodiment provides a recombinant plasmid containing the above DNA molecule.

[0059] Exemplarily, the recombinant plasmid of the present embodiment can be selected from any of the following:

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Recombinant strains.

[0064] It can be understood that the recombinant strain provided in the embodiment contains the recompliant plasmid described above.

[0065] 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.

[0066] The host cell can be a competent cell, and the competent cell is preferably Pichia pastoris, E. coli BL21 (DE3) or Transetta (DE3).

[0067] Exemplarily, the E. coli BL21 (DE3) is selected as the competent cell in the embodiment.

[0068] The recombinant strain of the embodiment can be prepared by using the existing preparation method, for example:

[0069] The recombinant plasmid described above is transformed into the E. coli BL21 (DE3) competent cell for culture to obtain a recombinant strain containing the tyrosine ammonia-lyase mutant plasmid. The naming rule is E. coli-TAL-corresponding mutant.

[0070] The prepared recombinant strain generally needs to be placed in a refrigerator (such as -80°C) for storage.

[0071] Use of the tyrosine ammonia-lyase mutant.

[0072] As described above, the tyrosine ammonia-lyase mutant of the embodiment can be used for catalyzing L-tyrosine to generate p-coumaric acid.

[0073] 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 of the embodiment can also be used for catalyzing L-tyrosine to generate p-coumaric acid.

[0074] The preparation method of p-coumaric acid will be described in detail below.

[0075] Exemplarily, the preparation method of p-coumaric acid in the embodiment includes:

[0076] (1) constructing a recombinant plasmid containing a coding tyrosine ammonia-lyase mutant;

[0077] (2) transforming the recombinant plasmid into a host cell to obtain a recombinant strain;

[0078] (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 obtaining p-coumaric acid by reaction.

[0079] The steps of the preparation method will be further described below.

[0080] In step (1), a recombinant plasmid containing a tyrosine ammonia-lyase mutant is prepared, including:

[0081] The expression vector containing the amino acid sequence shown in SEQ ID NO. 2 is used as a template, and a conventional reverse PCR-DpnI strategy is used to complete site-directed mutagenesis; multiple mutants are stacked in a "ladder" manner, that is, new sites are continuously introduced on the verified single-mutant recombinant plasmid, and the positive rate of each round of mutation is ensured to be >90%, and finally a high-activity mutant library containing 2-9 combined mutations is obtained. After sequencing verification, it is subcloned into a T7 promoter vector such as pET-28a(+) to obtain a mutant recombinant plasmid.

[0082] In step (2), the recombinant plasmid is transformed into host cells to obtain a recombinant strain, including:

[0083] 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 screening. A single colony is picked and cultured for expansion, and the recombinant plasmid is extracted. After sequencing confirmation (the target sequence is correct), a positive recombinant strain is obtained.

[0084] In step (3), using L-tyrosine as a substrate, the recombinant strain is used to catalyze L-tyrosine under the conditions of a temperature of 15-55°C and a pH value of 8.5-11.5, and p-coumaric acid is obtained by reaction, including:

[0085] The transformed single colony is inoculated in LB and TB liquid media, respectively, and after shaking culture, the bacterial cells are collected by centrifugation, and the wet bacterial cells are resuspended by a buffer (0.1 M glycine-sodium hydroxide, pH=9.5).

[0086] In a centrifuge tube, the substrate, wet bacterial cells and the above buffer are added, and the recombinant strain is used to catalyze L-tyrosine to generate p-coumaric acid under the conditions of a temperature of 15-55°C and a pH value of 8.5-11.5.

[0087] Preferably, the reaction temperature is 50°C, and the pH value is 9.5.

[0088] It can be understood that the L-tyrosine described in the embodiment is the substrate of the reaction.

[0089] Therefore, the tyrosine ammonia-lyase mutant provided by 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 an outstanding industrial application prospect in the efficient and green synthesis of p-coumaric acid.

[0090] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate / explain the present application and are not used to limit the scope of the present application.

[0091] In the following examples, the materials, reagents and instruments used are commercially available unless otherwise specified.

[0092] Example 1: Synthesis and recombinant expression of template pET-28a(+)-RsTAL.

[0093] In this example, the wild-type tyrosine ammonia-lyase is used as a template (amino acid sequence is WP_011339422.1, hereinafter referred to as WT), and the selected tyrosine ammonia-lyase gene (nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2) is synthesized by Sheng Wu Biotechnology Co., Ltd. to perform codon optimization and introduce BamH I (5' end) and EcoR I (3' end) restriction enzyme cutting sites to improve the expression efficiency in Escherichia coli.

[0094] In this example, the optimized gene fragment and the pET-28a (+) vector are simultaneously double-cut by two endonucleases, and then the cut gene fragment and the linearized vector are connected by T4 DNA ligase to obtain the recombinant expression plasmid pET-28a(+)-RsTAL.

[0095] The recombinant plasmid is transformed into E. coli BL21 (DE3) competent cells, and positive clones are selected by kanamycin (Kana) screening; a single colony is selected for sequencing verification to confirm that the amino acid sequence of the cloned tyrosine ammonia-lyase is SEQ ID NO: 2.

[0096] The single colony obtained by transformation is inoculated in LB medium and cultured at 37°C, 200 rpm for 12 hours. Then, the inoculum is transferred to TB medium at a final OD 600 of about 0.2, and the culture is continued at 37°C, 200 rpm. When the OD 600 of the bacterial solution reaches 0.6-0.8, an inducer IPTG (isopropyl-β-D-thiogalactopyranoside) is added to a final concentration of 0.2 mM, and the culture is induced at 16°C, 200 rpm for 12 hours.

[0097] After the above induction expression is completed, the culture is centrifuged at 4°C and 5000 rpm for 5 minutes, and the supernatant (containing medium components and a small amount of secreted protein) is discarded, and the recombinant engineering bacteria pET28a-RsTAL (after mutation)-E. coli BL21 (DE3) precipitate at the bottom is collected. According to the wet weight ratio of 1:10, 0.1 M glycine-sodium hydroxide buffer with pH = 9.5 is used for resuspension, and then physiological saline is used for washing twice to obtain wet bacteria, which is used for subsequent enzyme activity determination and application experiment.

[0098] Example 2: Construction of tyrosine ammonia lyase mutant.

[0099] In this embodiment, the plasmid pET-28a (+)-RsTAL is used as a template, and a mutation primer is designed, and the base to be mutated is placed in the middle of the primer. The PCR amplification system and the PCR reaction program are referred to the Vazyme 2xPhanta Flash Master Mix (P510-02) PCR instruction manual.

[0100] In this embodiment, the sequences of the upstream and downstream mutation primers used are shown in Table 1 as follows:

[0101] Table 1: Sequences of upstream and downstream mutation primers

[0102]

[0103] After the PCR program is completed, first, the band size of the amplification product is 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.

[0104] 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 an LB plate containing kanamycin (Kana), and incubated at 37°C overnight. A single colony on the plate is 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 the target gene mutation.

[0105] 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) by using the same method as described above.

[0106] 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; the multiple mutant C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V is denoted as M9.

[0107] The induced expression of the mutants is consistent with the induced expression of the templates described above.

[0108] Example 3: Screening of catalytic activity of mutants.

[0109] In this embodiment, the catalytic activity of the mutants in Example 2 is determined by taking L-tyrosine as a substrate.

[0110] The construction of the catalytic reaction system and the detection steps are as follows:

[0111] 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.

[0112] 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.

[0113] To ensure the accuracy and reliability of the HPLC test results, a standard curve of coumaric acid concentration-peak area was constructed before the experiment for the quantitative analysis of coumaric acid. Figure 1 A standard curve of coumaric acid is shown in the figure, in which the vertical coordinate is the peak area of coumaric acid standard determined by liquid chromatography. Figure 1 In this embodiment, the standard concentration range is set to 5-500 μg / mL, and the linear correlation coefficient R 2 >0.999, which meets the linear requirement of quantitative analysis.

[0114] Table 2: Test results of catalytic activity of mutants

[0115]

[0116] Note: In this embodiment, the yield is defined as: actual yield of target product (moles) / theoretical yield of target product (moles) x 100%.

[0117] In this 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.

[0118] In combination with the test results in Table 2, the wild-type strain (template, or WT) has the defect of low yield in the reaction of catalyzing L-tyrosine to generate coumaric acid. The key active sites of the template (WT) are mutated and modified in this embodiment, and mutants with significantly improved yield in catalyzing L-tyrosine under different concentrations are obtained.

[0119] 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).

[0120] Further, based on mutant M1, this embodiment performs superimposed mutation 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 coumaric acid.

[0121] The experiment was performed with 20 g / L L-tyrosine as the substrate, and after whole-cell catalysis, the reaction solution was diluted 50 times and subjected to chromatographic analysis. Figure 2 The HPLC chart of mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) catalyzing L-tyrosine substrate with a concentration of 20 g / L to generate p-coumaric acid is shown. According to the chromatographic results of Figure 2 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 identified as the catalytic product p-coumaric acid.

[0122] In combination with the test results of Table 2 and Figure 2 Based on mutant M1, the multi-mutants of the present example significantly improved the yield and yield of p-coumaric acid generated by catalyzing L-tyrosine relative to mutant M1 at the same concentration (10 g / L). Among them, multi-mutants M3-M9 are preferred embodiments of the present example.

[0123] Further, the WT of the present example significantly reduced the yield when catalyzing higher concentration of L-Tyr (20 g / L). At this concentration, the preferred mutants M3-M9 all effectively improved the yield of pCA relative to M1. Among them, mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) showed the highest yield, reaching 16.48±0.17%, and the content of p-coumaric acid was 90.92±0.91 g / L.

[0124] Therefore, compared with the prior art, the present example 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 the present example can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

[0125] Example 4:

[0126] In the present example, mutant M9 is used as an exemplary mutant to further determine the catalytic activity of the above-mentioned mutant on high-concentration L-Tyr.

[0127] The liquid chromatography results show that the optimal induction temperature of mutant M9 is 20-25°C, so the subsequent protein induction expression uses 20°C. At the same time, in the construction of the catalytic system, through time exploration experiments, it is determined that the yield reaches the highest at 10 h of reaction.

[0128] The whole cell catalysis was carried out with 100 g / L L-tyrosine as the substrate, and the reaction solution was diluted 200 times and subjected to chromatographic analysis. Figure 3 The HPLC chart of mutant M9 (C42I+I126V+G179A+L248F+A245D+V482L+M4T+A247T+D192V) catalyzing L-tyrosine substrate with a concentration of 100 g / L to generate p-coumaric acid was shown. According to the chromatographic results of Figure 3 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:

[0129] Table 3: Catalytic activity of M9 in 100 g / L L-tyrosine

[0130]

[0131] 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.

[0132] 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 capacity of M9 under high substrate concentration, solving the problem of low catalytic efficiency of WT for high concentration of L-tyrosine.

[0133] In summary, mutant M9 can better catalyze L-Tyr to generate pCA, and pCA has excellent yield and / or higher yield.

[0134] Example 5:

[0135] In this example, mutant M9 was used as an exemplary mutant, and the tyrosine ammonia lyase (TAL) activity detection kit (Solarbio®, Catalog No.: BC4065) was used to further determine the enzyme activity content according to the instruction manual.

[0136] The detection results are shown in Table 4:

[0137] Table 4: Determination of different tyrosine ammonia lyase enzyme activities

[0138]

[0139] According to the enzyme activity detection results in Table 4, the enzyme activity of the wild type strain (WT) is set as 100%, and the enzyme activity of M9 reaches 550%, that is, the activity of the tyrosine deaminase mutant of M9 is 5.5 times that of WT.

[0140] 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 provides a key enzymatic basis for achieving high yield under high concentration of substrate (100 g / L-Tyr).

[0141] In summary, in this embodiment, 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 modified, 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 this 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.

[0142] The above describes the technical solutions provided in the embodiments of the present application in detail, and the principles and implementation modes of the embodiments of the present application are described by applying specific examples; 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 ranges will be changed, and in summary, the content of the specification should not be understood as limiting 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: 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 as claimed in claim 4. The recombinant strain contains the recombinant plasmid as claimed in 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 generate 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 coding sequence of 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 obtain p-coumaric acid.

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