Tyrosine phenol lyase mutant, engineering bacterium and application of tyrosine phenol lyase mutant in synthesis of levodobar

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and aims to provide a tyrosine phenol lyase mutant which is higher in catalytic efficiency and more suitable for industrial production of levodopa and is derived from citrobacter freundii, and compared with a wild tyrosine phenol lyase type, the tyrosine phenol lyase mutant has T49R or / and M379R mutation; the amino acid sequence of the tyrosine phenol lyase wild type is shown as SEQ ID NO.2 in a sequence table. The mutant and the engineering strain provided by the invention have the huge advantages of high catalytic efficiency and high product yield.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and enzyme engineering technology, specifically relating to a tyrosine phenol-lyase (TPL) mutant derived from Citrobacter freundii, its encoding gene, recombinant engineered bacteria containing the gene, and their application in the efficient and highly stereoselective synthesis of levodopa (L-DOPA). Background Technology

[0002] Levodopa (L-3,4-dihydroxyphenylalanine, L-DOPA), as a direct precursor of dopamine in the human body, is the first-line drug for the clinical treatment of Parkinson's disease. It can effectively cross the blood-brain barrier and be converted into dopamine in the central nervous system, thereby relieving patients' motor dysfunction.

[0003] Among various synthetic routes for levodopa, enzymatic catalysis has attracted much attention due to its clean process, mild conditions, and excellent stereoselectivity. Tyrosine phenol-lyase (TPL, EC 4.1.99.2) can efficiently synthesize levodopa in a single reaction step using catechol, pyruvate, and ammonia as substrates. This not only avoids the heavy metal catalysts and complex protection-deprotection steps involved in traditional chemical synthesis but also possesses extremely high atom economy and regioselectivity, with product optical purity typically exceeding 99.9%.

[0004] However, natural TPL still faces several key bottlenecks in practical industrial applications. On the one hand, its natural substrate is L-tyrosine, while its affinity and catalytic efficiency for catechol, a non-natural substrate used to synthesize levodopa, are generally low. On the other hand, high concentrations of catechol in the reaction system can easily induce conformational changes in the enzyme, leading to severe substrate inhibition effects, thereby limiting the substrate feed concentration and final yield in the reaction system.

[0005] To overcome the aforementioned limitations, existing research has attempted to modify TPL from different microbial sources through protein engineering. For example, existing technologies (such as patents CN114250237A and CN119120437A) have reported the generation of several mutant enzymes with improved catalytic performance through directed evolution or semi-rational design of TPL derived from *Fusobacterium nucleatum*. Another approach (such as CN119570774A) focuses on TPL from *Erwinia herbicola*, improving its substrate conversion ability to some extent through combined mutations at key sites. *Citrobacter freundii* is also a known microbial source capable of producing TPL, possessing unique enzymatic properties. However, to date, there has been no systematic molecular modification research targeting TPL from this specific species, and there is a lack of effective mutation schemes that can significantly improve its catalytic efficiency, stability, and tolerance in L-DOPA synthesis. This represents a technological gap to be filled in this field. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a mutant of tyrosine phenol lyase derived from Citrobacter freundii with higher catalytic efficiency and more suitable for industrial production of levodopa, which has a T49R or / and M379R mutation compared with the wild type of tyrosine phenol lyase; the amino acid sequence of the wild type of tyrosine phenol lyase is shown in SEQ ID NO.2 in the sequence listing.

[0007] The present invention also provides an engineered strain containing E. coli BL21(DE3) cells containing the tyrosine phenol lyase mutant.

[0008] Compared with the prior art, the present invention has the following significant advantages.

[0009] 1. High catalytic efficiency: The TPL mutants obtained by this invention, especially the combined mutants, have a specific enzyme activity that is 50% to 100% higher than that of wild-type Citrobacter freundii TPL.

[0010] 2. High product yield: In fed-batch reaction, using the wet cell of the mutant engineered bacteria of this invention as a catalyst, the cumulative yield of levodopa can reach more than 150 g / L, and the conversion rate of the substrate catechol exceeds 99%. Attached Figure Description

[0011] Picture 1 This is a flowchart of the reaction process for the synthesis of levodopa catalyzed by the TPL mutant of this invention.

[0012] Picture 2This is a comparison chart of the yield of levodopa synthesized by wild-type and mutant TPL in the examples. Detailed Implementation

[0013] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0014] Example 1

[0015] Cloning and expression of the wild-type TPL gene in Citrobacter freundii This gene was synthesized based on the TPL gene of *Citrobacter freundii* (GenBank ID: CP020857.1). Synthesis was performed by Nanjing GenScript Gene Technology Co., Ltd. The synthesized sequence was cloned into the pET-28a(+) vector and transformed into *E. coli* BL21(DE3) competent cells (purchased from Beijing Zhuangmeng Co., Ltd.) to obtain wild-type engineered bacteria. The base sequence of the TPL wild-type gene is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence is shown in SEQ ID NO.2 of the sequence listing.

[0016] Single colonies were picked from the plate and inoculated into 5 mL of LB / Kan medium, and incubated overnight at 37°C. The next day, the colonies were transferred to 50 mL of fresh medium at a 1:50 ratio and incubated at 37°C with shaking until the OD600 reached 0.6-0.8. 0.1 mM IPTG was added, and the culture was induced at 37°C for 3-4 hours. After induction, 1 mL of culture was collected by centrifugation. The supernatant was discarded, and the bacterial cells were resuspended in 100 μL of 1×SDS-PAGE loading buffer and boiled in a water bath for 10 minutes to fully denature the proteins. SDS-PAGE analysis was then performed.

[0017] Example 2

[0018] Construction and screening of TPL mutants Using the wild-type TPL gene as a template, saturated mutant libraries at the T49, N185, and M379 sites were constructed using whole-plasmid amplification PCR technology, respectively, according to the following methods: PCR was performed according to standard procedures. In a sterile 200 μl centrifuge tube, add 5 μl of PCR buffer (10×), 5 μl of ldNTP, 2.5 μl of F primer (10 pmol / μl), 2.5 μl of R primer (10 pmol / μl), 1–10 ng of template DNA, and bring the volume to 50 μl with sterile water. Add 1 μl of KOD (TOYOBO), mix well, and place the solution in an Eppendorf PCR instrument. Set the PCR cycling parameters as follows: denaturation at 94°C for 30–40 s, annealing at 55°C for 1 min, extension at 72°C for 5 min, for a total of 30–35 cycles.

[0019] The primer pairs for the saturation mutant libraries at T49, N185, and M379 sites are as follows: T49 F: 5'-ATCCCGCGTGATGAGCGCNNKAATGCAGGAAGCGGGTTACA-3'.

[0020] T49 R: 5'-TGTAACCCGCCTTCCTGCATMNNGCTCCTCACGCGGGATCATAG-3'.

[0021] N185 F: 5'-GCAATATGTTGCCGGGAATNNKATTTCACCACCACCCG-3'.

[0022] N185 R: 5'-CGGGGTGGTTGGTGAAAMNNATTCCCGGCGCAACATATT-3'.

[0023] M379 F: 5'-GCTGAAAGCCGCTGGCGTNNKATTGTTGAACCGGTAGGC-3'.

[0024] M379 R: 5'-GCCTACCGGTTCAACAATMNNACCAGCGGCTTTCAGCT-3'.

[0025] After the reaction was complete, the PCR product was collected, chemically transformed into BL21(DE3) cells, and then plated onto Kans plates. The colonies on the plates were inoculated into 96-well plates, incubated at 37°C for 12 hours, and then induced with 0.1 mM IPTG. After incubation for another 20 hours, the plates were stored at -80°C.

[0026] The high-throughput salicylaldehyde colorimetric method is based on the following principle: Under strongly alkaline conditions, the remaining sodium pyruvate in the reaction solution undergoes a specific colorimetric reaction with salicylaldehyde, generating a yellow product with a characteristic absorption peak at 465 nm. Therefore, at the same initial substrate concentration and reaction time, the mutant with higher catalytic efficiency will consume more sodium pyruvate, resulting in less residual sodium pyruvate in the supernatant after the reaction terminates, and ultimately a lower absorbance value of the colorimetric reaction system.

[0027] The specific screening steps are as follows: A standardized enzymatic reaction system of 200 μL was established in a 96-well deep plate. This system contained: 50 mM Tris-HCl buffer (pH 8.0), 40 mM sodium pyruvate, 40 mM catechol, 50 g / L ammonium acetate, 1 mM pyridoxal phosphate (PLP), 1 g / L sodium sulfite, and 2 g / L EDTA·Na2. 20 μL of the crude enzyme solution or whole cell suspension of the induced and lysed candidate mutant strain was added to each well, and the reaction was carried out at 30℃ and 180 rpm for 30 minutes with shaking. After the reaction, 200 μL of 1 M hydrochloric acid solution was added to each well to terminate the reaction.

[0028] After terminating the reaction, centrifuge the 96-well deep-well plate at 4°C and 4000 × g for 15 minutes, and collect the supernatant for subsequent colorimetric analysis. In a new 96-well clear microplate, precisely add the following reagents sequentially to construct a 1 mL colorimetric system: 300 μL of 25% sodium hydroxide solution (w / v); 40 μL of the reaction supernatant obtained in step 2; 660 μL of ultrapure water; 10 μL of pure salicylaldehyde (or salicylaldehyde solution dissolved in anhydrous ethanol, volume ratio 1:3). After adding all the ingredients, use a micro-shaker to thoroughly mix the microplate for 30 seconds to ensure the reactants are evenly mixed.

[0029] The 96-well microplate was placed at room temperature (25°C) and kept in the dark for 120 minutes to ensure sufficient and stable colorimetric reaction. Then, the absorbance of each well was measured using a multi-mode microplate reader at a wavelength of 465 nm.

[0030] The absorbance of samples treated under the same conditions with strains carrying wild-type TPL was used as a control. Mutant clones with absorbance values ​​significantly lower than this control were identified as positive clones in the initial screening, suggesting faster sodium pyruvate conversion rates and potentially higher enzyme catalytic activity. These positive clones will be selected for the next round of precise secondary screening using high-performance liquid chromatography (HPLC).

[0031] Initial screening was performed using a salicylaldehyde colorimetric method, followed by secondary screening using HPLC quantitative analysis. Beneficial single-point mutations, including T49R, N185H, and M379R, were ultimately obtained and further combined to obtain the double mutant T49R / M379R. The amino acid sequence of the double mutant T49R / M379R is shown in SEQ ID NO.3 of the sequence listing.

[0032] Example 3

[0033] Comparison of inducible expression and enzyme activity assays of wild-type and single mutant T49R, N185H, M379R TPL Cultured separately to express plasmids containing wild-type and single mutant types. E. coli After collecting wet bacterial cells, the enzyme activity of BL21(DE3) was determined in a standard reaction system (containing catechol, sodium pyruvate, PLP, etc., pH 8.0, 15°C). The results showed that the specific enzyme activities of the T49R, N185H, and M379R single mutants were 1.5, 0.9, and 1.2 times that of the wild type, respectively, amounting to approximately 60, 36, and 48 U / g wet bacterial cells. The wild type had a specific activity of 40 U / g wet bacterial cells.

[0034] Example 4

[0035] Comparison of induction expression and enzyme activity assays of wild-type and double mutant T49R / M379R TPL Cultured separately to express plasmids containing wild-type and T49R / M379R mutant plasmids E. coli After collecting wet bacterial cells, the enzyme activity of BL21(DE3) was determined in a standard reaction system (containing catechol, sodium pyruvate, PLP, etc., pH 8.0, 15°C). The results showed that the specific enzyme activity of the optimal mutant T49R / M379R was 2.0 times that of the wild type, approximately 80 U / g wet bacterial cells.

[0036] Example 5

[0037] Application of T49R mutant in whole-cell catalytic synthesis of levodopa Expressing plasmids containing the T49R mutant E. coli After collecting wet bacterial cells, BL21(DE3) was added to a 500 mL reaction system with 20 g / L wet bacterial cells as a catalyst, initially with 5 g / L catechol and 7 g / L sodium pyruvate. The reaction was carried out at 15°C and pH 8.0, with 15 g / L catechol and 15 g / L sodium pyruvate added every 2 hours.

[0038] Results: The reaction using the T49R mutant achieved a cumulative levodopa concentration of 109 g / L within 8 hours, with a catechol conversion rate >99.5% and optical purity >99.9%. In contrast, under the same conditions, wild-type TPL only synthesized 85 g / L of levodopa.

[0039] Example 6

[0040] Application of the M379R mutant in the whole-cell catalytic synthesis of levodopa Expressing plasmids containing the M379R mutant E. coli After collecting wet bacterial cells, BL21(DE3) was added to a 500 mL reaction system with 20 g / L wet bacterial cells as a catalyst, initially with 5 g / L catechol and 7 g / L sodium pyruvate. The reaction was carried out at 15°C and pH 8.0, with 15 g / L catechol and 15 g / L sodium pyruvate added every 2 hours.

[0041] Results: The reaction using the T49R mutant achieved a cumulative levodopa concentration of 103 g / L within 8 hours, with a catechol conversion rate >99.5% and optical purity >99.9%. In contrast, under the same conditions, wild-type TPL only synthesized 85 g / L of levodopa.

[0042] Example 7

[0043] Application of T49R / M379R mutant in whole-cell catalytic synthesis of levodopa Expressing plasmids containing the T49R / M379R mutant E. coli After collecting wet bacterial cells, BL21(DE3) was added to a 500 mL reaction system with 20 g / L wet bacterial cells as a catalyst, initially with 5 g / L catechol and 7 g / L sodium pyruvate. The reaction was carried out at 15°C and pH 8.0, with 15 g / L catechol and 15 g / L sodium pyruvate added every 2 hours.

[0044] Results: The reaction using the T49R / M379R mutant achieved a cumulative levodopa concentration of 162 g / L within 8 hours, with a catechol conversion rate >99.5% and optical purity >99.9%. In contrast, under the same conditions, wild-type TPL only synthesized 85 g / L of levodopa.

Claims

1. A tyrosine phenol lyase mutant having a T49R or / and M379R mutation compared to the wild-type tyrosine phenol lyase; the amino acid sequence of the wild-type tyrosine phenol lyase is shown in SEQ ID NO.2 in the sequence listing.

2. The use of the tyrosine phenol lyase mutant of claim 1 in the synthesis of levodopa.

3. An engineered strain, comprising E. coli BL21(DE3) cells containing the tyrosine phenol lyase mutant of claim 1.

4. The use of the engineered strain described in claim 3 in the synthesis of levodopa.

5. The method for constructing the tyrosine phenol lyase mutant according to claim 1, characterized in that, The 49th amino acid T of the wild-type tyrosine phenol lyase was mutated to R, or / and the 379th amino acid M of the wild-type tyrosine phenol lyase was mutated to R.

6. The method for constructing the engineered strain according to claim 3, characterized in that, The tyrosine lyase mutant of claim 1 was obtained by transfecting it into E. coli BL21(DE3) competent cells.

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