Threonine aldolase mutant with enhanced enzyme activity and its application in producing L-serine

Abstract

The present invention belongs to the field of bioengineering, and specifically discloses a threonine aldolase mutant with improved enzyme activity and its application in the production of L-serine. The amino acid sequence of the mutant has a mutation of T140A or T140A / I118V compared to the wild-type threonine aldolase derived from Aeromonas. Experiments show that, after analysis of enzyme activity assays of induced expression and purification, the enzyme activity of the mutant is 1.6 and 2.9 times higher than that of the wild type, respectively, and the thermal stability is significantly improved. The beneficial mutants provided by the present invention can lay a good foundation for the industrial production of L-serine and downstream derivatives such as L-cysteine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the construction and application of a threonine aldolase mutant with enhanced enzyme activity. Background Technology

[0002] Over the past decade, biocatalysis has emerged as a promising cutting-edge technology in the industrial synthesis of natural medicines and fine chemicals. Compared to traditional methods, it offers highly precise stereoselectivity, allowing for precise control of product stereoconfiguration to meet the stringent configuration requirements of complex compounds. Furthermore, its ease of operation significantly reduces process complexity and cost, making it a highly competitive alternative to traditional methods. The construction of CC bonds has always held a fundamental position in chemical synthesis systems. In addition to chemical synthesis pathways, the scientific community has successfully discovered numerous enzymes capable of specifically catalyzing CC bond formation, such as aldolases, transketolases, transaldolases, and alcohol nitrile lyases. Among these, aldolases have attracted considerable attention in the field of enzymatic CC bond synthesis due to their broad substrate adaptability and excellent stereoselectivity. Aldolases catalyze reversible condensation reactions, promoting the reaction between nucleophilic donors (such as ketones, enols, and their analogues) and electrophilic acceptors (aldehydes or ketones) to form CC bonds. It is worth noting that aldolases exhibit strict structure specificity for substrate donors but diversity for substrate acceptors, enabling them to adapt to a variety of aldehydes or ketones with different structures. This characteristic gives aldolases great flexibility in catalytic synthesis, allowing them to synthesize products with a rich variety of structures.

[0003] Threonine aldolases (TAs, EC 4.1.2.5) are a class of enzymes that rely on pyridoxal 5′-phosphate (PLP) as a cofactor to catalyze a series of reversible aldol condensation reactions using glycine as a donor substrate. In the forward reaction, TAs catalyze the cleavage of L-threonine into acetaldehyde and glycine; in the reverse reaction, TAs can use glycine as a donor and aliphatic or aromatic aldehydes as acceptors to catalyze the synthesis of β-hydroxy-α-amino acids through aldol condensation. Based on their stereospecificity to the α-carbon chiral center of threonine, TAs can be divided into two main classes: L-TAs and D-TAs. L-TAs can be further divided into three categories: (i) L-TAs that specifically recognize L-threonine; (ii) L-allo-TAs that specifically recognize L-allo-threonine; and (iii) low-specificity L-TAs that can recognize both L-threonine and L-allo-threonine. Although these three types of enzymes are highly conserved in their primary structure, they differ significantly in substrate recognition and catalytic properties.

[0004] Threonine aldolases, capable of specifically forming C-C bonds, have important applications in organic chemical synthesis and are widely used in pharmaceutical and chemical industries to catalyze the synthesis of key precursors for pharmaceutical intermediates and fine chemicals. In recent years, with the development of synthetic biology and metabolic engineering, amino acids (TAs) have shown significant application potential in the industrial synthesis of amino acids, especially L-serine. In microbial cell factories, by introducing or modifying TAs, the efficient synthesis of L-serine from glycine and aldehyde intermediates can be achieved, bypassing the dependence on energy and intermediate metabolites in traditional synthetic pathways, thereby increasing yield and reducing byproduct accumulation. Furthermore, the stereoselectivity and substrate adaptability of TAs provide powerful tools for the targeted synthesis of high-value non-natural amino acids. Therefore, TAs are becoming important enzymatic tools for constructing efficient biosynthetic pathways and expanding the structural diversity of amino acid products. Summary of the Invention

[0005] Based on the above requirements, the primary objective of this invention is to provide a threonine aldolase mutant, which improves its catalytic activity and thermal stability, thus facilitating the catalytic production of L-serine and its derivatives and other metabolites.

[0006] This invention is achieved through the following technical approach: based on Aeromonas hydrophila... Aeromonas sp The wild-type threonine aldolase encoding gene sequence of CU5 was obtained using whole-genome synthesis technology after codon optimization by Genewiz Suzhou Co., Ltd., and then cloned into the pET21b expression vector. Nde I / Xho At site I, the corresponding recombinant plasmid was obtained and named pET21b-AsTA [gene sequence shown in SEQ ID NO.2]. Using error-prone PCR random mutagenesis, a mutant library of the Aeromonas threonine aldolase encoding gene AsTA was obtained, and it was subcloned into the pET21b expression vector using a classic restriction enzyme digestion and ligation construction strategy. Nde I / Xho At site I, a recombinant plasmid library containing the encoding gene of an Aeromonas threonine aldolase mutant was obtained. The recombinant plasmid library was then introduced into Escherichia coli BL21(DE3), and after primary screening in 96-well plates and secondary screening in shake flasks, mutants with enhanced enzyme activity and thermostability were finally selected, thus completing this invention.

[0007] This invention provides a strain derived from Aeromonas hydrophila. Aeromonas sp The threonine aldolase mutant of CU5, relative to the wild-type amino acid sequence shown in SEQ ID No. 1, has only a mutation at position 141 where threonine T is mutated to alanine A, or only a combination mutation exists where threonine T is mutated to alanine A at position 141 and isoleucine I is mutated to valine V at position 119.

[0008] The present invention further provides the coding gene for the Aeromonas threonine aldolase mutant. In a preferred embodiment, the nucleotide sequence of the coding gene for the Aeromonas threonine aldolase mutant is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 2.

[0009] The present invention also provides an expression vector containing the aforementioned coding gene. Specifically, it is a prokaryotic expression vector.

[0010] Preferably, it is the Escherichia coli expression vector pET21b and the Corynebacterium glutamicum expression vector pXMJ19.

[0011] The present invention contains recombinant bacteria with the expression vector described above.

[0012] Specifically, it is a recombinant engineered Escherichia coli or a recombinant engineered Corynebacterium glutamicum.

[0013] The present invention also provides the use of the threonine aldolase mutant, or its encoding gene, or the expression vector containing the above, or the recombinant bacteria in the catalytic synthesis of L-serine or its derivatives.

[0014] Specifically, L-serine is catalyzed by using formaldehyde and glycine as substrates.

[0015] The threonine aldolase mutant provided by this invention has significant advantages in the catalytic synthesis of L-serine and its derivatives. In the reported L-serine synthesis pathway, serine hydroxymethyltransferase (SHMT) is one of the key catalytic enzymes. However, this enzyme relies on PLP as a cofactor and requires the participation of tetrahydrofolate, resulting in a complex mechanism of action. Furthermore, it increases the complexity of the reaction system and the cost of separation and purification in actual production.

[0016] The beneficial effects of this invention are that, by mutating and screening threonine aldolase derived from Aeromonas hydrophila, mutant threonine aldolase with varying degrees of enhanced catalytic performance was obtained. This enzyme can effectively catalyze the formation of L-serine from formaldehyde and glycine without the need for the addition of tetrahydrofolate. It has advantages such as high atom economy, simple catalytic process, mild reaction conditions, and no reaction byproducts, and shows good application prospects in the synthesis of L-serine and its derivatives. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0018] Example 1: Screening and identification of threonine aldolase mutant libraries

[0019] Threonine aldolases are a class of glycine-dependent aldolases that use glycine as a donor substrate and rely on pyridoxal phosphate as a cofactor. They catalyze reversible alcohol-aldol condensation reactions and have significant application prospects in pharmaceutical and fine chemical synthesis. In previous studies, when screening based on our laboratory-preserved threonine aldolase gene database, we discovered that they originated from Aeromonas. Aeromonassp The wild-type threonine aldolase of CU5 (SEQ ID NO: 1) exhibits good C / C bond linkage ability, catalyzing the aldol condensation reaction of formaldehyde and glycine to form L-serine. In this embodiment, we based on... Aeromonassp The CU5 threonine aldolase encoding gene sequence (SEQ ID NO: 2) was obtained through whole-gene synthesis after codon optimization by Suzhou Genewiz Co., Ltd., and cloned into the pET21b expression vector to obtain the corresponding recombinant plasmid, named pET21b-AsTA. Using a fault-prone PCR random mutagenesis method, with the pET21b-AsTA recombinant plasmid as a template, primer pairs AsTA-For (5'-TCGACATATGcgctatattgatctgcgcagcgatac-3', SEQ ID No: 3) and AsTA-Rev (5'-CTAGCTCGAGttagcgcgcgcccagatattcggt-3', SEQ ID No: 4) were used to amplify the AsTA mutant library of the Aeromonas threonine aldolase encoding gene. Using a conventional restriction enzyme digestion and ligation construction strategy, this library was subcloned into the NdeI / XhoI site of the pET21b expression vector to obtain a recombinant plasmid library containing the Aeromonas threonine aldolase mutant encoding gene.

[0020] The error-prone PCR system used in this invention is as follows: 5 μL 10× EasyTaq buffer, 0.2 μM upstream primer P1, 0.2 μM upstream primer P2, 200 μM dNTPs, 0.8 mM MnCl2, 6 mM MgSO4, 50 ng template DNA, 1 μL EasyTaq DNA polymerase, and sterile water to bring the volume to 50 μL. The PCR reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 2 min, repeated 35 times; 72℃ extension for 5 min, and storage at 4℃.

[0021] Given that threonine aldolase from Aeromonas hydrophila can catalyze the aldol condensation of formaldehyde and glycine to produce L-serine, mutants with enhanced catalytic activity can be rapidly screened by measuring and monitoring the formaldehyde content in the reaction system. The formaldehyde content in the reaction system was determined using the acetylacetone spectrophotometric method described in the national standard HJ 601-2011; specifically, formaldehyde reacts with acetylacetone in the presence of excess ammonium salt to form a yellow compound, which exhibits maximum absorption at 414 nm. In a preferred embodiment, the recombinant plasmid library containing the encoding gene of the Aeromonas hydrophila threonine aldolase mutant was introduced into Escherichia coli BL21(DE3). The absorption peak changes at 414 nm were measured using 96-well plate primary screening and shake-flask secondary screening, ultimately identifying mutants with enhanced enzyme activity, named T140A and T140A / I118V, respectively.

[0022] Example 2: Activity assay of a threonine aldolase mutant library

[0023] The selected inducible expression vector is pET21b, which contains a strong T7 promoter-related sequence (including the operator sequence lacO and the conserved RBS sequence). When an inducer such as IPTG or lactose is added, it will cause the repressor protein to leave the operator sequence and initiate gene expression.

[0024] The inducible expression vector selected in this invention is pXMJ19. This plasmid itself contains a strong promoter tac-related sequence (including the operator sequence lacO and the conserved RBS sequence), which can replicate in Escherichia coli and Corynebacterium glutamicum. When an inducer such as IPTG or lactose is added, it will also cause the repressor protein to leave the operator sequence and initiate gene expression.

[0025] In this embodiment, recombinant *E. coli* cells induced by the expression vector pET21b were collected. The culture medium was removed by centrifugation, and the bacterial cells were resuspended in 10 mL of pre-chilled lysis buffer (20 mM Na₂HPO₃, 200 mM NaCl, pH 7.5). Cells were disrupted using an ultrasonic cell disruptor at 200 W, with a 2-second sonication interval of 1 second, for a total of 10 min. Subsequently, the cells were centrifuged at 8000 × g for 10 min in a high-speed refrigerated centrifuge, and the supernatant was collected for subsequent protein purification and enzyme activity assays.

[0026] Based on the catalytic characteristics of Aeromonas threonine aldolase, its enzyme activity was determined using the ability to produce L-serine products as a standard. The enzyme activity assay system consisted of 1 mL (100 mM HEPES-NaOH, pH 8.0), 100 mM glycine, 50 mM formaldehyde, 50 μM MPa / L, and 10 μL crude enzyme solution. The reaction system was subjected to a 30°C reaction. o The L-serine content in the system was detected by HPLC after incubating at C for 10 min. The study found that compared with the activity of wild-type unmutated threonine aldolase (38.6 µmol / min / mg), both mutants T140A and T140A / I118V showed significantly enhanced catalytic performance, with enzyme activities increased by 1.6 and 2.9 times, respectively.

[0027] Furthermore, the thermostability of both mutants was significantly improved after 40 days of testing. o After incubation at C for 30 min, the remaining enzyme activity was 31%, while under the same high-temperature treatment, the remaining enzyme activity of mutant T140A was 78%, and that of mutant T140A / I118V was 94%, all of which were much higher than that of wild-type non-mutated threonine aldol.

[0028] Example 3: Application of threonine aldolase mutant in the production of L-serine

[0029] This embodiment is based on a whole-cell transformation method, using basic raw materials such as formaldehyde and glycine, and efficiently catalyzing the synthesis of L-serine with the addition of PLP cofactor.

[0030] Recombinant Corynebacterium glutamicum (based on expression vector pXMJ19) containing different threonine aldolase encoding genes were inoculated into seed culture medium containing antibiotics and cultured on a shaker at 32°C and 200 r / min for 16–20 h to obtain seed culture. The inoculum was then transferred to a 3 L fermenter at a rate of 5–10%, and the culture temperature was set at 32°C, the stirring speed at 300–500 r / min, and the pH maintained neutral. During the culture, when dissolved oxygen fell below 30%, a stirrer-dissolved oxygen control system was activated to maintain sufficient dissolved oxygen supply. When the cell concentration (OD) reached a certain level...600 When the concentration reaches 20-40%, 0.4-0.6 mM IPTG is added for induction, and the culture temperature is lowered to below 25°C. Induction culture continues under neutral conditions for at least 20 hours. Finally, the bacterial culture is collected by centrifugation at 8000×g for 10 min, and the supernatant is removed to obtain recombinant bacterial sludge. The seed culture medium consists of: yeast extract 2.5 g / L, peptone 5 g / L, NaCl 5 g / L, and brain heart extract 37 g / L; the fermentation culture medium consists of: glucose 100 g / L, corn steep liquor 20 g / L, molasses 20 g / L, ammonium sulfate 10 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium citrate 1 g / L, calcium carbonate 5 g / L, and biotin 0.1 mg / L. The culture medium is adjusted to pH 7.0.

[0031] In this embodiment, a 50 mL Tris-HCl buffer system was used, with 30 g / L of the whole-cell bacterial sludge prepared in the above embodiment added, along with 250 mM formaldehyde, 250 mM glycine, and 0.4~0.6 mM PLP. The reaction solution was placed at 30~40 °C. o C. The reaction was carried out at a speed of 200-400 rpm. After the reaction was completed, the L-serine content in the reaction system was determined by HPLC.

[0032] The test results are shown in the table below. Compared with the wild-type enzyme, the two threonine aldolase mutants exhibited superior and improved catalytic performance. Under high concentrations of aldehyde substrates, they could efficiently catalyze the condensation of formaldehyde and glycine to produce L-serine. In particular, the T140A / I118V combined mutant showed even higher efficiency in catalyzing the production of L-serine from formaldehyde, yielding 26.06 g / L of L-serine with a catalytic conversion rate of 99%, demonstrating promising prospects for industrial applications.

[0033] Table 1. Analysis of L-serine production results based on threonine aldolase

[0034]

[0035] The above description is merely a preferred embodiment of the present invention, intended to illustrate the technical solution of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core technical concept of the present invention, those skilled in the art can make appropriate adjustments, changes, or equivalent substitutions based on the specification and claims of the present invention. All technical improvements, equivalent solutions, and modifications within the scope defined by the claims of the present invention should be considered within the scope of protection of the present invention. Furthermore, technical content not specifically described in the present invention, if it is a conventional technical means for those skilled in the art, is also considered part of the present invention.

Claims

1. A threonine aldolase mutant derived from Aeromonas, characterized in that: Compared with the wild-type amino acid sequence shown in SEQ ID No. 1, there is only a mutation from threonine T to alanine A at position 140, or there is only a combined mutation from threonine T to alanine A at position 140 and from isoleucine I to valine V at position 118.

2. The gene encoding the threonine aldolase mutant according to claim 1.

3. An expression vector containing the coding gene according to claim 2.

4. The expression vector according to claim 3, wherein It is a prokaryotic expression vector.

5. The expression vector according to claim 3, wherein They are the Escherichia coli expression vector pET21b and the Corynebacterium glutamicum expression vector pXMJ19.

6. A recombinant bacterium containing the expression vector according to any one of claims 3 to 5.

7. The recombinant bacterium according to claim 6, characterized in that The invention is a recombinant Escherichia coli engineering bacterium or a recombinant Corynebacterium glutamicum engineering bacterium.

8. Use of the threonine aldolase mutant according to claim 1, the encoding gene according to claim 2, or the expression vector containing the threonine aldolase mutant according to any one of claims 3 to 5, or the recombinant bacterium according to claim 6 or 7 in catalyzing the synthesis of L-serine.

9. The use according to claim 8, characterized in that It uses formaldehyde and glycine as substrates to catalyze the production of L-serine.

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