(S)-omega-transaminase mutant and application thereof in efficient preparation of L-homoserine

Abstract

This invention discloses an (S)-ω-transaminase mutant and its application in the efficient preparation of L-homoserine, belonging to the fields of genetic engineering and enzyme engineering technology. This invention utilizes transaminase from Vibrio fluvialis. Vf Using TA as the wild type, a (S)-ω-transaminase mutant with high enzyme activity, high temperature, and pH tolerance was constructed through single and combined mutations. Based on this, this invention utilizes a multi-enzyme cascade reaction between the (S)-ω-transaminase mutant and aldolase to efficiently catalyze the synthesis of L-homoserine. The mutant M1 constructed in this invention exhibits 4.13 times the enzyme activity of the wild-type enzyme at 30°C; the multi-enzyme cascade reaction with aldolase can achieve a homoserine yield of up to 106 g / L, with a conversion rate of 88%.

Description

Technical Field

[0001] This invention relates to a (S)-ω-transaminase mutant and its application in the efficient preparation of L-homoserine, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] ω-transaminase (ω Transaminases (TAs) (EC 2.6.1.X) are a class of enzymes that use pyridoxal 5′-phosphate (PLP) as an essential coenzyme to catalyze the reversible transfer of amino groups between amino donors and amino acceptors. Their most significant feature is their ability to recognize and catalyze substrates with non-α-amino groups, especially various keto esters, aromatic ketones, and even sterically hindered ketones, thereby generating high-value chiral amines and non-natural amino acids.

[0003] Unlike α-transaminases, which are strictly limited to a substrate spectrum of amino acids and their corresponding keto acids, ω-transaminases have an extremely broad substrate recognition range, accepting almost any compound with a suitable-sized substituent next to an amino or keto group. This characteristic makes them indispensable biocatalysts in synthetic chemistry. Based on the stereochemical characteristics of their catalytic products, ω-transaminases are clearly divided into two major categories: (S)-selective and (R)-selective. Although ω-transaminases have shown great potential in chiral synthesis, their industrial application still faces common challenges such as substrate limitation, unfavorable reaction equilibrium, poor operational stability, and product inhibition.

[0004] L-homoserine is a non-protein chiral amino acid that has attracted much attention in the fields of biotechnology, medicine, and chemical engineering. L-homoserine and its derivatives are key precursors for the synthesis of various physiologically active substances. The three functional groups in its molecular structure allow for diverse chemical modifications, leading to its widespread use in the synthesis of important intermediates such as O-acetyl-L-homoserine and O-succinyl-L-homoserine. Furthermore, it is used to prepare L-methionine, L-glufosinate, and various C4 compounds such as isobutanol, γ-butyrolactone, 1,4-butanediol, and 2,4-dihydroxybutyric acid. L-homoserine can also be used as an internal standard for neurotransmitter analysis and amino acid quantification, ensuring the accuracy and reliability of analytical results. In microbial quorum sensing research, its derivative, N-acylhomoserine lactone, plays a crucial role as a signaling molecule in regulating bacterial biofilm formation, toxin production, and other quorum behaviors.

[0005] It is evident that there is a huge market demand for L-homoserine, thus necessitating a more efficient and cost-effective synthetic method to meet this demand. Therefore, screening for a (S)-ω-transaminase mutant capable of efficiently preparing L-homoserine has extremely high practical and economic value. Summary of the Invention

[0006] To solve the above problems, the present invention uses transaminase from Vibrio fluvialis. Vf As a wild type, TA was used to construct (S)-ω-transaminase mutants with high enzyme activity, high temperature, and pH tolerance through single mutation and combination mutation. Based on this, the present invention uses the (S)-ω-transaminase mutant and aldolase to efficiently catalyze the synthesis of L-homoserine through a multi-enzyme chain reaction.

[0007] The first objective of this invention is to provide a (S)-ω-transaminase mutant, wherein the (S)-ω-transaminase mutant has one or more mutations at positions 20, 61, 62, 63, 68, 101, 190, 218, 221 or 246, based on the amino acid sequence corresponding to the parental (S)-ω-transaminase, or has one or more amino acid mutations at amino acid residues at equivalent positions in the parental (S)-ω-transaminase; The parental (S)-ω-transaminase has an amino acid sequence that is at least 80% identical to the amino acid shown in SEQ ID NO.1 and has transaminase activity.

[0008] In one embodiment, the parental (S)-ω-transaminase has an amino acid sequence that is at least 90% identical to the amino acid shown in SEQ ID NO.1 and has transaminase activity.

[0009] In one embodiment, the (S)-ω-transaminase with an amino acid sequence such as SEQ ID NO.1 is derived from Vibrio fluvialis ( Vibrio fluvialis ).

[0010] In one embodiment, the (S)-ω-transaminase mutant has an amino acid mutation at positions 62 and 221, based on the amino acid sequence as shown in SEQ ID NO.1.

[0011] In one embodiment, the methionine at position 20 is mutated to threonine; the glycine at position 61 is mutated to aspartic acid; the serine at position 62 is mutated to threonine; the leucine at position 63 is mutated to alanine; the threonine at position 68 is mutated to aspartic acid; the serine at position 101 is mutated to glycine; the asparagine at position 190 is mutated to glycine; the aspartic acid at position 218 is mutated to glutamine; the tyrosine at position 221 is mutated to glutamine; and the serine at position 246 is mutated to glutamic acid.

[0012] In one embodiment, the (S)-ω-transaminase mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has the following mutations: methionine at position 20 is mutated to threonine, glycine at position 61 is mutated to aspartic acid, serine at position 62 is mutated to threonine, threonine at position 68 is mutated to aspartic acid, serine at position 101 is mutated to glycine, and tyrosine at position 221 is mutated to glutamine (i.e., multiple mutant M1); or, Based on the amino acid sequence as shown in SEQ ID NO.1, serine at position 62 is mutated to threonine and tyrosine at position 221 is mutated to glutamine (i.e., multiple mutant M2); or, Based on the amino acid sequence such as SEQ ID NO.1, serine at position 62 is mutated to threonine, threonine at position 68 is mutated to aspartic acid, and tyrosine at position 221 is mutated to glutamine (i.e., multiple mutant M3).

[0013] A second objective of this invention is to provide nucleotides encoding the aforementioned (S)-ω-transaminase mutant.

[0014] A third objective of this invention is to provide plasmids containing the aforementioned nucleotides.

[0015] In one embodiment, the plasmid includes the pet-28a(+) series.

[0016] A fourth object of the present invention is to provide any of the (S)-ω-transaminase mutants or nucleotides containing the above (S)-ω-transaminase mutants or strains containing the above plasmids.

[0017] In one embodiment, the strain comprises prokaryotic or eukaryotic cells.

[0018] In one embodiment, the strain is *Escherichia coli*, including... E.coil BL21 (DE3).

[0019] A fifth object of the present invention is to provide the use of any of the (S)-ω-transaminase mutants or the nucleotides of the (S)-ω-transaminase mutants or the plasmids or strains described above in the synthesis of L-homoserine.

[0020] The sixth object of the present invention is to provide a method for the synthesis of L-homoserine catalyzed by a multi-enzyme-chain reaction, using the above-mentioned (S)-ω-transaminase mutant and aldolase to catalyze the synthesis of L-homoserine, comprising the steps of: (1) Pyruvate, formaldehyde and PBS buffer are mixed, and aldolase is added to react to obtain reaction solution 1; (2) Add sodium pyrophosphate buffer, L-alanine, PLP and (S)-ω-transaminase mutant to reaction solution 1 to obtain reaction solution 2 containing L-homoserine; The amino acid sequence of the aldolase is shown in SEQ ID NO.3.

[0021] In one embodiment, in step (1), the concentration of sodium pyruvate is 0.05~1 M, the concentration of formaldehyde is 0.05~1 M, the concentration of PBS buffer is 0.02~0.1 M, and the amount of aldolase added is 10~20 U; In step (2), the concentration of sodium pyrophosphate buffer is 0.05~0.1 M, the concentration of L-alanine is 0.1~2 M, the concentration of PLP is 0.5~2 mM, and the amount of (S)-ω-transaminase mutant added is 2~6 U.

[0022] In one embodiment, the reaction in step (1) is carried out at 25~40 °C for 8~12 h; In step (2), the reaction is carried out at 20~35 ℃ for 12~16 h.

[0023] Beneficial effects of the present invention This invention uses transaminase from Vibrio fluvialis. Vf As a wild type, TA was used to construct (S)-ω-transaminase mutants with high enzyme activity, high temperature, and pH tolerance through single mutation and combination mutation. Based on this, the present invention uses the (S)-ω-transaminase mutant and aldolase to efficiently catalyze the synthesis of L-homoserine through a multi-enzyme chain reaction.

[0024] Specifically: The mutant M1 constructed in this application exhibits 4.13 times the enzyme activity of the wild-type enzyme at 30℃; the yield of homoserine synthesized by the multi-enzyme-chain reaction with aldolase can reach 106 g / L, with a conversion rate of 88%. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of L-homoserine synthesis. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0027] In this invention, parent or parental (S)-ω-transaminase: as used herein, the term "parental (S)-ω-transaminase" refers to an (S)-ω-transaminase that has been modified to produce the (S)-ω-transaminase mutant of this invention. The term also refers to the polypeptide to which the mutant of this invention is compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can be, or even a variant thereof, prepared by any suitable means. For example, the parent protein can be a variant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered.

[0028] Therefore, parental (S)-ω-transaminases may have one or more (or one or more) amino acid substitutions, deletions, and / or insertions. Thus, parental (S)-ω-transaminases can be variants of parental (S)-ω-transaminases. The parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

[0029] Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0030] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0031] Raw materials used in the examples: The raw materials used in the examples, such as sodium pyruvate, formaldehyde, and L-alanine, are all commercially available.

[0032] Experimental methods: 1. Protein expression and purification The recombinant engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 rpm for 10 days. Seed culture was obtained after 12 hours; the seed culture was inoculated into fresh LB medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37℃ and 150 r / min for OD. 600 Once the concentration reaches 0.6-0.8, add isopropyl alcohol to the culture medium to a final concentration of 0.2 mM. β D isopropyl β-thiogalactoside D 1 After inducing expression of thiogalactopyranoside (IPTG) at 16℃ for 12 h, the cells were centrifuged at 4℃ and 8000 r / min for 10 min, the supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (PBS, 50 mM, H7.0). The cells were then re-vortexed with 10 mL of PBS for later use.

[0033] The wet bacterial cells were disrupted using ultrasonic disruption. Disruption conditions: 1 second disruption followed by 2 seconds pause, power 45%, until the solution became clear, with a total disruption time not exceeding 30 minutes; centrifugation at 8000 rpm for 10 minutes, and collection of the supernatant.

[0034] For protein purification, the supernatant after ultrafiltration through a 0.22 μm filter membrane was loaded into a Ni NTA gel columns were used, with flow rate controlled by gravity. Contaminating proteins were removed using PBS (50 mM, pH 7.0) buffer containing 30 mM imidazole. Target proteins were collected using PBS (50 mM, pH 7.0) buffer containing 300 mM imidazole. The gel column was then stored in 20% ethanol. The eluent was analyzed by SDS-PAGE. Imidazole was removed by ultrafiltration using a MWCO 10 kDa filter column.

[0035] 2. Enzyme activity detection The reaction system (1 mL) consisted of 20 mM 4-hydroxy-2-oxobutyric acid, 20 mM L-alanine, 1 mM pyridoxal phosphate (PLP), 200 μL enzyme solution, and PBS buffer (pH 7.5).

[0036] Reaction conditions: The reaction was carried out at 30℃ for 20 min, then boiled for 10 min to terminate the reaction, and centrifuged at 8000 r / min for 10 min. The supernatant was collected. The concentration of L-homoserine was detected by HPLC.

[0037] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol / L L-homoserine from a substrate per minute under certain conditions. One enzyme activity unit (U) is defined as such.

[0038] 3. Liquid phase method The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm). The detection wavelength was 338 nm. OPA derivatization was performed before sample loading. Mobile phase A was an aqueous sodium acetate solution (5 g / L, pH 7.2), and mobile phase B was acetonitrile:methanol:water = 45:45:10 (v / v). The flow rate was 0.6 mL / min, the injection volume was 4 μL, and the column temperature was 40℃. Each run lasted 15 minutes.

[0039] Example 1 1. Constructing transaminase Vf TA single mutant Transaminase from Vibrio fluvialis Vf As the wild type, TA has the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2.

[0040] With PET-28A(+) Vf Using TA as a template (the gene insertion site is between the restriction enzyme sites SacⅠ and SalⅠ), single-point mutations were performed to obtain recombinant plasmids expressing mutants M20T, G61N, S62T, L63A, T68D, S101G, N190G, D218Q, Y221Q, and S246E, respectively.

[0041] Recombinant plasmids were transformed into E.coil BL21(DE3) was expressed and purified to obtain mutants M20T, G61N, S62T, L63A, T68D, S101G, N190G, D218Q, Y221Q, and S246E.

[0042] 2. Transaminase Vf TA single mutant enzyme activity assay The single mutants M20T, G61N, S62T, L63A, T68D, S101G, N190G, D218Q, Y221Q, and S246E prepared in step 1 were tested for relative enzyme activity, and the results are shown in Table 1.

[0043] Table 1. Relative enzyme activities of single mutants

[0044] Example 2 Based on Example 1, Vf TA underwent multi-site combination mutations to construct multiple mutants M20T / G61N / S62T / T68D / S101G / Y221Q (named M1), S62T / Y221Q (named M2), and S62T / T68D / Y221Q (named M3).

[0045] The performance of M1, M2, and M3 was tested, as follows: (1) Enzyme activity The relative enzyme activities of M1, M2 and M3 are shown in Table 2.

[0046] Table 2 Relative enzyme activities of multiple mutants

[0047] The results showed that the enzyme activity of M1 was significantly increased. Mutations at the S62T and Y221Q sites may have a synergistic effect, which greatly increased the enzyme activity of M2. The G61N, S62T, and T68D sites are located in the same functional region and function through similar mechanisms, thus resulting in a certain degree of functional redundancy.

[0048] (2) Thermal stability Wild-type, M1, M2 and M3 were incubated at 40℃ and 50℃ for 120 min respectively, and the remaining enzyme activity was detected. The results are shown in Table 3.

[0049] Table 3 Thermal stability test

[0050] The results showed that the thermostability of the mutant was significantly improved. After incubation at 40℃ for 120 min, the wild-type enzyme activity was only 23%, while the mutant M1 retained 76% of its enzyme activity, still retaining most of its catalytic activity. After incubation at 50℃ for 120 min, the wild-type enzyme activity was only 15%, close to inactivation, while the mutant M1 retained 58% of its enzyme activity, and could continue to participate in the catalytic reaction.

[0051] (3) pH stability Wild-type, M1, M2 and M3 were incubated at pH 9.0 for 120 min and the remaining enzyme activity was measured. The results are shown in Table 4.

[0052] Table 4 pH tolerance performance test

[0053] The results showed that the pH stability of the mutant was significantly improved. After incubation at pH 9.0 for 120 min, only 28% of the wild-type enzyme activity remained, while 60% of the enzyme activity remained in mutant M1, indicating that the mutant has good stability in alkaline environment.

[0054] Example 3 The multiple mutants (M1, M2, and M3) obtained in Example 2 were respectively reacted with aldolase. Rt ALD (amino acid sequence as shown in SEQ ID NO.3) forms a multi-enzyme cascade reaction (reaction diagram as shown in the diagram). Figure 1 (As shown), using wild-type as a control, the catalytic synthesis of L-homoserine was performed using the following steps in a 20 mL catalytic system: (1) Mix 1 M sodium pyruvate, 1 M formaldehyde, and PBS buffer, and add 15 U of [unclear - possibly a specific ingredient or solution]. Rt ALD enzyme solution, reacted at 30℃ for 20 hours; (2) Add sodium pyrophosphate buffer (50 mM), 2 M L-alanine, 1 mM PLP and 4 UVf The TA enzyme solution was reacted at 30℃ for another 16 h, and the concentration of L-homoserine was detected by HPLC.

[0055] The results are shown in Table 5. The results indicate that the L-homoserine synthesis using the mutant in the cascade catalytic process was significantly improved compared to the wild type. The L-homoserine yield of M1 reached 106 g / L, with a conversion rate of 88.3%, which is about four times that of the wild type.

[0056] Table 5 L-homoserine yield

[0057] The sequence used in this application wild type Vf TA amino acid sequence SEQ ID NO.1 MQFSTFGEKFNRYSGITQLMDDLNDGLRTPGAIMLGGGNPAAIPEMLDYFKLTSEEMLADGSLIAAMTNYDGPQGKDVLVKALAKLLRDTYGWDISEKNLSLTNGSQSGFFYLFNLLAGRQPDGSFKKILLPLAPEYIGYGDAGIDEDIFVSYHPEIELLDNGLFKYHVDFEQLKVDESVAAICASRPTNPTGNVLTEEEVHKLDKLA RDNNIPIIDNAYGVPFPNIIFEDVEPFWNDNTILCMSLSKLGLPGVRCGIVASEAITQALTNMNGIISLAPGSVGPALAHRIIEKGDLLRLSQEVIKPFYRQ KSQRAVELLQQAIDDPRFRIHKPEGAVFLWLWFDELPITTMELYRRLKARGVLIVPGEYFFIGQKEDWAHAHQCLRMNYVQSDDAMQQGIAIIAEEVQKAYLEG* wild type Vf TA nucleotide sequence SEQ ID NO.2 Aldolase Rt ALD amino acid sequence SEQ ID NO.3 MTLPPNAFKIALRERRPQIGLWVAMADAYAAEIAGHAGFDWLVLDGEHGPNDLRSIMAQLQALHASPSEPVVRLPTGASWMIKQFLDIGARTLLIPMVDSAEAAELVRAVRYPPDGIRGMGAGIGRASRFNTV PGYVADAGKDICLLVQAETRAALADLERIAGVEGVDGIFIGPADLAADMGFAGDLEAPEVQAAIEAAIATIVKAGKPAGILTFNETLNRRYLELGATFVAVGADVTEFSTALQRLRRRYGPEQEDTQAGPRGY* Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A (S)-ω-transaminase mutant, characterized in that, The (S)-ω-transaminase mutant is defined as having one or more mutations at positions 20, 61, 62, 63, 68, 101, 190, 218, 221, or 246, based on the amino acid sequence corresponding to the parental (S)-ω-transaminase, or having one or more amino acid mutations at amino acid residues at equivalent positions in the parental (S)-ω-transaminase. The parental (S)-ω-transaminase has an amino acid sequence that is at least 80% identical to the amino acid shown in SEQ ID NO.1 and has transaminase activity.

2. The (S)-ω-transaminase mutant according to claim 1, characterized in that, The methionine at position 20 mutated to threonine; the glycine at position 61 mutated to aspartic acid; the serine at position 62 mutated to threonine; the leucine at position 63 mutated to alanine; the threonine at position 68 mutated to aspartic acid; the serine at position 101 mutated to glycine; the asparagine at position 190 mutated to glycine; the aspartic acid at position 218 mutated to glutamine; the tyrosine at position 221 mutated to glutamine; and the serine at position 246 mutated to glutamic acid.

3. The (S)-ω-transaminase mutant according to any one of claims 1 to 2, characterized in that, The (S)-ω-transaminase mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has the following mutations: methionine at position 20 is mutated to threonine, glycine at position 61 is mutated to aspartic acid, serine at position 62 is mutated to threonine, threonine at position 68 is mutated to aspartic acid, serine at position 101 is mutated to glycine, and tyrosine at position 221 is mutated to glutamine; or, Based on the amino acid sequence as shown in SEQ ID NO.1, the serine at position 62 is mutated to threonine and the tyrosine at position 221 is mutated to glutamine; or, Based on the amino acid sequence as shown in SEQ ID NO.1, serine at position 62 is mutated to threonine, threonine at position 68 is mutated to aspartic acid, and tyrosine at position 221 is mutated to glutamine.

4. Nucleotides encoding the (S)-ω-transaminase mutant of claim 1.

5. A plasmid containing the nucleotide of claim 4.

6. A strain expressing any of the (S)-ω-transaminase mutants of claims 1 to 3, or containing nucleotides of the (S)-ω-transaminase mutant of claim 4, or containing the plasmid of claim 5.

7. The use of the (S)-ω-transaminase mutant of any one of claims 1 to 3, or the nucleotide of the (S)-ω-transaminase mutant of claim 4, or the plasmid of claim 5, or the strain of claim 6, in the synthesis of L-homoserine.

8. A method for the synthesis of L-homoserine via a multi-enzyme-chain reaction catalysis, characterized in that, The synthesis of L-homoserine using aldolase and the (S)-ω-transaminase mutant of claim 1 as catalyzes the following steps: (1) Pyruvate, formaldehyde and PBS buffer are mixed, and aldolase is added to react to obtain reaction solution 1; (2) Add sodium pyrophosphate buffer, L-alanine, PLP and (S)-ω-transaminase mutant to reaction solution 1 to obtain reaction solution 2 containing L-homoserine; The amino acid sequence of the aldolase is shown in SEQ ID NO.

3.

9. The method according to claim 8, characterized in that, In step (1), the concentration of sodium pyruvate is 0.05~1 M, the concentration of formaldehyde is 0.05~1 M, the concentration of PBS buffer is 0.02~0.1 M, and the amount of aldolase added is 10~20 U; In step (2), the concentration of sodium pyrophosphate buffer is 0.05~0.1 M, the concentration of L-alanine is 0.1~2 M, the concentration of PLP is 0.5~2 mM, and the amount of (S)-ω-transaminase mutant added is 2~6 U.

10. The method according to claim 8, characterized in that, The reaction in step (1) is carried out at 25~40 ℃ for 8~12 h; the reaction in step (2) is carried out at 20~35 ℃ for 12~16 h.

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