L-threonine aldolase mutant and application thereof in synthesis of L-serine

Abstract

The invention provides an L-threonine aldolase mutant and application thereof in synthesis of L-serine, the amino acid sequence of the L-threonine aldolase mutant comprises at least one mutation selected from the following mutations compared with a wild type amino acid sequence as shown in SEQ ID NO: 2: 1) serine S at the 8th site is mutated into arginine R or cysteine C; the L-threonine aldolase mutant provided by the invention has the advantages that the L-threonine aldolase mutant has a synergistic regulation effect through combination of different mutation sites, the capability of synthesizing L-serine by L-threonine aldolase is obviously improved, and the L-threonine aldolase mutant has better thermal stability and formaldehyde tolerance.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and genetic engineering, specifically to an L-threonine aldolase mutant or its active fragment, genetically engineered bacteria containing the mutant or its active fragment, and their applications. Background Technology

[0002] L-Serine (chemical formula C3H7NO3) is a natural amino acid that exists as hexagonal plate-like or prismatic crystals at room temperature. It is readily soluble in water and has a melting point of 222-228℃. Its structural formula is as follows:

[0003] .

[0004] L-Serine, an important intermediate metabolite in living organisms, plays a crucial role in physiological metabolism. It is a precursor in the synthesis of substances such as glycine, nucleotides, choline, and phospholipids, and is widely used in medicine, food, and cosmetics. In the pharmaceutical field, it can be used in compound amino acid infusions and nutritional supplements; in cosmetics, it enhances moisturizing properties and possesses antibacterial and surface-active functions; in the food industry, it can impart unique flavors to food through the Maillard reaction, and its derivatives (such as cycloserine and azoserine) also have significant medicinal value.

[0005] Currently, the main methods for producing L-serine include protein hydrolysis extraction, chemical synthesis, precursor fermentation, and enzymatic methods. However, existing methods have significant limitations and cannot meet the demands of industrial-scale production: protein hydrolysis is cumbersome, the hydrolysis endpoint is difficult to control, L-serine loss is high, and environmental pollution is severe; chemical synthesis requires multiple purification steps to obtain optically pure products, making the process complex; although precursor fermentation is currently the mainstream method, subsequent separation is difficult, purification costs are high, and the requirements for production equipment are stringent; in traditional enzymatic methods, serine hydroxymethyltransferase (SHMT) is the main catalytic enzyme, but its reaction depends on pyridoxal phosphate (PLP) as a cofactor and requires tetrahydrofolate as a cofactor. Tetrahydrofolate has poor stability and high cost, and the reaction process requires nitrogen protection, which significantly increases the complexity and cost of production, limiting its industrial application.

[0006] To address the aforementioned issues, L-threonine aldolase (L-TA) has gradually become a research hotspot. L-TA is a PLP-dependent enzyme widely found in bacteria, fungi, and mammals. It catalyzes the aldol condensation reaction of glycine and formaldehyde to produce L-serine without the need for tetrahydrofolate, offering advantages such as a simple reaction pathway, high raw material economy, and environmental friendliness. Its catalytic mechanism involves using PLP as a cofactor, forming a Schiff base intermediate between the active site lysine residue and the substrate glycine, followed by a nucleophilic reaction to form a C-C bond with formaldehyde, ultimately releasing L-serine.

[0007] However, wild-type L-TA still faces three major problems that restrict its industrial application: first, insufficient catalytic activity and low reaction efficiency; second, poor tolerance to high-concentration substrates (such as formaldehyde), easily leading to substrate inhibition and inactivation; and third, low thermal stability, making it difficult to adapt to temperature fluctuations in industrial production. To address these issues, researchers have conducted targeted modification studies, but existing mutants have not yet overcome performance bottlenecks.

[0008] CN115109769A discloses a method for creating new mutants T202S and T202G by replacing the threonine at position 202 with serine or glycine using wild-type L-threonine aldolase derived from Agrobacterium as a template. These mutants achieve a conversion rate of over 99% after 3 hours of transformation with a formaldehyde concentration of 200 mM, which is significantly higher than the 54% of the wild type. However, the concentration of glycine, the substrate, is low at 1 M. This low concentration of glycine limits the yield of L-serine per unit of reaction system, requiring frequent feeding to maintain reaction efficiency. This not only increases operational complexity but also raises production energy consumption and costs, making it difficult to meet the needs of large-scale continuous industrial production.

[0009] CN120249261A discloses a mutant that uses wild-type L-threonine aldolase from the genus *Pseudohoeflea* as a template, replacing the 88th aspartic acid with alanine or leucine. Using this mutant, the yield of L-serine production increased from 56.7% to 72.3%. However, although this mutant achieved improved yield, the 72.3% yield still does not meet the economic requirements for industrial production and is insufficient to meet the demands of large-scale production.

[0010] CN120026014A discloses a mutant derived from wild-type L-threonine aldolase from Aeromonas sp. CU5, which is modified by changing threonine T at position 141 to alanine A, or by a combination of threonine T at position 141 to alanine A and isoleucine I at position 119 to valine V. The enzyme activity is increased by 1.6 and 2.9 times compared to the wild type, respectively. However, even with double mutation, the absolute value of enzyme activity is still insufficient to support efficient catalytic reaction, and the amount of L-serine produced per unit time is limited, which restricts the improvement of production efficiency.

[0011] Therefore, the targeted modification of L-TA through genetic engineering to obtain high-quality mutants with high catalytic activity, high substrate tolerance, and high thermal stability, thereby catalyzing the synthesis of L-serine from glycine and formaldehyde with high efficiency, has become a bottleneck that needs to be overcome in the current L-serine production technology and a key research direction to promote the low-cost mass production of L-serine in the industry. Summary of the Invention

[0012] Therefore, to address the shortcomings of existing technologies, this invention provides an L-threonine aldolase mutant and its application in the synthesis of L-serine. Compared with existing technologies, the L-threonine aldolase mutant provided by this invention exhibits better catalytic activity, substrate tolerance, and thermal stability.

[0013] The inventive concept of this invention is as follows: Starting from the amino acid sequence of wild-type L-threonine aldolase (SEQ ID NO: 2), an L-threonine aldolase mutant library is constructed through rational design of the enzyme molecule, thereby obtaining a series of mutants with enhanced catalytic activity, all of which show significantly improved activity compared to the parent enzyme. Based on this, various combinations of beneficial mutation sites unexpectedly yielded the L-threonine aldolase combinatorial mutant of this invention, which can further exert a synergistic regulatory effect on the basis of improved L-threonine aldolase, that is, while possessing better enzyme activity, it also exhibits improved thermal stability and substrate tolerance.

[0014] The objective of this invention is achieved through the following technical solution:

[0015] In a first aspect, the present invention provides an L-threonine aldolase mutant or an active fragment thereof, wherein the amino acid sequence of the L-threonine aldolase mutant, compared with the wild-type amino acid sequence shown in SEQ ID NO: 2, contains at least one mutation selected from the following:

[0016] (1) The serine S at position 8 is mutated to arginine R or cysteine ​​C (i.e. S8R or S8C).

[0017] (2) The glutamic acid E at position 138 is mutated to alanine A (i.e., E138A).

[0018] (3) The alanine A at position 170 is mutated to glycine G (i.e., A170G).

[0019] In this invention, the term "active fragment" refers to an amino acid sequence fragment derived from the L-threonine aldolase mutant according to the first aspect of the invention, formed by the deletion of some non-essential amino acid residues (such as N-terminal / C-terminal non-functional extension regions, flexible loops, etc.), but the fragment contains at least one of the mutations and retains the catalytic core functional region of L-threonine aldolase, and has the activity of catalyzing the reaction of glycine with formaldehyde to produce L-serine.

[0020] According to some embodiments of the present invention, the amino acid sequence of the L-threonine aldolase mutant contains a mutation compared with the wild-type amino acid sequence shown in SEQ ID NO: 2, said mutation being S8R, S8C, E138A or A170G.

[0021] According to some embodiments of the present invention, the amino acid sequence of the L-threonine aldolase mutant contains two mutations compared with the wild-type amino acid sequence shown in SEQ ID NO: 2, namely S8R / E138A, S8R / A170G, S8C / E138A or S8C / A170G.

[0022] According to some embodiments of the present invention, the amino acid sequence of the L-threonine aldolase mutant contains three mutations compared with the wild-type amino acid sequence shown in SEQ ID NO: 2, said mutations being S8R / E138A / A170G or S8C / E138A / A170G.

[0023] According to some embodiments of the present invention, the amino acid sequence shown in SEQ ID NO: 2 is derived from the amino acid sequence of L-threonine aldolase of wild-type Aeromonas.

[0024] SEQ ID NO: 2:

[0025] MRYIDLRSDTVTQPTDAMRQCMLHAEVGDDVYGEDPGVNALEAYGADLLGKEAALFVPSGTMSNLLAVMSHCQRGEGAVLGSAAHIYRYEAQGSAVLGSVALQPVPMQADGSLALADVRAAIAPDDVYFTPTRLVCLENTHNGKVLPLPYLREMRELVDEHGLQLHLDG ARLFNAVVASGHTVRELVAPFDSVSICLSKGLGAPVGSLLVGSHAFIARARRLRKMVGGGMRQAGILAQAGLFALQQHVVRLADDHRRARQLAEGLAALPGIRLDLAQVQTNMVFLQLTSGERAPLLAFMKARGILFSGYGELRLVTHLQIHDDDIEEVIDAFTEYLGA

[0026] According to some embodiments of the present invention, the amino acid sequence of the L-threonine aldolase mutant comprises or consists of the following amino acid sequences:

[0027] (1) The amino acid sequence shown in any one of SEQ ID NO: 13-22; or

[0028] (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the amino acid sequences shown in SEQ ID NO: 13-22.

[0029] According to some specific embodiments of the present invention, the amino acid sequence of the L-threonine aldolase mutant comprises or consists of the amino acid sequences shown in any one of SEQ ID NO: 13-22.

[0030] SEQ ID NO: 13

[0031] MRYID LRRDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLENT HNGKV LPLPY LREMR ELVDE HGLQL HLDGA RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0032] SEQ ID NO: 14

[0033] MRYID LRCDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLENT HNGKV LPLPY LREMR ELVDE HGLQL HLDGA RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0034] SEQ ID NO: 15

[0035] MRYID LRSDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLANT HNGKV LPLPY LREMR ELVDE HGLQL HLDGA RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0036] SEQ ID NO: 16

[0037] MRYID LRSDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLENT HNGKV LPLPY LREMR ELVDE HGLQL HLDGG RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0038] SEQ ID NO: 17

[0039] MRYID LRRDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLANT HNGKV LPLPY LREMR ELVDE HGLQL HLDGA RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0040] SEQ ID NO: 18

[0041] MRYID LRRDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLENT HNGKV LPLPY LREMR ELVDE HGLQL HLDGG RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0042] SEQ ID NO: 19

[0043] MRYID LRCDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLANT HNGKV LPLPY LREMR ELVDE HGLQL HLDGA RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0044] SEQ ID NO: 20

[0045] MRYID LRCDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLENT HNGKV LPLPY LREMR ELVDE HGLQL HLDGG RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0046] SEQ ID NO: 21

[0047] MRYID LRRDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLANT HNGKV LPLPY LREMR ELVDE HGLQL HLDGG RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0048] SEQ ID NO: 22

[0049] MRYID LRCDT VTQPT DAMRQ CMLHA EVGDD VYGED PGVNA LEAYG ADLLG KEAALFVPSG TMSNL LAVMS HCQRG EGAVL GSAAH IYRYE AQGSA VLGSV ALQPV PMQAD GSLAL ADVRAAIAPD DVYFT PTRLV CLANT HNGKV LPLPY LREMR ELVDE HGLQL HLDGG RLFNA VVASG HTVRELVAPF DSVSI CLSKG LGAPV GSLLV GSHAF IARAR RLRKM VGGGM RQAGI LAQAG LFALQ QHVVRLADDH RRARQ LAEGL AALPG IRLDL AQVQT NMVFL QLTSG ERAPL LAFMK ARGIL FSGYG ELRLVTHLQI HDDDI EEVID AFTEY LGA

[0050] In a second aspect, the present invention provides an isolated nucleic acid molecule whose nucleotide sequence comprises or consists of a nucleotide sequence encoding or an active fragment thereof as described in the first aspect of the present invention.

[0051] Preferably, the nucleotide sequence is obtained by base substitution based on the nucleotide sequence encoding wild-type L-threonine aldolase.

[0052] Thirdly, the present invention provides an expression vector comprising the isolated nucleic acid molecule described in the second aspect of the invention. The expression vector selected in this invention can stably exist and autonomously replicate in various hosts, including prokaryotic and eukaryotic cells. The expression vector can be a plasmid vector for prokaryotic expression systems, including but not limited to the pET series, such as pET28a and pET3a; the pBAD series, such as pBAD-HisA, pBAD30, pGEX-2T, pXMJ19, and pecxk99e, etc., or a plasmid vector for fungal expression systems, including but not limited to pPIC9K, pPIC9, PRS304, pUG6, pSH47, pUC110, pPZP-HYG2, and pFC330, etc. The expression vector is preferably pET28a or pPIC9K.

[0053] Fourthly, the present invention provides a host cell or genetically engineered bacterium comprising the isolated nucleic acid molecule according to the second aspect of the present invention or the expression vector according to the third aspect of the present invention. The host cell or genetically engineered bacterium includes, but is not limited to, *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, or *Bacillus subtilis*, preferably *Escherichia coli* or *Pichia pastoris*, more preferably *Escherichia coli* BL21(DE3). The host cell or genetically engineered bacterium is obtained by directly inserting the isolated nucleic acid molecule according to the second aspect of the present invention into the chromosome of the host cell or genetically engineered bacterium, or by introducing the expression vector according to the third aspect of the present invention into the initial host cell or genetically engineered bacterium using the calcium chloride method or electroporation transformation method.

[0054] Fifthly, the present invention provides a catalyst for catalyzing the reaction of glycine and formaldehyde to produce L-serine, comprising an L-threonine aldolase mutant or its active fragment as described in the first aspect of the present invention, or a host cell or genetically engineered bacterium as described in the fourth aspect of the present invention. The catalyst includes three forms: whole-cell catalyst, free protein catalyst, or immobilized enzyme catalyst. The whole-cell catalyst refers to the whole cells or bacterial cells obtained after enrichment culture and induced expression of the target protein by the host cells or genetically engineered bacterium described in the fourth aspect of the present invention; the free protein catalyst is a crude enzyme solution obtained by ultrasonically or high-pressure homogenization of the whole cells or bacterial cells, followed by centrifugation, and also includes pure enzyme obtained through protein purification. The immobilized enzyme catalyst involves selecting different immobilization carriers to immobilize the free protein catalyst, thereby obtaining different forms of immobilized L-threonine aldolase mutants or their active fragments.

[0055] In a sixth aspect, the present invention provides a method for preparing an L-threonine aldolase mutant or its active fragment according to the first aspect of the present invention, comprising the following steps:

[0056] Designed site-directed mutagenesis primers, using the nucleotide sequence of wild-type Aeromonas L-threonine aldolase (as shown in SEQ ID NO: 1) as a template for site-directed mutagenesis and construction of mutant expression plasmids, and transformation of competent host cells with mutant expression plasmids to obtain recombinant mutant strains; fermentation culture of recombinant mutant strains to induce expression, thereby obtaining L-threonine aldolase mutants or their active fragments.

[0057] In a seventh aspect, the present invention provides a method for preparing an L-threonine aldolase mutant or an active fragment thereof according to the first aspect of the present invention, the method comprising culturing a host cell or genetically engineered bacteria according to the fourth aspect of the present invention to obtain a fermentation product, and obtaining the L-threonine aldolase mutant or an active fragment thereof from the fermentation product.

[0058] Eighthly, the present invention provides a method for preparing L-serine, comprising using glycine and formaldehyde as raw materials, and using an L-threonine aldolase mutant or its active fragment as described in the first aspect of the present invention, a host cell or genetically engineered bacteria as described in the fourth aspect of the present invention, or a catalyst as described in the fifth aspect of the present invention to catalyze the reaction of glycine and formaldehyde to produce L-serine.

[0059] Preferably, the preparation method further includes, before the catalytic reaction of the raw materials, incubating the L-threonine aldolase mutant or its active fragment, the host cell or genetically engineered bacteria or the catalyst at 40°C-45°C for 1-3 hours, more preferably at 45°C for 3 hours.

[0060] According to some embodiments of the present invention, the reaction in the preparation method can be carried out under conventional process conditions, such as a reaction temperature of 20-40°C, for example 25-37°C;

[0061] Preferably, the reaction system can be a buffer system, such as phosphate buffer, with a pH of 5.0-9.0, for example, pH 6.0-8.0, more preferably pH 6.5-7.5.

[0062] According to some embodiments of the present invention, the concentration of the L-threonine aldolase mutant or its active fragment in the reaction system is 10-80 g / L, preferably 25 g / L.

[0063] According to some embodiments of the present invention, MgSO4, for example, at a concentration of 1-5 g / L, preferably 2 g / L, and / or MnCl2, for example, at a concentration of 1-5 g / L, preferably 2 g / L, is also used in the reaction system.

[0064] Compared to L-threonine aldolase derived from wild-type microorganism Aeromonas, the L-threonine aldolase mutant or its active fragment constructed in this invention exhibits significantly improved enzyme activity, better stability, and formaldehyde tolerance. Furthermore, the L-threonine aldolase mutant or its active fragment of this invention can be incubated at a higher temperature, such as 45°C, before being used in the reaction. This incubation process helps remove host cell-derived amino acid hydrolases, racemic enzymes, and other miscellaneous enzymes, thereby reducing substrate consumption.

[0065] In a ninth aspect, the present invention provides the use of the L-threonine aldolase mutant or its active fragment as described in the first aspect of the present invention, the host cell or genetically engineered bacteria as described in the fourth aspect of the present invention, or the catalyst as described in the fifth aspect of the present invention in the preparation of L-serine.

[0066] Specifically, serine is preferably produced by the following preparation method:

[0067] Using glycine and formaldehyde as raw materials, the product L-serine is obtained by catalyzing the reaction of the raw materials with the L-threonine aldolase mutant or its active fragment as described in the first aspect of the present invention, the host cell or genetically engineered bacteria as described in the fourth aspect of the present invention, or the catalyst as described in the fifth aspect of the present invention.

[0068] Preferably, the method includes incubating the L-threonine aldolase mutant or its active fragment, the host cell or genetically engineered bacteria or the catalyst at 40°C-45°C for 1-3 hours before the catalytic reaction, more preferably at 45°C for 3 hours.

[0069] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0070] The inventors discovered beneficial mutation sites that significantly enhance L-threonine aldolase activity. Combining these beneficial mutation sites unexpectedly yielded the L-threonine aldolase mutant or its active fragment of the present invention, which, based on the improved L-threonine aldolase, exerts a synergistic regulatory effect, significantly increasing activity while maintaining better thermostability, and achieving high yields; the highest yield of L-serine produced in a fermenter can reach 109.3 g / L.

[0071] Furthermore, the L-threonine aldolase mutant or its active fragment of the present invention can be incubated at a high temperature, such as 45°C, before being used in a reaction. This incubation process helps to remove miscellaneous enzymes such as amino acid hydrolases and racemic enzymes from the host cell itself, thereby reducing the consumption of substrates. Therefore, the L-threonine aldolase mutant or its active fragment of the present invention has promising prospects for industrial development and application. Attached Figure Description

[0072] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0073] Figure 1 The enzyme activity measured by the enzyme activity detection method in Example 4 of the present invention is shown, and the relative enzyme activity of each L-TA at other temperatures is calculated with the maximum enzyme activity as 100%.

[0074] Figure 2 As shown in Example 4 of the present invention, the thermal stability of each L-TA is compared according to the relative enzyme activity of each L-TA, wherein the columns of each L-TA are presented in the order of temperature 37°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C.

[0075] Figure 3 As shown in Example 4 of the present invention, the formaldehyde tolerance of each L-TA was compared based on the relative enzyme activity of each L-TA. Detailed Implementation

[0076] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. The specific embodiments listed herein are merely examples, and the present invention is not limited to the specific embodiments described below.

[0077] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be included within its scope. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without a specified manufacturer are commercially available conventional products.

[0078] To better illustrate the present invention, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that the present invention can be practiced without certain specific details. In other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0080] definition

[0081] Throughout this discussion, the standard single-letter codes for amino acids are used, as well as the standard substitution notation. Specifically:

[0082] 1) Amino acid residues are represented using the recognized IUPAC nomenclature, represented by single-letter symbols. DNA nucleic acid sequences also use the recognized IUPAC nomenclature.

[0083] 2) Mutant identification: The mutated amino acid in the L-TA mutant is represented by "the amino acid that was replaced at the original amino acid position". For example, S8R indicates that the amino acid at position 8 is replaced by arginine (Arg) instead of serine (Ser) in the original L-threonine aldolase. The position number corresponds to the amino acid sequence number in SEQ ID NO:2.

[0084] In expressions separated by the " / " symbol at different positions, the " / " symbol means "and". For example, S8R / E138A means that the amino acid at position 8 is replaced by arginine (Arg) instead of serine (Ser) in the original L-threonine aldolase, and the amino acid at position 138 is replaced by alanine (Ala) instead of glutamate (Glu) in the original L-threonine aldolase.

[0085] The term "wild-type" refers to a gene or gene product isolated from its naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in their "normal" or "wild-type" form. Conversely, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits modifications to its nucleotide sequence (e.g., substitution, truncation, or insertion), post-translational modifications, and / or functional characteristics (e.g., altered properties) compared to a wild-type gene or gene product. Note that naturally occurring mutants can be isolated and identified by the fact that they possess altered properties compared to a wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, this can be achieved by substituting the codon of methionine (ATG) with the codon of arginine (CGT) at the relevant position in the polynucleotide encoding the mutant, and by substituting methionine (M) with arginine (R). Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art.

[0086] Wild-type L-threonine aldolase

[0087] L-Threonine aldolase is a catalytic enzyme that synthesizes L-serine from glycine and formaldehyde. In this document, the terms "wild-type enzyme" and "wild-type L-Threonine aldolase" have the same meaning, referring to L-Threonine aldolase derived from Aeromonas hydrophila, whose amino acid sequence is shown in SEQ ID NO: 2.

[0088] Correspondingly, the terms "L-threonine aldolase mutant", "mutant L-threonine aldolase", "mutant L-TA", and "mutant enzyme" have the same meaning, all referring to mutants formed by altering the amino acids of the wild-type enzyme.

[0089] Determination of the catalytic activity of L-threonine aldolase

[0090] 1. Determination of enzyme properties

[0091] Enzyme activity definition: 1U enzyme activity is defined as the amount of enzyme required to generate 1μM L-serine per minute at 37℃. The reaction system contains: 200mM formaldehyde, 1M glycine, 2g / L MgSO4, 0.035g / L PLP, 25 g / L wet bacterial cells, reacted at 37℃ for 30min, and the L-serine content was determined by HPLC.

[0092] The enzymatic properties of L-TA may change after mutation. To explore the optimal enzyme activity conditions, the enzymatic properties of the unmutated WT (original strain) and the mutant were analyzed and compared under the same conditions.

[0093] 2. Thermal stability

[0094] Temperature stability determination: The sample was kept at 25℃~60℃ for 30 min, and the residual enzyme activity in the sample was determined according to the enzyme activity detection method. The enzyme activity of the untreated sample was defined as 100% to calculate the relative enzyme activity, and a temperature-relative enzyme activity curve was plotted.

[0095] 3. Recombinant vectors and transformants of L-threonine aldolase mutants

[0096] Depending on the host cell, the nucleotide sequence encoding the L-threonine aldolase mutant can be constructed into different types of recombinant vectors, or it can be directly integrated into the host bacterial chromosome. Examples of usable vectors include plasmid DNA, bacteriophage DNA, retrotransposon DNA, and artificial chromosome DNA.

[0097] The host used for the transformant of the present invention is not particularly limited as long as it can express the target L-threonine aldolase after the above-mentioned recombinant vector or the nucleotide sequence encoding the L-threonine aldolase mutant is introduced. For example, bacteria such as Escherichia coli, yeast, animal cells, insect cells, plant cells, etc. can be used.

[0098] There are no particular limitations on any method for introducing recombinant vectors into bacteria, as long as it involves introducing DNA into bacteria. For example, methods using calcium ions and electroporation can be cited.

[0099] There are no particular limitations on any method for integrating nucleotide sequences encoding L-threonine aldolase mutants into bacteria, as long as it involves introducing DNA into bacteria. Examples include homologous recombination and gene editing.

[0100] Example

[0101] Unless otherwise specified, the experimental methods described in the following examples were performed according to conventional methods and conditions, or according to the product instructions.

[0102] Plasmid pET28a, EcoRI, HindIII and other restriction endonucleases, E. coli DH5α, BL21(DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, T4 ligase, and DNA polymerase (Q5 High-Fidelity DNA Polymerase) were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0103] Kanamycin sulfate and BCA protein content assay kits were purchased from Biosharp; Ni-NTA protein purification kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0104] All chemical reagents were of analytical grade (nationally certified). The plasmid extraction procedure followed the instructions for the plasmid mini-extraction kit.

[0105] The DNA gel recovery procedure is performed according to the DNA gel recovery kit instructions; the DNA fragment ligation procedure is performed according to the T4 ligase instructions.

[0106] The protein purification procedure should be performed according to the instructions for the Ni-NTA protein purification kit.

[0107] The procedure for determining protein content should be performed according to the instructions for the BCA Protein Content Assay Kit.

[0108] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0109] LB medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10, pH natural (LB solid medium with an additional 20 g / L agar powder), sterilized at 121°C for 20 min;

[0110] TB medium (g / L): potassium dihydrogen phosphate 2.31, yeast extract 24, tryptone 12, glycerol 2, dipotassium hydrogen phosphate 16.43, sterilized at 121℃ for 20 min.

[0111] Example 1: Construction of L-TA genetically engineered bacteria

[0112] The sequence information of the L-threonine aldolase gene from Aeromonas was retrieved from the NCBI database (SEQ ID NO: 1). Based on the codon preference rules of Escherichia coli, the gene sequence was codon optimized. EcoRI and HindIII restriction sites were designed and added at both ends of the gene before it was sent to Sangon Biotech (Shanghai) Co., Ltd. for artificial synthesis.

[0113] SEQ ID NO: 1

[0114]

[0115] The plasmid pET28a and the synthesized L-TA gene fragment were digested with EcoRI and HindIII, respectively. The reaction mixture consisted of 1 μg of plasmid or L-TA gene fragment, 5 μl of 10× buffer, 1 μl of EcoRI, 1 μl of HindIII, and water to a final volume of 50 μl. Digestion was carried out at 37°C for 5 h. The fragment was detected by 1% agarose gel electrophoresis and the 6350 bp nucleotide fragment was recovered using a DNA gel purification kit.

[0116] The L-TA gene DNA fragment was ligated to the pET28a vector using T4 DNA ligase. The ligation system consisted of 0.5 μl pET28a, 5 μl Solution I, and 4.5 μl L-TA gene DNA fragment. Ligation was carried out overnight at 16°C.

[0117] The ligation product was transformed into E. coli DH5α competent cells by heat shock transformation. The transformation product was plated on LB plates containing 50 μg / ml kanamycin sulfate and cultured overnight at 37°C. Positive transformants were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced transformants were plasmid vectors containing the L-threonine aldolase gene, namely L-TA-pET28a.

[0118] Transformants with correct sequencing were transferred to 5 ml of LB medium and cultured at 37°C with shaking at 200 rpm for 16 h. 2 ml of the bacterial culture was then centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. Plasmids were extracted from the wet bacterial cells using a plasmid mini-extraction kit. The plasmid was transformed into *E. coli* BL21(DE3) using the heat shock method and cultured overnight at 37°C. Positive transformants were selected and sequenced again; the correctly sequenced transformants were identified as L-TA genetically engineered bacteria W1.

[0119] Example 2 Construction of L-TA mutant genetically engineered bacteria

[0120] Based on the L-TA genetically engineered bacterium W1, the inventors designed a list of single mutations and combined mutations, as shown in Table 1:

[0121]

[0122] Mutation primers were designed based on the mutation sites, and the primer sequences are shown in Table 2:

[0123]

[0124] Single-point mutation: Using L-TA FP / S8R RP, L-TA FP / S8C RP, L-TA FP / E138A RP, and L-TA FP / A170G RP as primers, and the L-TA-pET28a plasmid from Example 1 as a template, the left-side fragments of each mutation site were amplified by PCR using high-fidelity Q5 DNA polymerase polymerase. Using S8R FP / L-TA RP, S8C FP / L-TA RP, E138A FP / L-TA RP, and A170G FP / L-TA RP as primers, the right-side fragments of the mutation sites were amplified using L-TA-pET28a as a template. Then, using L-TA FP / L-TA RP as primers, overlap extension PCR was performed using the amplified left and right fragments corresponding to the mutations as templates to amplify the full-length L-TA single-mutant fragments W2 to W5.

[0125] Combinatorial mutation: Using the amplified single-mutant DNA gene fragment as a template, following the primer combination method described above, the left fragment of the mutation site is amplified first, and then the right fragment of the mutation site is amplified. Then, using L-TA FP / L-TA RP as primers, overlapping extension PCR is performed with the left and right fragments corresponding to the amplified mutation as templates to amplify the full-length L-TA double-mutant fragments W6 to W9. Similarly, using the amplified double-mutant DNA gene fragment as a template, the full-length L-TA triple-mutant fragments W10 and W11 are amplified.

[0126] The PCR reaction system (50 μl) consists of: 10 μl of 5×Q5 reaction buffer, 1 μl of 10 mM dNTP, 2.5 μl of each primer, 0.5 μl of Q5 enzyme, and template concentration depending on sample concentration. Add water to a final volume of 50 μl.

[0127] The final PCR products were detected by 1% (w / w) agarose gel electrophoresis and purified and recovered using a DNA gel recovery kit.

[0128] The plasmid pET28a was digested with EcoRI / Hind III. The reaction mixture consisted of 1 μg plasmid, 5 μl 10× buffer, 1 μl EcoRI, 1 μl Hind III, and water to a final volume of 50 μl. Digestion was carried out at 37°C for 5 h. The 6350 bp linear fragment was recovered by 1% (w / v) agarose gel electrophoresis and purified using a DNA gel recovery kit.

[0129] The above-mentioned double-digested linear fragment and the amplified and recovered mutant fragment were recombined using the Onestep cloning and recombination kit. The recombinant products were transformed into E. coli DH5α competent cells. The transformed E. coli were plated on LB plates containing 50 μg / ml kanamycin sulfate and cultured overnight at 37°C. Positive transformants were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the W2-W11 mutant plasmid vectors of the correctly sequenced transformants.

[0130] Transfectants with correct sequencing were transferred to 5 ml of LB medium and cultured at 37°C with shaking at 200 rpm for 16 h. 2 ml of the bacterial culture was then centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. Plasmids were extracted from the wet bacterial cells using a plasmid mini-extraction kit. The plasmids were transformed into *E. coli* BL21(DE3) using the heat shock method and cultured overnight at 37°C. Positive transformants were selected and sequenced again; the correctly sequenced transformants were identified as the W2–W11 mutant genetically engineered bacteria.

[0131] Example 3 Expression and purification of primitive L-TA and mutant L-TA

[0132] The original L-TA genetically engineered strain W1 and the mutant genetically engineered strains W2–W11 were inoculated into 100 ml of LB liquid medium containing 50 μg / ml kanamycin sulfate and cultured overnight at 37°C and 200 rpm. 4 ml of the culture was then transferred to 400 ml of fermentation medium containing 50 μg / ml kanamycin sulfate and cultured at 37°C and 220 rpm until OD reached. 600 The concentration was increased to 0.8-1.0, then 0.5 mM IPTG was added, and the mixture was incubated overnight at 20°C to express the recombinant protein.

[0133] The fermentation broth was centrifuged at 10,000 r / min for 15 min at 4 °C, and the bacterial precipitate was collected. The precipitate was washed and resuspended with buffer (20 mM potassium phosphate buffer, pH 7.0). The precipitate was then sonicated in an ice-water bath (100 W, 3 s / 5 s, 30 min) and centrifuged at 12,000 r / min for 20 min at 4 °C. The supernatant was collected to obtain the crude enzyme solution.

[0134] The crude enzyme solution was purified using a Ni-NTA protein purification kit, and the purification effect was verified by SDS-PAGE. The protein concentration was determined by the BCA method.

[0135] Example 4 L-TA enzyme activity assay

[0136] Reaction system: 200 mM formaldehyde, 1 M glycine, 2 g / L MgSO4, 0.035 g / L PLP, 25 g / L wet bacterial cells, reacted at 37℃ for 30 min, and the content of L-serine was determined by HPLC.

[0137] Take 50 μl of purified W1 and W2-W11 proteases from Example 3, add them to a 1.5 ml EP tube, add the substrate, and calculate the L-TA enzyme activity. The results are shown in Table 3.

[0138] HPLC analysis method: C18 (1.6×250mm 5μm), wavelength 360nm, mobile phase A: acetonitrile, mobile phase B: 0.05mol / L acetic acid-sodium acetate buffer solution, A:B=15:85 (volume ratio), flow rate 1 ml / min, temperature 37℃.

[0139]

[0140] The results showed that the enzyme activity of the mutant was improved, reaching up to 3.052 times that of the original W1 enzyme activity, indicating that site-directed mutagenesis improved the enzyme's catalytic activity.

[0141] Example 5: Determination of optimal temperature and thermal stability of L-TA

[0142] The purified W1-W11 proteases from Example 3 were reacted with substrate reaction solutions at different temperatures (25-60°C), and then the enzyme activity was measured according to the enzyme activity detection method in Example 4. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at other temperatures was calculated. The results are as follows: Figure 1 As shown.

[0143] The sample was incubated at 25℃~60℃ for 30 minutes, and then the residual enzyme activity was measured according to the enzyme activity detection method in Example 4. The relative enzyme activity was calculated with the enzyme activity of the untreated sample defined as 100%. A temperature-relative enzyme activity curve was constructed. When the residual enzyme activity reached 85% or higher, it was defined as having good thermal stability. The relative enzyme activity results are as follows: Figure 1 As shown, the optimal temperature for the L-TA mutant is the same as that for the wild type, which is 37℃. When the temperature is below or above 37℃, the catalytic activity of the L-TA mutant changes less compared to the wild type, indicating that its operating temperature range is wider than that of the wild type.

[0144] The enzyme was placed in 20 mM potassium phosphate buffer at pH 7.0 and incubated at 37°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 3 hours, respectively, and then cooled in an ice-water bath. The residual enzyme activity was then measured at 37°C according to the enzyme activity detection method in Example 4. The highest enzyme activity was defined as 100%. The enzyme activity measured under different conditions was compared with this value to calculate the relative enzyme activity. The thermal stability was compared based on the relative enzyme activity of each L-TA. The results are as follows: Figure 2As shown, the L-TA mutant exhibits significantly higher thermostability than the original enzyme, especially the W10 mutant, which showed virtually no significant change in enzyme activity after incubation at 45℃ for 3 hours. Furthermore, at 60℃, the relative enzyme activity of the W10 mutant L-TA remained above 85%, while the relative enzyme activity of the other mutant L-TA strains also remained above 75%, whereas the relative enzyme activity of the wild type was below 70%.

[0145] Example 6: L-TA Formaldehyde Tolerance Test

[0146] The enzyme was placed in a 20 mM potassium phosphate buffer solution at pH 7.0 and incubated at 37°C for 1 h at formaldehyde concentrations of 0, 1, 2, 3, and 4 g / L, respectively. Residual enzyme activity was then measured at 37°C according to the enzyme activity detection method in Example 4. The highest enzyme activity was defined as 100%. The enzyme activity measured under different conditions was compared with this value to calculate the relative enzyme activity. Formaldehyde tolerance was compared based on the relative enzyme activity of each L-TA. The results are as follows: Figure 3 As shown, the L-TA mutant exhibits significantly higher formaldehyde tolerance than the original enzyme, especially the W10 mutant, which shows virtually no change in enzyme activity after incubation at a formaldehyde concentration of 4 g / L for 1 h, while the relative enzyme activity of the wild type is already below 50%.

[0147] Example 7: Fermentation of original L-TA and mutant in a tank.

[0148] Activate 1 ml of glycerol culture containing the original W1 and W10 / W11 mutants on an agar slant and incubate at 37°C for 16 h. Transfer a loopful of bacterial growth from the agar slant to 100 ml of LB seed culture medium and incubate at 37°C with shaking at 200 rpm for 10–16 h. Inoculate the seed culture at a volume ratio of 5% into a 15 L fermenter containing 8 L of fermentation medium. Maintain the temperature at 37°C ± 1°C. During fermentation, supplement with ammonia to maintain the pH at 7.0 ± 0.1, control the aeration rate at 0.5 vvm–3.0 vvm, and control the rotation speed at 200 rpm–800 rpm. Maintain dissolved oxygen at 30% by adjusting the rotation speed and aeration rate. After 7–8 h of fermentation, cool down to 20–30°C, add 0.5 mM IPTG, and induce for approximately 16 h. Fermentation ends when enzyme activity no longer increases.

[0149] The fermentation broth was centrifuged at 6000 rpm for 20 min, the supernatant was discarded, and the cells were washed and resuspended in buffer (20 mM potassium phosphate buffer, pH 7.0). The cells were then homogenized twice at 700-800 bar using an autoclave, followed by centrifugation at 12000 rpm for 20 min at 4°C. The supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution was incubated at 45°C for 3 h to remove host cell amino acid hydrolases, racemic enzymes, and other contaminating enzymes, thus reducing substrate consumption.

[0150] Example 8: L-TA conversion to produce L-serine

[0151] The reaction system was 1L, containing 1.5M glycine, 1.5M formaldehyde (33%), 2g / L MgSO4, 0.035g / L PLP, and 25g / L crude enzyme solution obtained in Example 6. The reaction was carried out at 37℃ and 200rpm for 24h. The pH was controlled at 7.5 during the reaction process. The concentration of L-serine was measured by sampling, and the conversion rate was calculated. The results are shown in Table 4.

[0152]

[0153] The concentration of L-serine in the transformation solution of the original L-TA was 35.5 g / L, while the concentration of L-serine in the transformation solution of the mutant L-TA was above 100 g / L, with a product generation rate of over 90%. This indicates that site-directed mutagenesis of L-TA improved the enzyme's catalytic activity and product generation rate. The product generation rate of the W10 mutant strain reached over 97% after the reaction, which facilitates subsequent product extraction.

[0154] In summary, the L-threonine aldolase mutant constructed in this invention exhibits significantly higher activity than the original L-threonine aldolase (its amino acid sequence is shown in SEQ ID NO: 2). Using this enzyme mutant, after heat treatment at 45°C for 3 hours, it catalyzes the reaction of glycine and formaldehyde, and after 24 hours of reaction under PLP, it can generate 109.3 g / L of L-serine with no substrate residue, demonstrating promising prospects for industrial development and application.

[0155] The above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An L-threonine aldolase mutant or its active fragment, wherein, The amino acid sequence of the L-threonine aldolase mutant contains at least one mutation selected from the following, compared to the amino acid sequence shown in SEQ ID NO: 2: (1) The serine S at position 8 is mutated to arginine R or cysteine ​​C (i.e. S8R or S8C). (2) The glutamic acid E at position 138 is mutated to alanine A (i.e., E138A). (3) The alanine A at position 170 is mutated to glycine G (i.e., A170G).

2. The L-threonine aldolase mutant or its active fragment according to claim 1, wherein, The amino acid sequence of the L-threonine aldolase mutant contains one mutation compared to the amino acid sequence shown in SEQ ID NO: 2; preferably, the mutation is S8R, S8C, E138A or A170G. The amino acid sequence of the L-threonine aldolase mutant contains two mutations compared to the amino acid sequence shown in SEQ ID NO: 2; preferably, the mutations are S8R / E138A, S8R / A170G, S8C / E138A, or S8C / A170G; or The amino acid sequence of the L-threonine aldolase mutant contains three mutations compared to the amino acid sequence shown in SEQ ID NO: 2; preferably, the mutations are S8R / E138A / A170G or S8C / E138A / A170G.

3. The L-threonine aldolase mutant or its active fragment according to claim 1 or 2, wherein, The amino acid sequence of the L-threonine aldolase mutant contains or is composed of the following amino acid sequences: (1) The amino acid sequence shown in any one of SEQ ID NO: 13-22; or (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the amino acid sequences shown in SEQ ID NO: 13-22; Preferably, the L-threonine aldolase mutant comprises or consists of the amino acid sequences shown in any one of SEQ ID NO: 13-22.

4. An isolated nucleic acid molecule whose nucleotide sequence comprises or consists of a nucleotide sequence encoding or consisting of an L-threonine aldolase mutant or an active fragment thereof according to any one of claims 1 to 3.

5. An expression vector comprising the isolated nucleic acid molecule according to claim 5; Preferably, the expression vector is a plasmid vector, such as a plasmid vector for a prokaryotic expression system or a plasmid vector for a fungal expression system; More preferably, the plasmid vector for the prokaryotic expression system is selected from the pET series, and more preferably from the pET series, such as pET28a, pET21b or pET3a; and more preferably from the pBAD series, such as pBAD HisA, pBAD30, pGEX 2T, pXMJ19 or pecxk99e; More preferably, the plasmid vector for the fungal expression system is pPIC9K, pPIC9, PRS304, pUG6, pSH47, pUC110, pPZP HYG2 or pFC330; More preferably, the expression vector is pET28a or pPIC9K.

6. A host cell or genetically engineered bacterium comprising the isolated nucleic acid molecule according to claim 4 or the expression vector according to claim 5; Preferably, the host cell or genetically engineered bacteria is Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, or Bacillus subtilis; More preferably, the host cell or genetically engineered bacteria is Escherichia coli or Pichia pastoris; More preferably, the host cell or genetically engineered bacterium is Escherichia coli BL21 (DE3).

7. A catalyst for catalyzing the reaction of glycine and formaldehyde to produce L-serine, comprising an L-threonine aldolase mutant or its active fragment according to any one of claims 1 to 3, or a host cell or genetically engineered bacterium according to claim 6.

8. Use of the L-threonine aldolase mutant or its active fragment according to any one of claims 1 to 3, the host cell or genetically engineered bacteria according to claim 6, or the catalyst according to claim 7 in the preparation of L-serine.

9. A method for preparing the L-threonine aldolase mutant or its active fragment according to any one of claims 1 to 3, comprising culturing the host cell or genetically engineered bacteria according to claim 6 to obtain a fermentation product, and obtaining the L-threonine aldolase mutant or its active fragment from the fermentation product.

10. A method for preparing L-serine, comprising using an L-threonine aldolase mutant or its active fragment according to any one of claims 1 to 3, a host cell or genetically engineered bacterium according to claim 7, or a catalyst according to claim 8 to catalyze the reaction of glycine and formaldehyde to produce L-serine; Preferably, the preparation method includes, before catalyzing the reaction of glycine and formaldehyde, incubating the L-threonine aldolase mutant or its fragment, the host cell or genetically engineered bacteria according to any one of claims 1 to 3, or the catalyst according to claim 6, at 40°C-45°C for 1 to 3 hours, more preferably at 45°C for 3 hours.

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