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

Abstract

The invention discloses an L-threonine aldolase mutant and application of the L-threonine aldolase mutant in synthesis of L-serine. The mutant is obtained by performing single mutation on the 88th amino acid of a wild type L-threonine aldolase amino acid sequence shown as SEQ ID No.2. The invention further discloses a preparation method of the L-threonine aldolase mutant. The invention provides the L-threonine aldolase mutant with better catalytic performance, aldol condensation reaction of formaldehyde and glycine can be efficiently catalyzed to prepare L-serine under the condition of not adding a tetrahydrofolic acid precursor, the yield of the L-serine is increased to 72.3% from 56.7% before mutation, the yield is remarkably increased, and the L-threonine aldolase mutant has the advantages that the L-threonine aldolase mutant has a good application prospect. Good application prospects are shown in the synthesis of the L-serine.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and more specifically relates to an L-threonine aldolase mutant and application thereof in L-serine synthesis. (II) Background technology

[0002] L-serine (L-Ser, chemical formula C3H7NO3), also known as L-2-amino-3-hydroxypropionic acid, is an important intermediate metabolite in organisms. It has many important physiological functions and is a synthetic precursor for glycine and other amino acids, nucleotides, choline, and phospholipids. It is currently widely used in the fields of medicine, food, and cosmetics. For example, L-serine can be added to high-end cosmetics to improve the moisturizing properties of cosmetics. It also has certain antibacterial and surfactant effects. L-serine can react with sugars to produce a special flavor in food through the Maillard reaction at high temperature.

[0003] The production methods of L-serine include protein hydrolysis extraction, chemical synthesis, conversion and microbial fermentation. The main disadvantage of the chemical synthesis method is that the reaction steps are cumbersome, and multi-step purification is required to obtain optically pure chiral L-serine. The protein hydrolysis process is relatively cumbersome, the hydrolysis end point is difficult to accurately judge, the L-serine loss rate is high, and the environmental pollution is serious. At present, the production method of L-serine is mainly the precursor fermentation method, but this method is difficult to separate later, resulting in high costs, and there are also high requirements for production equipment. The bioenzyme method has the advantages of mild reaction conditions, high stereoselectivity and environmental friendliness, and has been widely used in the industrial production of chiral chemicals such as pharmaceutical intermediates and fine chemicals. At present, the enzyme involved in the biosynthesis of L-serine is mainly serine hydroxymethyltransferase (SHMT), which has a relatively complex catalytic process. It not only requires PLP as a coenzyme, but also requires tetrahydrofolate as a cofactor, and tetrahydrofolate is extremely unstable and expensive, resulting in an increase in production costs. Therefore, it is still necessary to seek a method that can produce L-serine relatively simply and efficiently. In recent years, many researchers at home and abroad have achieved the production of L-serine by microbial fermentation using renewable raw materials as substrates by modifying Escherichia coli and Corynebacterium glutamicum, but key indicators such as output and yield are still difficult to meet the requirements of industrial production.

[0004] L-threonine aldolase (L-TA) is ubiquitous in nature and exists in bacterial, fungal, and mammalian species. Currently, approximately 5,000 sequences annotated as L-TA can be found in databases such as NCBI and Swiss-Prot. However, only a few genes have been studied in terms of their structure, mechanism, and biochemistry. L-TA can use pyridoxal-5'-phosphate (PLP) as a cofactor and covalently bind to the Apo-enzyme through the active-site lysine residue to form a Schiff base intermediate; after the substrate glycine enters the active center, the active lysine residue dissociates from PLP and forms a new Schiff base intermediate with glycine. Then, the α-carbon of glycine is deprotonated, and the nucleophilic acceptor formaldehyde that enters the active center forms a new C-C bond with the α-carbon; finally, PLP recombines with the active lysine residue to form a Schiff base intermediate, and the product L-serine is released.

[0005] So far, the enzyme serine hydroxymethyltransferase, which has been studied extensively for the production of L-serine, has a complex reaction process, and most of them are applied to the fermentation method for the production of L-serine. In addition, other reported enzyme cascades for the production of L-serine involve multiple enzymes, which is a complex process and has a low yield.

[0006] Therefore, exploring a highly active L-threonine aldolase that can efficiently catalyze the synthesis of L-serine from glycine and formaldehyde without the addition of tetrahydrofolic acid has important application prospects. (III) Summary of the Invention

[0007] The object of the present invention is to provide an L-threonine aldolase mutant and its application in the synthesis of L-serine. The L-threonine aldolase mutant can efficiently catalyze the synthesis of L-serine from glycine and formaldehyde without the addition of tetrahydrofolic acid, solving the problems that the current production of L-serine cannot fully meet the market demand, and its current main production method is the precursor fermentation method, which has difficulties in subsequent separation, high purification costs, and high requirements for production equipment.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides an L-threonine aldolase mutant, which is obtained by single mutation of the 88th amino acid of the wild-type L-threonine aldolase amino acid sequence shown in SEQ ID No.2.

[0010] Furthermore, preferably, the L-threonine aldolase mutant is obtained by replacing the 88th aspartic acid of the amino acid sequence shown in SEQ ID No.2 with alanine (D88A, amino acid sequence as shown in SEQ ID No.3) or leucine (D88L, amino acid sequence as shown in SEQ ID No.4).

[0011] In a second aspect, the present invention provides a gene encoding the L-threonine aldolase mutant.

[0012] In a third aspect, the present invention provides a recombinant expression vector comprising the gene encoding the L-threonine aldolase mutant, preferably plasmid pET-21b(+).

[0013] In a fourth aspect, the present invention provides a recombinant expression transformant comprising the gene encoding the L-threonine aldolase mutant. The recombinant expression transformant can be prepared by transforming the recombinant expression vector into a host cell. The host cell is various conventional host cells in the art, as long as the recombinant expression vector can stably self-replicate and can effectively express the target protein after induction by an inducer, preferably Escherichia coli E. coli BL21(DE3).

[0014] In a fifth aspect, the present invention provides an application of the L-threonine aldolase mutant in the synthesis of L-serine.

[0015] Furthermore, the method of the application is as follows: using the wet cells obtained by fermentation culture of the recombinant genetic engineering bacteria expressing the L-threonine aldolase mutant or the crude enzyme solution after ultrasonic disruption of the wet cells as a catalyst, using formaldehyde and glycine as substrates, adding pyridoxal phosphate (PLP), KCl and mercaptoethanol, and using a buffer solution with a pH of 6-10 as a reaction medium to form a reaction system, reacting at 30-60 °C and 200-600 rpm (preferably 60 °C and 250 rpm) to obtain a reaction solution containing L-serine.

[0016] Furthermore, the addition amount of the catalyst in the reaction system is 10-80 g / L (preferably 50 g / L) based on the weight of the wet cells or the wet cells before ultrasonic disruption; the final concentration of formaldehyde added is 10-300 mM (preferably 100 mM); the final concentration of glycine added is 10-200 g / L (preferably 50 g / L); the final concentration of pyridoxal phosphate added is 10-200 mg / L (preferably 125 mg / L); the final concentration of KCl added is 5-15 g / L (preferably 7.5 g / L); the final concentration of mercaptoethanol added is 1-5 mL / L (preferably 3.5 mL / L).

[0017] Furthermore, the buffer solution is Tris-HCl buffer solution (0.1 M, pH 9.0).

[0018] Further, the catalyst is prepared by the following method: inoculate the recombinant expression transformant of the L-threonine aldolase mutant into a test tube containing LB liquid medium with 100 mg / L ampicillin, and culture it overnight in a constant temperature shaker at 37 °C and 220 rpm; inoculate the bacterial liquid into the LB liquid medium containing 100 mg / L ampicillin at an inoculation amount of 1% (v / v), and culture it in a constant temperature shaker at 37 °C and 220 rpm until the OD 600 reaches 0.6; add IPTG with a final concentration of 0.1 mM, place it in a constant temperature shaker at 28 °C and 180 rpm for induction expression for 16 h, then centrifuge at 4 °C and 4000 rpm for 10 min to obtain wet bacterial cells;

[0019] Resuspend the wet bacterial cells in PBS buffer (0.1 M, pH 7.0) at a concentration of 50 g / L, ultrasonically disrupt them at 300 W for 15 min, with a working time of 2 s and an interval of 3 s, centrifuge at 4 °C and 4000 rpm for 15 min, collect the supernatant to obtain a crude enzyme solution.

[0020] The catalyst described in the present invention can also be a purified solution, a freeze-dried bacterial powder of wet bacterial cells, a crude enzyme solution or a pure enzyme solution.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] The present invention provides a mutant of L-threonine aldolase with better catalytic performance. Without the addition of a tetrahydrofolate precursor, it can efficiently catalyze the aldol condensation reaction of formaldehyde and glycine to prepare L-serine. The yield of L-serine is increased from 56.7% before mutation to 72.3%, and the yield is significantly improved, showing good application prospects in the synthesis of L-serine. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram showing the interaction mode of WT and glycine-PLP complex in Example 1.

[0024] Figure 2 It is a bar graph showing the biomass after induction culture of WT, 88A, and 88L in Example 3.

[0025] Figure 3 It is a SDS-PAGE diagram of whole cells after induction culture of WT, 88A, and 88L in Example 3; lane M represents DNA marker, lanes 1 and 2 represent WT, lanes 3 and 4 represent 88A, and lanes 5 and 6 represent 88L.

[0026] Figure 4SDS-PAGE of the supernatants after induction and culture of WT, 88A, and 88L in Example 3; lane M represents DNA marker, lanes 1 and 2 represent WT, lanes 3 and 4 represent 88A, and lanes 5 and 6 represent 88L.

[0027] Figure 5 Reaction formula for the production of L-serine by L-threonine aldolase catalyzing formaldehyde and glycine.

[0028] Figure 6 Column chart of the yields of L-serine synthesized by L-threonine aldolase and mutants catalyzing formaldehyde and glycine. (V) Specific implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0030] Materials and reagents used in the embodiments of the present invention: E. coli BL21(DE3) and plasmid pET-21b(+) are commercially available. The plasmid extraction kit and DNA purification and recovery kit are both purchased from TOROIVD Technology Co., Ltd.; the SDS-PAGE precast gel is purchased from Nanjing Kingsrui Biotechnology; the DNA marker is purchased from Beijing TransGen Biotech Co., Ltd.; the usage methods of the above reagents refer to the product manuals.

[0031] Primer synthesis and sequence sequencing work are completed by Beijing Tsingke Biotechnology Co., Ltd. (Hangzhou).

[0032] Glycine is purchased from Shanghai Titan Technology, and the rest are purchased from Sinopharm Chemical Reagent Co., Ltd.

[0033] Example 1: Construction of the starting strain

[0034] The sequence annotated as threonine aldolase family protein (NCBI accession number WP_133285361.1) from Pseudohoeflea suaedae in NCBI was cloned and denoted as the L-threonine aldolase Ps-LTA fragment (the gene sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2). It was inserted between the NdeI and HindIII restriction sites of the expression plasmid pET-21b(+) to obtain pET21b-Ps-LTA. After sequencing verification, pET21b-Ps-LTA was transferred into the expression host Escherichia coli E. coli BL21(DE3) to obtain the starting strain E. coli BL21(DE3)-pET21b-Ps-LTA (strain abbreviation WT), and the corresponding protein was denoted as wild-type Ps-LTA.

[0035] Example 2: Mutant Construction and Screening

[0036] 1. Selection of Mutation Sites

[0037] The structure of wild-type Ps-LTA protein was predicted by AlphaFold2, and the interaction mode between wild-type Ps-LTA and the substrate glycine-PLP complex was simulated by Pymol software (such as Figure 1 ), and the amino acid residues around the glycine-PLP complex with van der Waals distance may be related to the binding of formaldehyde molecules and enzyme activity, and were used as candidate mutation sites, including 8 sites: S5, D6, N7, A29, Y30, D88, E287, and R238.

[0038] 2. Construction of Site-Directed Saturation Mutation Library

[0039] The plasmid pET21b-Ps-LTA from Example 1 was extracted as a template, and saturation mutation primers were designed for the 8 selected mutation sites (Table 1), and a saturation mutation library was constructed and screened.

[0040] PCR System: 1 μL of Phanta Max Super-Fidelity DNA Polymerase, 1 μL of dNTP Mix, 25 μL of 2×Phanta Max buffer, 1 μL each of forward and reverse primers, 1 μL of template, 20 μL of ddH2O, and the total system was 50 μL.

[0041] PCR Program: First, pre-denature at 95°C for 3 min; then cycle 35 times according to the following program: denature at 95°C for 15 s, anneal at 60°C for 15 s, extend at 72°C for 4.5 min; final extension at 72°C for 5 min, and maintain at 4°C.

[0042] Table 1 Primers Used for Mutant Construction

[0043]

[0044] In Table 1, N represents the base A / C / G / T, M represents the base A / C, and K represents the base G / T.

[0045] 3. Screening of Mutants

[0046] (1) Preparation of Wet Bacterial Cells of Genetically Engineered Bacteria Expressing Mutants

[0047] The above PCR products were verified by 1% agarose nucleic acid electrophoresis. The successfully verified PCR products were digested with DpnⅠ enzyme in a metal bath at 37°C for 2 h. Transformation into Escherichia coli host: 2 μL of the digested PCR products were transferred into BL21(DE3) competent cells pre-cooled on ice, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, then incubated on ice for 2 min. 900 μL of LB medium without antibiotic was added, and the mixture was cultured at 37°C and 220 rpm for 1.5 h. The supernatant of 800 μL was discarded by centrifugation at 9 000 rpm for 2 min. After resuspending the cells, they were spread on an LB solid plate (containing 100 mg / L ampicillin) and cultured overnight in an incubator at 37°C. The monoclonal strains were transferred from the antibiotic-resistant plate to LB liquid medium and cultured overnight in a constant temperature shaker at 37°C and 220 rpm. Then, 1 mL of the overnight cultured bacterial solution was added to a 250 mL flask containing 100 mL of LB liquid medium with 100 mg / L ampicillin resistance, and cultured in a constant temperature shaker at 37°C and 220 rpm for about 2.5 h until the OD 600 reached 0.6. IPTG with a final concentration of 0.1 mM was added for induction, and the mixture was induced to express at 28°C and 180 rpm in a constant temperature shaker for 16 h. Then, it was centrifuged at 4°C and 4 000 rpm for 10 min to collect the wet bacterial cells.

[0048] (2) Screening of mutant enzyme activity

[0049] Formulation of 2× reaction premix: glycine 20 g / L, PLP 0.25 g / L, KCl 15 g / L, mercaptoethanol 7 mL / L. The above components were dissolved in 0.1 M Tris-HCl buffer (pH 9.0) and made up to 1 L for standby.

[0050] In a 1.5 mL centrifuge tube, 500 μL of 2× reaction premix, 490 μL of 0.1 M Tris-HCl buffer (pH 9.0), 0.1 g of wet bacterial cells, and 10 μL of formaldehyde with a volume concentration of 37% (corresponding to a final concentration of 100 mM formaldehyde in a 1 mL reaction system) were added in sequence. The mixture was oscillated and reacted at 60°C and 600 rpm for 2 h, centrifuged at 12 000 rpm for 5 min, and the supernatant was derivatized with DNFB (2,4-dinitrofluorobenzene). Then, the content of L-serine in the product was detected by HPLC, and the relative enzyme activity was calculated (using the wild type as a control). Some results are shown in Table 3.

[0051] Definition of enzyme activity: The amount of enzyme required to generate 1 μmol of L-serine per minute is defined as 1 U.

[0052] HPLC detection conditions: Thermo Fisher liquid chromatograph, using a C18 chromatographic column (4.6*250mm, 5μm), injection volume 10 μL, flow rate 0.8 mL / min, column temperature 30 °C, UV detection wavelength 260 nm, gradient elution in Table 2, mobile phase A: pure acetonitrile; mobile phase B: 826 mL ddH2O, 170 mL acetonitrile, 2 mL triethylamine, 2 mL acetic acid.

[0053] Table 2 Liquid chromatography gradient elution program

[0054] Time (min) Mobile phase A, % Mobile phase B, % 0 18 82 3 20 80 5 35 65 8 35 65 10 50 50 12 50 50 13 80 20 15 70 30 18 18 82 23 18 82

[0055] Table 3 Relative enzyme activities of some mutants

[0056]

[0057]

[0058] The dominant mutants D88A (amino acid sequence shown in SEQ ID No. 3) and D88L (amino acid sequence shown in SEQ ID No. 4) of L-threonine aldolase Ps-LTA were obtained by mutation. The corresponding recombinant genetic engineering bacteria were E. coli BL21(DE3)-pET21b-Ps-LTA-D88A (abbreviated as 88A) and E. coli BL21(DE3)-pET21b-Ps-LTA-D88L (abbreviated as 88L).

[0059] Example 3: Fermentation of recombinant genetic engineering bacteria expressing mutants

[0060] 1. Wet bacterial cells

[0061] The starting strain WT constructed in Example 1 and the dominant mutant recombinant genetic engineering bacteria 88A and 88T screened in Example 2 were respectively inoculated into test tubes containing 5 mL of LB liquid medium with 100 mg / L ampicillin resistance and cultured overnight in a constant temperature shaker at 37 °C and 220 rpm. Then, 1 mL of the overnight culture broth was added to a 250 mL shake flask containing 100 mL of LB liquid medium with 100 mg / L ampicillin resistance and cultured in a constant temperature shaker at 37 °C and 220 rpm for about 2.5 h until the OD 600 reached 0.6. IPTG was added at a final concentration of 0.1 mM for induction, and the mixture was placed in a constant temperature shaker at 28 °C and 180 rpm for 16 h for induced expression. After the induced expression was completed, the biomass of the broth (OD 600 , Figure 2 ) was first measured, and then the broth was poured into a 50 mL centrifuge tube and centrifuged at 4 °C and 4000 rpm for 10 min. The supernatant and whole cells (precipitate) were obtained and subjected to SDS-PAGE respectively, as shown in Figure 3 ,4 As shown, the wet bacterial cells are stored in a refrigerator at -20 °C for future use.

[0062] 2. Crude enzyme solution

[0063] Resuspend 1 g of the wet bacterial cells from step 1 in 20 mL of PBS buffer (0.1 M, pH 7.0), and disrupt them by ultrasonic treatment at 300 W for 15 min, with a working time of 2 s and an interval of 3 s. The disrupted bacterial cell suspension is centrifuged at 4 °C and 4000 rpm for 15 min, and the supernatant is collected to obtain the crude enzyme solution. Both the disrupted bacterial cell suspension and the crude enzyme solution can be used for the reaction of catalyzing the formation of L-serine from formaldehyde and glycine.

[0064] Example 4: Reaction of Ps-LTA mutant to catalyze the formation of L-serine from formaldehyde and glycine

[0065] Refer to Figure 5 The schematic diagram of the principle of the condensation reaction of L-threonine aldolase mutant to catalyze the formation of L-serine from formaldehyde and glycine was used for the synthesis reaction, and the crude enzyme solution of the wild type / mutant prepared by the method of Example 3 was used as the catalyst.

[0066] The final concentration composition of the 10 mL reaction system: 4 mL of the crude enzyme solution of the wild type / mutant (the addition amount of the crude enzyme solution is 50 g / L based on the weight of the wet bacterial cells before disruption), 100 mM formaldehyde, 50 g / L glycine, 0.125 g / L PLP, 7.5 g / L KCl, 3.5 mL / L mercaptoethanol, and Tris-HCl buffer (0.1 M, pH 9.0).

[0067] The reaction solution was reacted at 60 °C and a rotation speed of 250 rpm. After the reaction was completed, a sample was taken for derivatization with DNFB (2,4-dinitrofluorobenzene), and then the content of L-serine was detected by HPLC described in Example 1. The results are shown in Figure 6 as shown.

[0068] As Figure 6 shown, when the formaldehyde concentration is 100 mM, the L-serine yield of the wild type Ps-LTA is 5.95 g / L, while the L-serine yields of the mutants D88A and D88L are 7.48 g / L and 7.49 g / L respectively, and the L-serine yield rate is increased from 56.7% to 72.3%.

[0069] The above-mentioned are only the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. The above embodiments of the present invention can also be variously changed. All simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the protection of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. An L-threonine aldolase mutant, characterized in that, The L-threonine aldolase mutant is obtained by single mutation of the amino acid at position 88 in the wild-type L-threonine aldolase amino acid sequence shown in SEQ ID No.

2.

2. The L-threonine aldolase mutant according to claim 1, wherein The L-threonine aldolase mutant is obtained by replacing the aspartic acid at position 88 in the amino acid sequence shown in SEQ ID No. 2 with alanine or leucine.

3. A gene encoding the L-threonine aldolase mutant according to claim 1.

4. A recombinant expression vector containing the gene encoding the L-threonine aldolase mutant according to claim 1.

5. A recombinant expression transformant containing the gene encoding the L-threonine aldolase mutant according to claim 1.

6. An application of the L-threonine aldolase mutant according to claim 1 in the synthesis of L-serine.

7. The application according to claim 6, wherein The method of the application is as follows: using the wet cells obtained by fermentation culture of the recombinant expression transformant of the L-threonine aldolase mutant or the crude enzyme solution after ultrasonic disruption of the wet cells as a catalyst, using formaldehyde and glycine as substrates, adding pyridoxal phosphate, KCl and mercaptoethanol, and using a buffer solution with a pH of 6-10 as a reaction medium to form a reaction system, and reacting at 30-60 °C and 200-300 rpm to obtain a reaction solution containing L-serine.

8. The application according to claim 7, characterized in that, In the reaction system, the addition amount of the catalyst is 10-80 g / L based on the weight of the wet cells or the wet cells before ultrasonic disruption; the final concentration of formaldehyde added is 10-300 mM; the final concentration of glycine added is 10-200 g / L; the final concentration of pyridoxal phosphate added is 10-200 mg / L; the final concentration of KCl added is 5-15 g / L; the final concentration of mercaptoethanol added is 1-5 mL / L.

9. The application according to claim 7, characterized in that, The buffer solution is 0.1 M Tris-HCl buffer solution with a pH of 9.

0.

10. The application according to claim 7, characterized in that The catalyst is prepared by the following method: inoculate the recombinant expression transformant of L-threonine aldolase mutant into a test tube containing LB liquid medium with 100 mg / L ampicillin, and culture overnight in a constant temperature shaker at 37 °C and 220 rpm; inoculate the bacterial liquid into LB liquid medium with 100 mg / L ampicillin at an inoculation amount of 1% (v / v), and culture in a constant temperature shaker at 37 °C and 220 rpm until the OD 600 reaches 0.6; add IPTG with a final concentration of 0.1 mM, place it in a constant temperature shaker at 28 °C and 180 rpm for induction expression for 16 h, then centrifuge at 4 °C and 4000 rpm for 10 min to obtain wet bacterial cells; resuspend the wet bacterial cells in 0.1 M PBS buffer at pH 7.0 at a amount of 50 g / L, ultrasonically disrupt for 15 min under the condition of 300 W, work for 2 s and interval for 3 s, centrifuge at 4 °C and 4000 rpm for 15 min, collect the supernatant to obtain the crude enzyme solution.

Images