A cystathionine-gamma-synthase mutant and use thereof

Abstract

This invention relates to a highly active cystathionine-γ-synthase mutant and its applications. To overcome the problem of low L-methionine synthesis efficiency caused by low cystathionine-γ-synthase activity, this invention provides a highly active cystathionine-γ-synthase mutant and applies this enzyme to the biosynthesis of L-methionine. The cystathionine-γ-synthase mutant MetB... M This enzyme was obtained by mutations in Q5R, F87I, E136G, V231E, F321V, and A346T into the wild-type cystathionine-γ synthase derived from *E. coli* W3110. The enzyme's Km and Kcat concentrations are 0.13 mmol / L and 138.2 s, respectively. ‑1 The Kcat / Km ratio is 1063.1 (mmol / L). ‑1 S ‑1 The specific activity was 195.3 U / mg. Both substrate binding efficiency and enzyme activity were higher than wild-type MetB. Applying the cystathionine-γ-synthase mutant to L-methionine synthesis increased its yield by 11-fold, making it widely applicable to L-methionine production.

Description

Technical fields:

[0001] This invention relates to a highly active cystathionine-γ-synthase mutant and its applications, belonging to the field of metabolic engineering. Background technology:

[0002] L-Methionine, also known as L-methionine, is the only sulfur-containing amino acid among the eight essential amino acids. It belongs to the aspartic acid family along with L-threonine, L-isoleucine, and L-lysine. L-methionine plays a crucial role in many bodily functions. Besides its role in protein biosynthesis, it also participates in methyl transfer, phosphorus metabolism, and the bioavailability of selenium and zinc. L-methionine can also be used directly to treat medical conditions such as allergies and rheumatic fever. However, higher animals cannot synthesize L-methionine themselves and must obtain it from external sources. Currently, most industrially produced L-methionine is added to animal feed to promote animal growth and reproduction and reduce production costs. Due to the unique biological and chemical activities of L-methionine and its derivatives, it is widely used in the food, pharmaceutical, agricultural, cosmetic, and feed additive industries. Currently, L-methionine is mainly synthesized chemically, but this method has drawbacks such as toxic raw materials, high energy consumption, demanding reaction conditions, complex extraction processes, and environmental pollution. In contrast, microbial fermentation has low production costs, mild conditions, and is environmentally friendly. In recent years, this method has been widely used in the production of various amino acids.

[0003] In the L-methionine biosynthesis pathway, cystathionine-γ-synthase encoded by metB is a key enzyme. This enzyme catalyzes the synthesis of cystathionine and succinate from L-cysteine ​​and O-succinyl-homoserine. Cystathionine then forms L-homocysteine ​​under the action of β-cystathionase, and subsequently, L-homocysteine ​​is converted to L-methionine under the catalysis of homocysteine ​​methyltransferase. Insufficient cystathionine-γ-synthase activity significantly limits the efficient synthesis of L-methionine. Summary of the Invention:

[0004] To overcome the problem of low L-methionine synthesis efficiency caused by low cystathionine-γ-synthase activity, this invention provides a highly active cystathionine-γ-synthase mutant and applies this enzyme to L-methionine biosynthesis.

[0005] One of the technical solutions of this invention to solve the above problems is to provide a highly active cystathionine-γ-synthase mutant, MetB. M The amino acid sequence is shown in SEQ ID NO.1, and the cystathionine-γ-synthase mutant MetB... MIt was obtained by mutations of Q5R, F87I, E136G, V231E, F321V and A346T in wild-type cystathionine-γ synthase derived from Escherichia coli W3110 as shown in SEQ ID NO.3.

[0006] The second technical solution provided by this invention is the cystathionine-γ-synthase mutant MetB. M The encoding gene;

[0007] Furthermore, the gene encoding the cystathionine-γ-synthase mutant is metB. M The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing.

[0008] The third technical solution provided by this invention is the cystathionine-γ-synthase mutant MetB. M Its applications, especially in the production of L-methionine.

[0009] Beneficial effects:

[0010] The metB described in this invention M MetB, a gene-encoded cystathionine-γ synthase M It has the following characteristics: the enzyme's Km and Kcat are 0.13 mmol / L and 138.2 s, respectively. -1 The Kcat / Km ratio is 1063.1 (mmol / L). -1 S -1 Its specific activity was 195.3 U / mg. Both its substrate binding efficiency and enzyme activity were higher than those of wild-type MetB. Attached image description:

[0011] Figure 1 Schematic diagram of the screening principle for highly active cystathionine-γ-synthase mutants;

[0012] Figure 2 Strain biomass. Detailed implementation method:

[0013] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0014] In this invention, the mutant MetB M The screening process is as follows:

[0015] A library of mutant cystathionine-γ-synthase encoding genes, named `metB`, was obtained using error-prone PCR and ligated into pUC19 to obtain a recombinant plasmid library containing the `metB` mutant (named `pUC-metBs`). Then, using *E. coli* W3110 as the starting strain, the cystathionine-γ-synthase encoding gene `metB` and the succinyl-CoA encoding gene `sucCD` were sequentially knocked out (resulting in strain MET02). The `pUC-metBs` library was transformed into MET02 and plated on solid selection medium. Larger colonies were selected and inoculated onto 96-well cell culture plates using liquid selection medium. Fast-growing strains were selected, plasmids were extracted, and the cystathionine-γ-synthase mutant encoding gene was amplified by PCR. Sequencing of the PCR product revealed the following amino acid mutations relative to the wild-type cystathionine-γ-synthase from *E. coli* W3110: Q5R, F87I, E136G, V231E, F321V, and A346T.

[0016] In this invention, the following definitions are used:

[0017] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0018] The IUPAC nomenclature, a widely accepted system for naming amino acid residues, is used, employing three-letter / single-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0019] 2. Identification of cystathionine-γ-synthase mutants

[0020] The mutated amino acid in the cystathionine-γ-synthase mutant is represented by "original amino acid + position + substituted amino acid". For example, Q5R indicates that the amino acid at position 5 is replaced by Arg instead of Gln in the wild-type cystathionine-γ-synthase, and Q5 indicates that the amino acid at position 5 is Gln. The position number corresponds to the amino acid sequence number of the wild-type cystathionine-γ-synthase in SEQ ID NO.3.

[0021] In this invention, metB represents the wild-type cystathionine-γ-synthase synthase encoding gene (SEQ ID NO.4), and MetB represents the wild-type cystathionine-γ-synthase (SEQ ID NO.3); metB M The gene is a mutant of cystathionine-γ-synthase (SEQ ID NO.2); MetB M This is a cystathionine-γ-synthase mutant (SEQ ID NO.1). The amino acid profiles before and after the mutation are shown in the table below:

[0022] MetB Q5, F87, E136, V231, F321, A346 <![CDATA[MetB M ]]> Q5R, F87I, E136G, V231E, F321V, A346T

[0023] The cystathionine-γ-synthase mutant MetBM It possesses the following enzymatic properties: Km, Kcat, and specific activity are 0.13 mmol / L, 138.2 s, and 138.2 s, respectively. -1 The concentration was 195.3 U / mg, and the Kcat / Km ratio was 1063.1 (mmol / L). -1 S -1 .

[0024] The gene or amino acid sequences involved in this invention are as follows:

[0025] MetB, a mutant of cystathionine-γ-synthase synthase. M SEQ ID NO.1:

[0026] MTRKRATIAVRSGLNDDEQYGCVVPPIHLSSTYNFTGFNEPRAHDYSRRGNPTRDVVQRALAELEGGAGAVLTNTGMSAIHLVTTVILKPGDLLVAPHDCYGGSYRLFDSLAKRGCYRVLFVDQGDEQALRAALAGKPKLVLVESPSNPLLRVVDIAKICHLAREVGAVSVVDNTFLSPALQNPLALGADLVL HSCTKYLNGHSDVVAGVVIAKDPDVVTELAWWANNIGETGGAFDSYLLLRGLRTLVPRMELAQRNAQAIVKYLQTQPLVKKLYHPSLPENQGHEIAARQQKGFGAMLSFELDGDEQTLRRFLGGLSLVTLAESLGGVESLISHAATMTHAGMTPEARAAAGISETLLRISTGIEDGEDLIADLENGFRAANKG.

[0027] Cystathione-γ-synthase mutant gene metB M SEQ ID NO.2:

[0028]

[0029] Wild-type cystathionine-γ-synthase MetB, SEQ ID NO.3:

[0030] MTRKQATIAVRSGLNDDEQYGCVVPPIHLSSTYNFTGFNEPRAHDYSRRGNPTRDVVQRALAELEGGAGAVLTNTGMSAIHLVTTVFLKPGDLLVAPHDCYGGSYRLFDSLAKRGCYRVLFVDQGDEQALRAALAEKPKLVLVESPSNPLLRVVDIAKICHLAREVGAVSVVDNTFLSPALQNPLALGADLVLHSCTKYLNGHSDVVAGVVIAKDPDVVTELAWWANNIGVTGGAFDSYLLLRGLRTLVPRMELAQRNAQAIVKYLQTQPLVKKLYHPSLPENQGHEIAARQQKGFGAMLSFELDGDEQTLRRFLGGLSLFTLAESLGGVESLISHAATMTHAGMAPEARAAAGISETLLRISTGIEDGEDLIADLENGFRAANKG.

[0031] Wild-type cystathionine-γ-synthase synthase-encoding gene metB, SEQ ID NO.4:

[0032]

[0033] The present invention will be further explained and illustrated below through specific embodiments.

[0034] Example 1: Construction of metB knockout bacteria MET01

[0035] (1) Overlapping segment U metB -D metB Construction

[0036] Using the wild-type Escherichia coli W3110 genome as a template, the upstream and downstream homologous arms of metB were amplified using primers metB-1 / metB-2 and metB-3 / metB-4, respectively. Then, the fusion fragment U of the upstream and downstream homologous arms of metB was obtained by overlap PCR. metB -D metB .

[0037] (2) Construction of pGRB-metB plasmid

[0038] Based on the metB sequence, 20bp forward and reverse sequences pG-metB-1 / pG-metB-2 of gRNA were designed and synthesized. After annealing, the two sequences were ligated into plasmid pGRB using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.). The recombinant plasmid pGRB-metB was obtained by transformation of E. coli DH5α, screening on LB solid medium containing 100 μg / mL ampicillin, and sequencing.

[0039] (3) Construction of metB knockout bacteria MET01

[0040] The recombinant plasmid pGRB-metB and the fusion fragment U metB -D metB Electroporation was performed into E. coli W3110 competent cells containing the pREDcas9 plasmid. After recovery, the cells were plated on LB agar plates containing 100 μg / mL spectinomycin and ampicillin and incubated overnight at 32°C. The next day, colony PCR was performed using primers metB-1 / metB-4 to identify positive transformants. The transformants were activated, and arabinose was added to a final concentration of 0.2 mmol / L. The cells were then incubated overnight at 32°C with shaking to induce pGRB-metB loss. Following this, the cells were incubated overnight at 42°C with shaking to induce pREDcas9 plasmid loss, resulting in the metB knockout strain MET01.

[0041] Example 2: Construction of sucCD knockout bacteria MET02

[0042] like Figure 1As shown, the sucCD-encoded succinyl-CoA synthase catalyzes the conversion of succinate-CoA to succinate. Therefore, after knocking out the sucCD gene in strain MET01, the strain cannot grow on the selection medium due to succinate deficiency. Cystathion-γ-synthase catalyzes the conversion of L-cysteine ​​and O-succinyl-homoserine to cystathionine and succinate. Therefore, if metB is reintroduced into the MET01 strain with the sucCD gene knocked out, succinate can be provided for the TCA cycle, and the strain can resume growth. Moreover, the higher the cystathion-γ-synthase activity, the greater the amount of succinate produced, and the faster the strain grows. Using this principle, strains with fast growth (large colonies) can be screened to obtain cystathion-γ-synthase mutants with high activity.

[0043] (1) Overlapping segment U sucCD -D sucCD Construction

[0044] Using the wild-type Escherichia coli W3110 genome as a template, the upstream and downstream homologous arms of sucCD were amplified using primers sucCD-1 / sucCD-2 and sucCD-3 / sucCD-4, respectively. Then, the fusion fragment U of the upstream and downstream homologous arms of sucCD was obtained by overlap PCR. sucCD -D sucCD .

[0045] (2) Construction of pGRB-sucCD plasmid

[0046] Based on the sucCD sequence, 20bp forward and reverse sequences pG-sucCD-1 / pG-sucCD-2 of gRNA were designed and synthesized. After annealing, the two sequences were ligated into plasmid pGRB using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.). The recombinant plasmid pGRB-sucCD was obtained by transformation of E. coli DH5α, screening on LB solid medium containing 100 μg / mL ampicillin, and sequencing.

[0047] (3) Construction of sucCD knockout bacteria MET02

[0048] The recombinant plasmid pGRB-sucCD and the fusion fragment U sucCD -D sucCDElectroporation was performed into MET01 competent cells containing the pREDcas9 plasmid. After recovery, the cells were plated on LB agar plates containing 100 μg / mL spectinomycin and ampicillin and incubated overnight at 32°C. The next day, colony PCR was performed using primers sucCD-1 / sucCD-4 to identify positive transformants. The transformants were activated, and arabinose was added to a final concentration of 0.2 mmol / L. The cells were then incubated overnight at 32°C with shaking to induce the loss of pGRB-sucCD. The cells were then incubated overnight at 42°C with shaking to induce the loss of the pREDcas9 plasmid, yielding strain MET02.

[0049] Example 3: metB M Filtering

[0050] Using the wild-type *Escherichia coli* W3110 genome as a template, the metB mutant library was amplified by error-prone PCR (using the error-prone PCR kit, Beijing Tianenze Gene Technology Co., Ltd.) with primers ER-1 / ER-2. The error-prone PCR reaction system was 30 μL: 3 μL Error-Prone PCR Mix, 3 μL dNTPs, 3 μL 5 mmol / L LmnCl2, 1 μL template, 10 μL each of ER-1 and ER-2, 1 μL Taq DNA polymerase, and deionized water to a final volume of 30 μL. PCR conditions were: 94℃ for 3 min, 1 cycle; 94℃ for 1 min s, 45℃ for 90 s, 72℃ for 1 min, 30 cycles.

[0051] The amplified metB mutant was then recombine with the expression plasmid pUC19 (digested with Hind III) to obtain a recombinant plasmid library containing the metB mutant (named pUC-metBs).

[0052] pUC-metBs were transformed into MET02 competent cells. MET02 cells transformed with pUC plasmids containing wild-type metB (named pUC-metB) served as a control (CK). After resuscitation, the cells were plated on solid selection medium containing 100 μg / mL ampicillin and incubated at 37°C. The next day, 480 large single colonies were picked from the plates and transferred to 96-well cell culture plates containing liquid selection medium containing ampicillin (100 μg / mL), and incubated at 37°C with shaking for 24 h. OD was measured using a microplate reader. 600 Select OD 600 The top 20 strains (named M1-M20) were inoculated at a 1% inoculation rate into shake tubes containing 5 mL of liquid selection medium for secondary screening. After incubation at 37°C with shaking for 24 h, the OD was measured using a spectrophotometer. 600 The result is as follows Figure 2 As shown. Where the OD of M4 is... 600 The value was the highest (1.89), which was 1.17 times higher than that of the control strain.

[0053] Plasmids were extracted from M4, and their mutant metB was analyzed. M Sequencing was performed (the plasmid was named pUC-metB). M The nucleotide sequence result is shown in SEQ ID NO.2, and the corresponding amino acid sequence is shown in SEQ ID NO.1.

[0054] The enzyme was found to have the following amino acid mutations relative to the wild-type cystathionine-γ synthase from Escherichia coli W3110: Q5R, F87I, E136G, V231E, F321V, A346T.

[0055] Solid screening medium: glucose 10g / L, MgSO4 0.24g / L, KH2PO4 2.5g / L, (NH4)2SO4 5g / L, FeSO4 2g / L, agar 20g / L, deionized water 1000mL, pH 6.5-7.0.

[0056] Liquid screening medium: glucose 10 g / L, MgSO4 0.24 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 5 g / L, FeSO4 2 g / L, deionized water 1000 mL, pH 6.5-7.0.

[0057] Example 4: Enzymatic Characterization of Cystatin-γ Synthase

[0058] pUC-metB and pUC-metB respectively M Using the template, metB and metB were amplified by PCR using primers CGS-1 and CGS-2. M After electrophoresis recovery, the plasmids were ligated into the Sac I-digested expression vector pET-28a using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.) to obtain plasmids p28-metB and p28-metB. M The recombinant strains DE-CGS and DE-CGS were obtained by transforming them into E. coli BL21(DE3) competent cells, respectively. M .

[0059] DE-CGS and DE-CGS respectively MSeed cultures were inoculated into LB liquid medium and induced for expression with 0.1 mmol / L IPTG for 4 h. 1 mL of culture was centrifuged at 10000 g for 1 min at 4 °C to collect bacterial cells. The bacterial cell pellet was washed three times with 1 mL buffer (200 mmol / L Tris-HCl, pH 8.1) and then resuspended in 1 mL buffer. The bacterial suspension was sonicated using an ultrasonic homogenizer under the following conditions: 350 W power, 5 sec working time, 10 sec interval, 5 cycles, operated on ice. The homogenate was centrifuged at 8000 g at 4 °C, and the supernatant was collected. Recombinant cystathionine-γ-synthase MetB and its mutant MetB were isolated and purified by Ni-NTA affinity chromatography. M .

[0060] An appropriate amount of recombinant enzyme solution was taken to determine its enzyme activity. The reaction conditions were as follows: 50 mmol / L Tris-HCl solution (pH 7.8) containing 20 mmol / L O-succinyl-homoserine, 0.025-20 mmol / L L-cysteine ​​(the L-cysteine ​​concentration for enzyme activity determination was 20 mmol / L), 10 μL cystathionine-γ-synthiase solution, and 20 μmol / L pyridoxal phosphate. The reaction was terminated with acetone after incubation at 25℃ for 30 min. The amount of L-cysteine ​​consumed was determined by high performance liquid chromatography (HPLC). Enzyme activity was defined as the amount of enzyme required to convert 1 μmol of L-cysteine ​​in 1 minute at 25℃ and pH 7.8, which is one unit of enzyme activity (U).

[0061] Detection of L-cysteine ​​in the reaction solution: After centrifugation at 8000×g for 10 min, the supernatant was collected and diluted 10 times with deionized water. The reaction solution was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene. The L-cysteine ​​content was determined by high performance liquid chromatography (HPLC). The detection conditions were: Agilent C18 (150 mm × 4.6 mm, 5 μm), acetonitrile / sodium acetate binary gradient elution, column temperature 33 ℃, and detection wavelength 360 nm.

[0062] Determination of MetB and MetB at different L-cysteine ​​concentrations M The enzyme's Km value was calculated using the double reciprocal method to determine the enzymatic reaction rate; simultaneously, enzyme activity was measured at an L-cysteine ​​concentration of 20 mmol / L. Specific results are shown in the table below.

[0063] MetB 0.25 mmol / L <![CDATA[111.9s -1 ]]> <![CDATA[447.6(mmol / L) -1 S -1 )]]> 158.1 <![CDATA[MetB M ]]> 0.13 mmol / L <![CDATA[138.2s -1 ]]> <![CDATA[1063.1(mmol / L) -1 S -1 ]]> 195.3

[0064] The results above show that MetB M The Km and Kcat values ​​of this enzyme differ somewhat from those of MetB. A smaller Km indicates higher substrate specificity; a larger Kcat indicates a faster rate of substrate conversion by the enzyme. (MetB) MThe specific activity is 23.5% higher than that of MetB. Therefore, the cystathionine-γ-synthase mutant obtained in this invention exhibits higher affinity for the substrate and higher catalytic efficiency than the wild-type cystathionine-γ-synthase.

[0065] Example 5: Application of highly active cystathionine-γ-synthase in L-methionine synthesis

[0066] (1) pUC-metB and pUC-metB respectively M Using the template, metB and metB were amplified using primers pS-1 / pS-2, respectively. M The plasmids pSTV28, digested with BamHI, were ligated into the recombinant DNA cloning kit ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.) to obtain plasmids pSTV-metB and pSTV-metB. M pSTV-metB and pSTV-metB respectively. M The bacteria were transformed into E. coli W3110 to obtain recombinant strains LMT-1 and LMT-2.

[0067] (2) LMT-1 and LMT-2 were inoculated into 30 mL of basic culture medium and cultured at 37 °C and 220 rpm for 24 h.

[0068] The basic culture composition is as follows: glucose 15g / L, MgSO4 0.3g / L, KH2PO4 2g / L, (NH4)2SO4 4g / L, MgSO4 1g / L, FeSO4·7H2O 10mg / L, MnSO4 10mg / L, deionized water 1000mL, pH 6.5-7.0.

[0069] (3) Detection of L-methionine in fermentation broth

[0070] After centrifuging the fermentation broth at 8000×g for 10 min, the supernatant was collected and diluted with deionized water. The fermentation broth was then derivatized with 0.8% (V / V) 2,4-dinitrofluorobenzene. The L-methionine content was determined by high performance liquid chromatography (HPLC) under the following conditions: Agilent C18 (150 mm × 4.6 mm, 5 μm), acetonitrile / sodium acetate binary gradient elution, column temperature 33 ℃, and detection wavelength 360 nm.

[0071] The L-methionine yields of LMT-1 and LMT-2 were 0.02 g / L and 0.25 g / L, respectively, indicating that the cystathionine-γ-synthase mutant MetB obtained in this invention... M The ability to synthesize L-methionine was significantly improved compared to the wild type.

[0072] List of primer sequences used in embodiments of this invention:

[0073]

[0074]

[0075] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A cystathionine-γ-synthase mutant, characterized in that, The amino acid sequence of the cystathionine-γ-synthase is shown in SEQ ID NO.

1.

2. The encoding gene of the cystathionine-γ-synthase mutant according to claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence is shown in the sequence listing SEQ ID NO.

2.

4. The application of the cystathionine-γ-synthase mutant according to claim 1, characterized in that, This relates to the application of the cystathionine-γ-synthase mutant in the production of L-methionine.

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