A mutant of 3-phosphoglycerate dehydrogenase with enhanced enzyme activity and thermostability

Abstract

This invention belongs to the field of bioengineering, specifically disclosing a 3-phosphoglycerate dehydrogenase mutant with improved enzyme activity and thermostability. Compared to the wild-type 3-phosphoglycerate dehydrogenase from *Corynebacterium glutamicum*, the mutant contains one mutation from among P85Q, D365Y, A389T, M196V, A183V, and I231V. Enzyme activity assays after induced expression and purification showed that the mutant's enzyme activity was 1.5–3.6 times higher than the wild type, and its thermostability was significantly improved. Therefore, the beneficial mutant provided by this invention lays a good foundation for the industrial fermentation production of L-serine and L-cysteine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and specifically discloses a 3-phosphoglycerate dehydrogenase mutant with improved enzyme activity and thermal stability. Background Technology

[0002] L-Serine is a non-essential amino acid widely used in the food, pharmaceutical, cosmetic, and chemical industries, possessing significant commercial value. With increasing market demand for L-serine, traditional production methods are too costly and complex to achieve industrial-scale production. Microbial fermentation, however, is gaining increasing attention due to its abundant raw materials, low cost, and environmentally friendly nature. In vivo, L-serine plays important physiological roles, participating in the metabolism of thymine, purines, choline, and one-carbon units. The L-serine synthesis pathway begins with 3-phosphoglycerate, an intermediate metabolite in glycolysis, and then... serA The gene-encoded 3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) catalyzes the production of 3-phosphohydroxypyruvate, which is then converted to L-serine by phosphoserine aminotransferase and phosphoserine phosphorylase. 3-PGDH is a key rate-limiting enzyme in the L-serine synthesis pathway, subject to strong feedback inhibition by L-serine, thus limiting the synthesis level of L-serine. Therefore, improving the enzymatic catalytic properties of 3-PGDH is of great significance for the production of L-serine and its derivative, L-cysteine.

[0003] In recent years, research on modifying 3-PGDH using protein engineering strategies to enhance its catalytic activity has received widespread attention. Studies have shown that the binding site of L-serine is located in the ACT domain of 3-PGDH, and the allosteric effect of this enzyme is regulated by changes in the ACT domain. Grant et al. found that mutating amino acid residues at positions 344 and 346 of 3-PGDH in *E. coli* to alanine weakens the enzyme's sensitivity to serine. Furthermore, knocking out 197 amino acids at the C-terminus of PGDH in *Corynebacterium glutamicum* relieves the feedback inhibition of L-serine, increasing the accumulation of L-serine. However, current research on 3-PGDH, both domestically and internationally, mainly focuses on relieving feedback inhibition, with limited research on modifying its enzymatic properties. Therefore, this study uses protein engineering strategies to perform directed evolution and screening of 3-PGDH to improve its activity and thermostability, providing an important foundation for the industrial fermentation production of L-serine and its derivative, L-cysteine. Summary of the Invention

[0004] Based on the above requirements, the primary objective of this invention is to provide a 3-phosphoglycerate dehydrogenase mutant to improve the enzyme's catalytic activity and thermal stability.

[0005] This invention is achieved through the following technical approach, in order to C.glutamicum Using the genome as a template, a Cg-3-PGDH gene-encoding mutant library was obtained using a fault-prone PCR random mutagenesis method. This library was then subcloned into an expression vector using a Golden Gate-based vector construction strategy, resulting in a recombinant plasmid library containing the Cg-3-PGDH mutant gene. Subsequently, the recombinant plasmid library was introduced into *E. coli* BL21(DE3) that knocks out the serine degradation pathway. Primary screening was performed using 96-well plates, followed by secondary screening in test tubes and shake flasks. Finally, mutants with enhanced enzyme activity and thermostability were selected, thus completing this invention.

[0006] This invention provides a wild-type Corynebacterium glutamicum The 3-phosphoglycerate dehydrogenase mutant of ATCC13032, relative to the amino acid sequence shown in SEQ ID No. 2, has one or more mutations at positions 85, 183, 196, 231, and 389. Preferably, it has one or more of the following mutations relative to the amino acid sequence shown in SEQ ID No. 2: proline P is mutated to glutamine Q at position 85, alanine A is mutated to valine V at position 183, methionine M is mutated to valine V at position 196, isoleucine I is mutated to valine V at position 231, aspartic acid D is mutated to tyrosine Y at position 365, or alanine A is mutated to threonine T at position 389.

[0007] More preferably, relative to the amino acid sequence shown in SEQ ID No. 2, only position 85 is mutated from proline P to glutamine Q, only position 183 is mutated from alanine A to valine V, only position 196 is mutated from methionine M to valine V, only position 231 is mutated from isoleucine I to valine V, only position 365 is mutated from aspartic acid D to tyrosine Y, only position 389 is mutated from alanine A to threonine T, only the combination mutation of only position 85 being mutated from proline P to glutamine Q and position 231 being mutated from isoleucine I to valine V, and only the combination mutation of only position 365 being mutated from aspartic acid D to tyrosine Y and position 389 being mutated from alanine A to threonine T.

[0008] This invention further provides the encoding gene of the 3-phosphoglycerate dehydrogenase mutant. In a preferred embodiment, the nucleotide sequence of the 3-phosphoglycerate dehydrogenase encoding gene is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1. This invention also provides an expression vector containing the encoding gene of the 3-phosphoglycerate dehydrogenase mutant and a host cell. In a preferred embodiment, the expression vector includes, but is not limited to, pACYC184 and pXMJ19, by constructing a recombinant plasmid containing the encoding gene of the 3-PGDH mutant and introducing it into microbial chassis cells. In a specific embodiment, the microbial chassis cells can be selected from Corynebacterium, Enterobacter, or Yeast, preferably Escherichia coli. Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) and brewer's yeast ( Saccharomyces cerevisiae ).

[0009] The present invention further provides the application of the 3-phosphoglycerate dehydrogenase mutant or its encoding gene, and the host cell in L-serine or downstream L-cysteine ​​products.

[0010] The beneficial effect of this invention is that, through the... C.glutamicum Directed evolution and screening of 3-phosphoglycerate dehydrogenase from the source yielded 3-phosphoglycerate dehydrogenase mutants with varying degrees of enhanced catalytic activity. Therefore, the several 3-phosphoglycerate dehydrogenase mutants provided by this invention lay a solid foundation for the efficient fermentation production of L-serine and downstream L-cysteine ​​products, and have better prospects for industrial application. Attached Figure Description

[0011] Figure 1 A schematic diagram of the microbial L-serine biosynthesis pathway.

[0012] Figure 2 Enzyme activity assay of 3-phosphoglycerate dehydrogenase mutant. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but this should not be construed as limiting the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0014] Example 1 C. glutamicum Construction of 3-phosphoglycerate dehydrogenase from the source

[0015] The nucleotide sequence of 3-phosphoglycerate dehydrogenase derived from wild-type C. glutamicum is shown in SEQ ID NO: 1, and the amino acid sequence it encodes is shown in SEQ ID NO: 2.

[0016] This invention uses EasyTaq DNA polymerase (Beijing TransGen Biotech, China), which has low fidelity, with wild-type... C.glutamicum Using the genome as a template, a 3-PGDH coding gene mutant library was obtained via error-prone PCR using primers P1 (5'-CACCAGGTCTCAACATATGAGCCAGAATGGCCGTCCGG-3') and P2 (5'-CACCAGGTCTCAGGTGGTCAAGATCAACCTGGAAGGAAGTAGCAC-3'). By adding certain concentrations of magnesium and manganese ions to the PCR reaction system, the fidelity of the PCR amplification process was further reduced, controlling the presence of 2-3 point mutations in the obtained 3-PGDH coding gene. The error-prone PCR system used in this invention consisted of: 5 μL 10× EasyTaq buffer, 0.2 μM upstream primer P1, 0.2 μM upstream primer P2, 200 μM dNTPs, 0.8 mM MnCl2, 6 mM MgSO4, 50 ng template DNA, 1 μL EasyTaq DNA polymerase, and sterile water to a final volume of 50 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 2 min, repeated 35 times; 72℃ extension for 5 min, and storage at 4℃. The above ligation system was then introduced into *E. coli* BL21(DE3) competent cells using the standard *E. coli* heat shock transformation method to obtain the recombinant plasmid library.

[0017] Example 2: Screening and Identification of 3-Phosphoglycerate Dehydrogenase Variant Library

[0018] 3-PGDH (3-Phosphoglycerate dehydrogenase, 3-PGDH) is produced by serA The gene encodes a catalyst that catalyzes the reaction of NAD+ and 3-phosphoglycerate to produce 3-phosphohydroxypyruvate and NADH. Therefore, monitoring the OD in the reaction solution... 340 The rate of change in NADH was determined by the change in the value of the enzyme, and then the enzyme activity of 3-phosphoglycerate dehydrogenase was measured and screened. The recombinant plasmid mutant library plasmid from Example 1 was introduced into *E. coli* BL21(DE3) with the serine degradation pathway knocked out, and the activity of the crude enzyme solution was measured using a microplate reader. The *E. coli* with the serine degradation pathway knocked out refers to... E.coliKnockout of sdaA and sdaA in BL21(DE3) sdaB .

[0019] The specific screening steps are as follows: The recombinant plasmid mutant library described in Example 1 was plated on LB agar plates containing ampicillin, a selective antibiotic. Single colonies were picked with sterile toothpicks and transferred to 96-well shallow plates containing 200 μL of LB medium. The plates were incubated at 37°C for approximately 12 h, then transferred to 96-well deep plates containing 600 μL of LB medium until OD=0.6. 0.4 mM IPTG was added, and the plates were induced overnight at 16°C and 800 rpm. The cells were collected by centrifugation, resuspended in 200 μL of lysis buffer containing 3 mg / ml lysozyme, and reacted at 37°C for 2 h. The supernatant was then centrifuged and the enzyme activity was measured. The mutants with increased enzyme activity obtained from the initial screening were inoculated into test tubes containing 5 ml of LB medium and incubated at 37°C until OD=0.6. 0.4 mM IPTG was added... IPTG induction was performed overnight at 16°C. Cells were collected by centrifugation, sonicated, and the supernatant was centrifuged again to determine enzyme activity. Sanger sequencing was used to identify mutation sites. After three rounds of screening, six different beneficial 3-PGDH mutants were obtained: P85Q, D365Y, A389T, M196V, A183V, and I231V. The LB medium consisted of 1% yeast extract, 2% tryptone, and 1% NaCl.

[0020] Example 3: Enzyme activity assay of 3-phosphoglycerate dehydrogenase

[0021] Collect the induced bacterial cells, centrifuge to remove the culture medium, and resuspend the bacterial pellet in 10 mL of pre-chilled lysis buffer (20 mM Na2HPO3, 200 mM NaCl, pH 7.5). Disrupt the cells using an ultrasonic cell disruptor at 200 W, sonicating for 2 seconds followed by a 1-second interval for 10 min. Then, centrifuge at 8000 × g for 10 min in a high-speed refrigerated centrifuge. Collect the supernatant for subsequent protein purification and enzyme activity assays.

[0022] Based on the catalytic properties of 3-phosphoglycerate dehydrogenase, the enzyme activity of 3-PGDH was calculated by continuously detecting the NADH production rate at 340 nm absorbance. The reaction system was as follows: 50 mM Tris-HCl (pH 8.8), 5 mM NAD+, 5 mM 3-phosphoglycerate disodium salt (3-PGA), 5 mM hydrazine, 5 mM EDTA, 1 mM dithiothreitol, and 10-300 ng of enzyme protein. The amount of enzyme required to produce 1 μmol of NADH per minute at 30 °C was defined as one enzyme activity unit (U).

[0023] Analysis of the activity of 3-PGDH, such as... Figure 2 As shown in the figure. The results showed that, compared with the unmutated Cg-3-PGDH, the six 3-phosphoglycerate dehydrogenase mutants all exhibited significantly enhanced enzyme activity. The specific enzyme activities of mutants P85Q, D365Y, A389T, M196V, A183V, and I231V were 5.3 U / mg, 4.1 U / mg, 7.0 U / mg, 2.7 U / mg, 3.0 U / mg, and 3.2 U / mg, respectively. Compared with the wild type, the enzyme activities of the six mutants increased by 2.5 times, 1.95 times, 3.36 times, 1.38 times, 1.42 times, and 1.52 times, respectively. The four double mutants P85-A389T, A183-I231V, I231V-D365Y, and A183-D365Y obtained through combined mutations had lower enzyme activities than the wild type. The enzyme activities of two mutants, P85Q-A183V and A389T-I231V, were increased by about 1.3 times, while the enzyme activities of the other four mutants, P85Q-I231V, P85Q-D365Y, A183V-A389T, and D365Y-A389T, were increased by 1.5-2.5 times.

[0024] The thermostability of the Cg-3-PGDH mutant was also significantly improved. After incubation at 40℃ for 10 min, the wild-type Cg-3-PGDH showed approximately 8% residual enzyme activity. However, after incubation at 50℃ and 60℃ for 10 min, the wild-type enzyme completely lost its catalytic activity. After incubation at 40℃ for 10 min, the residual enzyme activity was as follows: P85Q mutant 54%; I231V mutant 25%; D365Y mutant 34%; A389T mutant 25%; double mutant P85Q-D365Y 94%; double mutant P85Q-I231V 50%; double mutant A389T-A183V 40%; while the residual enzyme activity of mutants A183V and M196V was essentially zero.

[0025] In summary, the 3-phosphoglycerate dehydrogenase mutant obtained by this invention has high enzyme activity and thermostability, and can be used for the efficient biosynthesis of L-serine and its downstream product L-cysteine, with broad prospects for industrial application.

Claims

1. A 3-phosphoglycerate dehydrogenase mutant derived from Corynebacterium glutamicum, characterized in that, Compared to the amino acid sequence shown in SEQ ID No. 2: (1) The 85th position is mutated from proline P to glutamine Q; or (2) The 85th position is mutated from proline P to glutamine Q and the 231st position is mutated from isoleucine I to valine V; or (3) The 85th position is mutated from proline P to glutamine Q and the 365th position is mutated from aspartic acid D to tyrosine Y.

2. The encoding gene of the 3-phosphoglycerate dehydrogenase mutant as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene was obtained by mutation based on the nucleotide sequence shown in SEQ ID No.

1.

4. An expression vector containing the encoding gene of the 3-phosphoglycerate dehydrogenase mutant as described in claim 2 or 3.

5. The expression vector as described in claim 4, characterized in that, It is a prokaryotic expression vector.

6. The expression vector as described in claim 5, characterized in that, The launch carrier is pACYC184 or pXMJ19.

7. A host cell containing the gene encoding the 3-phosphoglycerate dehydrogenase mutant as described in claim 2 or 3, or its expression vector.

8. The host cell as described in claim 7, characterized in that, The host cell is selected from Corynebacterium, Enterobacter, or Yeast.

9. The host cell as described in claim 8, characterized in that, The host cell is Escherichia coli (E. coli) Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) or brewer's yeast ( Saccharomyces cerevisiae ).

10. The use of the encoding gene of the 3-phosphoglycerate dehydrogenase mutant as described in claim 2 or 3, or the use of the host cell as described in claim 7, 8, or 9 in the preparation of L-serine or downstream L-cysteine ​​products.

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