Threonine aldolase mutant with improved enzymatic activity and application of threonine aldolase mutant in production of L-serine

Abstract

The invention belongs to the field of bioengineering, and particularly discloses a threonine aldolase mutant with improved enzymatic activity and application of the threonine aldolase mutant in production of L-serine. Compared with wild threonine aldolase derived from aeromonas, the amino acid sequence of the mutant has the mutation of T140A or T140A / I118V. Experiments show that the enzyme activity of the mutant is increased by 1.6 and 2.9 times compared with that of a wild type through enzyme activity determination and analysis of induced expression and purification, and the thermal stability is remarkably improved. The beneficial mutant provided by the invention can lay a good foundation for industrial production of derivative products such as L-serine and downstream L-cysteine.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the construction of a threonine aldolase mutant with improved enzyme activity and its application. Background Art

[0002] In the past decade, biocatalysis has emerged in the field of industrial synthesis of natural medicines and fine chemicals, becoming a cutting-edge technology with great potential. Compared with traditional methods, it can precisely control the stereoconfiguration of products with its highly accurate stereoselectivity, which meets the strict requirements of complex compounds for configuration. It is also easy to operate, greatly reducing the complexity and cost of the process, and is a highly competitive alternative to traditional methods. The construction of CC bonds has always occupied a cornerstone position in the chemical synthesis system. In addition to the chemical synthesis path, the academic community has successfully discovered many biological enzymes that can specifically catalyze the formation of CC bonds, such as aldolase, transketolase, transaldolase, and alcohol nitrile lyase. Among them, aldolase has attracted much attention in the field of enzymatic CC bond synthesis due to its wide substrate adaptability and excellent stereoselectivity. Aldolase can catalyze reversible condensation reactions, prompting nucleophilic donors (such as ketones, enol compounds and their analogs) to react with electrophilic acceptors (aldehydes or ketones), thereby constructing CC bonds. It is worth noting that aldolase exhibits strict structural specificity for substrate donors, but is diverse in substrate acceptors and can adapt to a variety of aldehyde or ketone substrates with different structures. This characteristic gives aldolase great flexibility in catalytic synthesis and can be used to synthesize products with rich and diverse structures.

[0003] Threonine aldolase (TAs, EC 4.1.2.5) is a class of enzymes that rely on pyridoxal 5′-phosphate (PLP) as a cofactor and can catalyze a series of reversible aldol condensation reactions with glycine as a donor substrate. In the forward reaction, TAs can catalyze the cleavage of L-threonine into acetaldehyde and glycine; in the reverse reaction direction, TAs can use glycine as a donor and aliphatic or aromatic aldehydes as acceptors to catalyze the synthesis of β-hydroxy-a-amino acids through aldol condensation reactions. According to their stereospecificity for the chiral center of the α-carbon of threonine, TAs can be divided into two major categories: L-TA and D-TA. Among them, L-TA can be further divided into three categories: (i) L-TA that specifically recognizes L-threonine; (ii) L-allo-TA that specifically recognizes L-allo-threonine; (iii) low-specificity L-TA that can recognize both L-threonine and L-allo-threonine. Although these three types of enzymes are highly conserved in primary structure, they have significant differences in substrate recognition and catalytic properties.

[0004] Threonine aldolase can specifically form CC bonds and has important applications in organic chemical synthesis. It is widely used in the fields of pharmaceuticals and chemicals to catalyze the synthesis of key precursors of drug intermediates and fine chemicals. In recent years, with the development of synthetic biology and metabolic engineering, TAs have shown important application potential in the industrial synthesis of amino acids, especially L-serine. In microbial cell factories, by introducing or modifying TAs, efficient synthesis of glycine and aldehyde intermediates to L-serine can be achieved, bypassing the dependence on energy and intermediate metabolites in traditional synthetic pathways, thereby increasing yields and reducing the accumulation of by-products. In addition, the stereoselectivity and substrate adaptability of TAs also provide powerful tools for the directional synthesis of high-value non-natural amino acids. Therefore, TAs are becoming an important enzyme tool for building efficient biosynthetic pathways and expanding the structural diversity of amino acid products. Summary of the invention

[0005] Based on the above needs, the primary purpose of the present invention is to provide a threonine aldolase mutant so that its catalytic activity and thermal stability are improved, which is beneficial to the catalytic production of metabolites such as L-serine and its derivatives.

[0006] The present invention is realized by the following technical ideas: according to Aeromonas Aeromonas sp The wild-type threonine aldolase encoding gene sequence of CU5 was obtained by whole gene synthesis technology after codon optimization by Suzhou Genewise Co., Ltd. and cloned into the pET21b expression vector Nde I / Xho I site, and the corresponding recombinant plasmid was obtained, named pET21b-AsTA [the gene sequence is shown in SEQ ID NO.2]. Using the error-prone PCR random mutagenesis method, the AsTA mutation library of the Aeromonas threonine aldolase encoding gene was obtained, and the mutant library was subcloned into the pET21b expression vector using the classic restriction enzyme ligation construction strategy. Nde I / Xho I site, and obtain a recombinant plasmid library containing a gene encoding a mutant of Aeromonas threonine aldolase. Then, the recombinant plasmid library is introduced into Escherichia coli BL21 (DE3), and a 96-well plate primary screening and a shake flask secondary screening are performed to finally screen and obtain mutants with improved enzyme activity and thermal stability, thereby completing the present invention.

[0007] The present invention provides a kind of Aeromonas sp . The threonine aldolase mutant of CU5, relative to the wild-type amino acid sequence shown in SEQ ID No.1, has only the 141st position mutated from threonine T to alanine A, or has only a combined mutation of the 141st position mutated from threonine T to alanine A and the 119th position mutated from isoleucine I to valine V.

[0008] The present invention further provides a gene encoding the Aeromonas threonine aldolase mutant. In a preferred embodiment, the nucleotide sequence of the gene encoding the Aeromonas threonine aldolase mutant is obtained by mutation based on the nucleotide sequence shown in SEQ ID No.2.

[0009] The present invention also provides an expression vector containing the coding gene, specifically, a prokaryotic expression vector.

[0010] Preferably, it is the Escherichia coli expression vector pET21b and the Corynebacterium glutamicum expression vector pXMJ19.

[0011] The present invention provides a recombinant bacterium containing the expression vector as described above.

[0012] Specifically, it is a recombinant Escherichia coli engineered bacterium or a recombinant Corynebacterium glutamicum engineered bacterium.

[0013] The present invention also provides the use of the threonine aldolase mutant, or its coding gene, or the expression vector containing the threonine aldolase, or the recombinant bacteria in catalyzing the synthesis of L-serine or its derivatives.

[0014] Specifically, formaldehyde and glycine are used as substrates to catalyze the production of L-serine.

[0015] The application of the threonine aldolase mutant provided by the present invention in the catalytic synthesis of L-serine and its derivatives has obvious advantages. In the reported L-serine synthesis pathway, serine hydroxymethyltransferase (SHMT) is one of the key catalytic enzymes, but the enzyme relies on PLP as a cofactor and requires the participation of tetrahydrofolate, with a complex mechanism of action, and increases the complexity of the reaction system and the cost of separation and purification in actual production.

[0016] The beneficial effect of the present invention is that by mutating and screening the threonine aldolase from Aeromonas, threonine aldolase mutants with improved catalytic performance to varying degrees are obtained, the enzyme can effectively catalyze formaldehyde and glycine to produce L-serine, and does not require the addition of tetrahydrofolate, has the advantages of high atom economy, simple catalytic process, mild reaction conditions, and no reaction by-products, and shows good application prospects in the synthesis of L-serine and its derivatives. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific examples, but it should not be understood as limiting the present invention. The experimental methods used in the examples are conventional methods well known to those skilled in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0018] Example 1 Screening and identification of threonine aldolase mutant library Threonine aldolase is a type of glycine-dependent aldolase that uses glycine as a donor substrate and relies on pyridoxal phosphate as a cofactor. It can catalyze reversible alcohol-aldehyde condensation reactions and has important application prospects in the fields of pharmaceutical and fine chemical synthesis. In previous studies, we screened the threonine aldolase gene database resources stored in the laboratory and found that it originated from Aeromonas. Aeromonassp The wild-type threonine aldolase of CU5 (SEQ ID NO: 1) exhibits good CC bonding ability and can catalyze the aldol condensation reaction of formaldehyde and glycine to form L-serine. Aeromonassp . The CU5 threonine aldolase encoding gene sequence (SEQ ID NO: 2) was obtained by total gene synthesis technology after codon optimization by Suzhou Jinweizhi Company, and was cloned into the pET21b expression vector to obtain the corresponding recombinant plasmid, named pET21b-AsTA. Using the error-prone PCR random mutagenesis method, the pET21b-AsTA recombinant plasmid was used as a template, and the primer pair AsTA-For (5'-TCGACATATGcgctatattgatctgcgcagcgatac-3', SEQ ID No: 3) and AsTA-Rev (5'-CTAGCTCGAGttagcgcgcgcccagatattcggt-3', SEQ ID No: 4) was used to amplify the AsTA mutant library of the Aeromonas threonine aldolase encoding gene. It was subcloned into the NdeI / XhoI site of the pET21b expression vector using the traditional restriction ligation construction strategy to obtain a recombinant plasmid library containing the Aeromonas threonine aldolase mutant encoding gene.

[0019] The error-prone PCR system used in the present invention is: 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 MgSO 4, 50 ng template DNA, 1 μL EasyTaq DNA polymerase, add sterile water to make up to 50 μL system. PCR reaction program is: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 2 min, 35 cycles; 72℃ extension for 5 min, 4℃ storage.

[0020] In view of the fact that the threonine aldolase in Aeromonas origin can catalyze formaldehyde and glycine to generate L-serine by aldol condensation, the threonine aldolase mutant with improved catalytic activity can be quickly screened by measuring and monitoring the content of formaldehyde in the reaction system. The acetylacetone spectrophotometry described in national standard HJ 601-2011 is adopted to measure the formaldehyde content in the reaction system, i.e., formaldehyde generates a yellow compound with acetylacetone in the presence of excessive ammonium salt, and the colored substance has maximum absorption at a wavelength of 414nm. In a preferred specific embodiment, the recombinant plasmid library containing the aeromonas threonine aldolase mutant encoding gene of the above-mentioned construction is imported into Escherichia coli BL21 (DE3). By 96-well plate primary screening and shake bottle rescreening, the absorption peak variation at a wavelength of 414nm is measured, and finally the mutant with improved enzyme activity is screened, which is named as T140A and T140A / I118V respectively.

[0021] Example 2 Activity determination of threonine aldolase mutant library The selected inducible expression vector is pET21b, which contains T7 strong promoter-related sequences (including the operator sequence lacO and the conserved RBS sequence). When inducers such as IPTG or lactose are added, the repressor protein will leave the operator sequence and initiate gene expression.

[0022] The inducible expression vector selected in the present invention is pXMJ19, which contains a strong promoter tac-related sequence (including the operator sequence lacO and the conserved RBS sequence) and can be replicated in Escherichia coli and Corynebacterium glutamicum. When inducers such as IPTG or lactose are added, the repressor protein will also be prompted to leave the operator sequence and initiate gene expression.

[0023] In this example, the recombinant E. coli cells induced by expression vector pET21b were collected, the culture medium was removed by centrifugation, and the cell pellet was resuspended in 10 mL of pre-cooled lysis buffer (20 mM Na 2 HPO 3 , 200 mM NaCl, pH 7.5). The cells were disrupted using an ultrasonic cell disruptor, with a power of 200 W, 2 s of ultrasound followed by 1 s of rest, and 10 min of ultrasound. The cells were then centrifuged at 8000 × g for 10 min in a high-speed refrigerated centrifuge, and the supernatant was collected for subsequent protein purification and enzyme activity determination.

[0024] According to the catalytic properties of Aeromonas threonine aldolase, the enzyme activity was determined using the ability to generate L-serine products as a standard. The enzyme activity assay system consisted of 1 mL (100 mM HEPES-NaOH, pH 8.0), 100 mM glycine, 50 mM formaldehyde, 50 μM PLP and 10 μL crude enzyme solution. The reaction system was heated for 30 min. o The mixture was incubated at 4 °C for 10 min, and the L-serine content in the system was detected by HPLC. The study found that compared with the activity of wild-type unmutated threonine aldolase (38.6 µmol / min / mg), the two mutants T140A and T140A / I118V showed significantly enhanced catalytic performance, with their enzyme activities increased by 1.6 and 2.9 times, respectively.

[0025] In addition, the thermal stability of the two mutants was significantly improved. o C for 30 min, the residual enzyme activity was 31%, while under the same high temperature treatment conditions the residual enzyme activity of mutant T140A was 78%, and the residual enzyme activity of mutant T140A / I118V was 94%, both of which were much higher than the wild unmutated threonine aldehyde condensation.

[0026] Example 3 Application of Threonine Aldolase Mutants in the Production of L-Serine This example is based on the whole-cell transformation method, using basic raw materials such as formaldehyde and glycine, and adding PLP cofactor to efficiently catalyze the synthesis of L-serine.

[0027] Recombinant Corynebacterium glutamicum (based on the expression vector pXMJ19) containing different threonine aldolase encoding genes was inoculated into a seed culture medium containing antibiotics and cultured in a shaking incubator at 32°C and 200 r / min for 16-20 h to obtain seed liquid. Subsequently, the inoculation amount was transferred to a 3 L fermenter at 5-10%, the culture temperature was set to 32°C, the stirring speed was 300-500 r / min, and the pH was maintained at a neutral level. During the culture process, when the dissolved oxygen was lower than 30%, the stirring-dissolved oxygen joint control system was activated to maintain sufficient dissolved oxygen supply. When the bacterial concentration (OD 600) reached 20-40, 0.4-0.6 mM IPTG was added for induction, and the culture temperature was lowered to below 25°C, and the induction culture was continued under neutral conditions for more than 20 h. Finally, the bacterial liquid was centrifuged at 8000×g for 10 min to collect the bacteria, and the recombinant strain mud was obtained after removing the supernatant. The components of the seed culture medium are: yeast powder 2.5 g / L, peptone 5 g / L, NaCl 5 g / L, brain heart infusion 37 g / L; the components of the fermentation medium are: glucose 100 g / L, corn steep liquor 20 g / L, molasses 20 g / L, ammonium sulfate 10 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, sodium citrate 1 g / L, calcium carbonate 5 g / L, biotin 0.1 mg / L, and the culture medium was adjusted to pH 7.0.

[0028] In this example, 50 mL of Tris-HCl buffer system was used, 30 g / L of whole-cell bacterial sludge prepared in the above example was added, 250 mM formaldehyde, 250 mM glycine, and 0.4-0.6 mM PLP were added, and the reaction solution was placed at 30-40 o C. The reaction was carried out at a rotation speed of 200-400 rpm. After the reaction was completed, the L-serine content in the reaction system was determined by HPLC.

[0029] The test results are shown in the table below. Compared with the wild-type enzyme, the two threonine aldolase mutants showed better and improved catalytic performance. Under high concentration aldehyde substrate conditions, they can efficiently catalyze the condensation of formaldehyde and glycine to produce L-serine. In particular, the T140A / I118V combined mutant has a higher efficiency in catalyzing formaldehyde to produce L-serine, with an L-serine yield of 26.06 g / L and a catalytic conversion rate of 99%, which has good industrial application prospects.

[0030] Table 1. Analysis of L-serine production results based on threonine aldolase

[0031] The above contents are only preferred embodiments of the present invention, which are intended to illustrate the technical solutions of the present invention, rather than to limit the protection scope of the present invention. Without departing from the core technical concept of the present invention, those skilled in the art may, based on the specification and claims of the present invention, make appropriate adjustments, changes or equivalent substitutions thereto, and all technical improvements, equivalent solutions and variations within the scope defined by the claims of the present invention shall be deemed to be the protection scope of the present invention. In addition, technical contents not specifically described in the present invention, if they belong to conventional technical means of those skilled in the art, shall also be deemed to be part of the present invention.

Claims

1. A threonine aldolase mutant derived from Aeromonas, characterized in that: Compared with the wild-type amino acid sequence shown in SEQ ID No. 1, there is only a mutation from threonine T to alanine A at position 141, or there is only a combined mutation from threonine T to alanine A at position 141 and from isoleucine I to valine V at position 119.

2. The gene encoding the threonine aldolase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is obtained by performing mutation on the basis of the nucleotide sequence shown in SEQ ID No.

2.

4. An expression vector containing the coding gene as claimed in claim 2.

5. The expression vector according to claim 4, characterized in that It is a prokaryotic expression vector.

6. The expression vector according to claim 4, characterized in that They are the E. coli expression vector pET21b and the Corynebacterium glutamicum expression vector pXMJ19.

7. A recombinant bacterium containing the expression vector according to any one of claims 4 to 6.

8. The recombinant bacterium according to claim 7, characterized in that The invention is a recombinant Escherichia coli engineering bacterium or a recombinant Corynebacterium glutamicum engineering bacterium.

9. Use of the threonine aldolase mutant according to claim 1, the coding gene according to claim 2 or 3, or the expression vector containing any one of claims 4 to 6, or the recombinant bacterium according to claim 7 or 8 in catalyzing the synthesis of L-serine or its derivatives.

10. The use according to claim 9, characterized in that Using formaldehyde and glycine as substrates, it catalyzes the production of L-serine.

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