A 6'-sialyltransferase mutant and uses thereof

Abstract

This invention belongs to the field of genetic engineering, specifically relating to a 6'-sialyltransferase mutant and its applications. The 6'-sialyltransferase mutant described in this invention is derived from a mutation of the 6'-sialyltransferase Pd2,6ST gene of *Bacillus damselae*. Compared to the wild-type 6'-sialyltransferase, the 6'-sialyltransferase mutant constructed in this invention exhibits higher substrate CTP tolerance, making it more suitable for the production of sialyl lactose and also adaptable to ectopic regeneration systems to achieve CTP regeneration.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a 6′-sialyltransferase mutant and its applications. Background Technology

[0002] In the production of sialyl lactose, the use of high concentrations of the substrate 5'-cytidine triphosphate disodium salt (CTP) significantly inhibits the catalytic efficiency of the 6′-sialyl transferase Pd2,6ST (GenBank: AB012285.1). When the substrate concentration reaches 800 mM, the enzyme's conversion rate decreases to 77% of its original value. Therefore, to further meet the needs of large-scale industrial production, it is necessary to find a 6′-sialyl transferase that is tolerant to high concentrations of CTP.

[0003] The application of semi-rational design methods to modify 6′-sialyltransferases is increasingly common. Based on the crystal structure or homology model of the 6′-sialyltransferase protein, potential key amino acid residue sites are identified, and site-directed mutagenesis is performed to alter the enzyme's activity and stability. Li Ding et al., based on the protein crystal structure of 6′-sialyltransferase from *Cyclophorus melanogaster* sp. JT-ISH-224, obtained mutants by altering residues near the receptor substrate binding pocket. The mutant A366G showed increased enzyme activity of 21-115% and more than doubled expression levels compared to the wild-type enzyme (LiDing, et al. *CARBOHYD RES*, 2015, 408:127-133). This demonstrates the effectiveness of this method in modifying 6′-sialyltransferases. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by constructing a modified 6′-sialic acid transferase mutant with high concentration of substrate CTP tolerance based on the Pd2-6ST gene of damselae.

[0005] Another technical problem to be solved by this invention is the application of the above-mentioned 6′-sialyltransferase mutant in the production of sialyl lactose and in the CTP ectopic regeneration system.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A 6′-sialyltransferase mutant, wherein the mutant is derived from the wild-type 6′-sialyltransferase with sequence SEQ ID NO:1.

[0008] The mutation described here is to mutate leucine at position 447 into alanine.

[0009] The amino acid sequence of the 6′-sialyltransferase mutant is shown in SEQ ID NO:5.

[0010] The nucleotide sequence encoding the above-mentioned 6′-sialyltransferase mutant is shown in SEQ ID NO:9.

[0011] A recombinant vector containing the nucleotide sequence of the above-mentioned 6′-sialyltransferase mutant.

[0012] A host cell containing the nucleotide sequence of the above-mentioned recombinant vector or the above-mentioned 6′-sialyltransferase mutant.

[0013] The method for constructing the above-mentioned 6′-sialyltransferase mutant includes the following steps:

[0014] (1) Construct a recombinant plasmid containing the encoding gene of the 6′-sialyltransferase, wherein the recombinant plasmid is used as a host of Escherichia coli;

[0015] (2) Using the recombinant plasmid constructed in step (1) as a template, and using the oligonucleotide sequence with the mutation site as a primer pair, a PCR product containing the base sequence shown in SEQ ID NO:9 was obtained by circular PCR amplification. Then, the PCR product was digested with DpnI enzyme to obtain a circular recombinant plasmid.

[0016] (3) The circular recombinant plasmid obtained in step (2) is transformed into the host cell BL21(DE3) competent cells to obtain genetically engineered bacteria containing 6′-sialic acid transferase mutant.

[0017] In step (1), the recombinant plasmid is pET-28a-Pd2,6ST.

[0018] In step (2), the circular recombinant plasmid is pET-28a-Pd2,6ST-L447A.

[0019] The application of the above-mentioned 6′-sialyltransferase mutant in the production of sialyl lactose.

[0020] The results showed that although the conversion efficiency of 6′-sialyltransferase decreased significantly as the final concentration of substrate CTP increased from 40 mM to 80 mM, the conversion rates of the 6′-sialyltransferase mutant M4 were 18.03% and 26.40% higher than those of the wild-type 6′-sialyltransferase WT, respectively, at the same substrate concentration (40 mM and 80 mM). This indicates that the modified 6′-sialyltransferase exhibits better substrate tolerance.

[0021] Application of the above-mentioned 6′-sialyltransferase mutant in the CTP ectopic regeneration system.

[0022] The results showed that the 6′-sialyltransferase mutant could adapt to the ectopic regeneration system, and the reaction could generate about 40 g / L of sialyl lactose in a 2 L system after 4-5 h.

[0023] Beneficial effects: Compared with the prior art, the 6′-sialyltransferase mutant of the present invention has higher substrate tolerance than the wild-type 6′-sialyltransferase, which can be better applied to the preparation of sialyl lactose and can also be adapted to the ectopic regeneration system to realize CTP regeneration. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 The image shows the recombinant plasmid pET-28a-Pd2,6ST-E428Y.

[0026] Figure 2 Map of recombinant plasmid pET-28a-Pd2,6ST-A444E

[0027] Figure 3 Image of recombinant plasmid pET-28a-Pd2,6ST-S445G

[0028] Figure 4 Map of recombinant plasmid pET-28a-Pd2,6ST-L447A

[0029] Figure 5 PCR validation of transformants of wild-type 6′-sialyltransferase and its mutants, where M: 5000bp Marker; 1: transformant of wild-type 6′-sialyltransferase; 2: transformant of E428Y; 3: transformant of A444E; 4: transformant of S445G; 5: transformant of L447A.

[0030] Figure 6 This is a flowchart of the CTP ex-situ regeneration system. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0032] In the following examples, the DpnI enzyme was purchased from Takara; the pET-28a vector and Escherichia coli host BL21(DE3) were purchased from Novagen; and the 5'-cytidine triphosphate disodium was purchased from Yuanye Biotechnology.

[0033] Example 16′-Modification of sialyl transferase mutants M1-M4.

[0034] The wild-type 6′-sialyltransferase gene was synthesized by General Biotech after codon optimization of the nucleotide sequence of the Photobacterium damselae gene (GenBank: AB012285.1), and its nucleotide sequence is shown in SEQ ID NO:1.

[0035] The crystal structure of 6′-sialyltransferase (PDB:2Z4T) was searched in the PDB protein database. Using AutoDock software, it was molecularly docked with the substrate cytosine triphosphate (CTP). Based on intermolecular interactions and hydrogen bonds, potential key sites were identified around the acceptor-substrate binding pocket, and site-directed mutagenesis was performed at positions 428, 444, 445, or 447. Specifically, amino acid E at position 428 was modified to amino acid Y (E428Y), amino acid A at position 444 was modified to amino acid E (A444E), amino acid S at position 445 was modified to amino acid G (S445G), or amino acid L at position 447 was modified to amino acid A (L447A). This yielded a 6′-sialyltransferase with the following four amino acid sequences:

[0036] The amino acid sequence of 6′-sialyltransferase mutant M1 is shown in SEQ ID NO:2; the amino acid sequence of 6′-sialyltransferase mutant M2 is shown in SEQ ID NO:3; the amino acid sequence of 6′-sialyltransferase mutant M3 is shown in SEQ ID NO:4; and the amino acid sequence of 6′-sialyltransferase mutant M4 is shown in SEQ ID NO:5.

[0037] The corresponding base sequences were artificially synthesized according to the amino acid sequences of the 6′-sialyltransferase mutants described above. Specifically, the nucleotide sequence encoding SEQ ID NO:2 is shown in SEQ ID NO:6; the nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:7; the nucleotide sequence encoding SEQ ID NO:4 is shown in SEQ ID NO:8; and the nucleotide sequence encoding SEQ ID NO:5 is shown in SEQ ID NO:9.

[0038] The preparation of 6′-sialyltransferase mutants M1-M4 was carried out as follows:

[0039] (1) Construction of recombinant plasmid pET-28a-Pd2,6ST: First, based on the original amino acid sequence (GenBank: AB012285.1), the gene was synthesized by codon optimization using E. coli as the expression host, and the optimized 6′-sialyltransferase gene (nucleotide sequence as shown in SEQ ID NO:1) was obtained. The above 6′-sialyltransferase gene and pET-28a vector were double digested with EcoRI and XhoI enzymes, respectively, and then the fragments were recovered. The recovered target gene fragment was ligated with pET-28a vector using T4 ligase to obtain recombinant vector pET-28a-Pd2,6ST. Recombinant vector pET-28a-Pd2,6ST was transformed into E. coli host BL21(DE3). The transformed products were sequenced to verify whether they were correct gene clones. Strains with correct sequencing were selected, and recombinant plasmid pET-28a-Pd2,6ST was extracted.

[0040] (2) Construct a recombinant vector containing the coding gene of the 6′-sialyltransferase mutant.

[0041] Using the recombinant plasmid pET-28a-Pd2,6ST constructed in step (1) as a template, and the oligonucleotide sequence with the mutation site as the primer pair (428-F, 428-R), circular PCR amplification was performed using PrimeStar HSDNA Polymerase to obtain the PCR product containing the mutated gene base sequence. Then, the template was digested with DpnI enzyme to obtain the circular recombinant plasmid pET-28a-Pd2,6ST-E428Y. Figure 1 ).

[0042] (3) Construction of genetically engineered bacteria containing 6′-sialic acid transferase mutant.

[0043] The circular recombinant plasmid obtained in step (2) was transformed into BL21(DE3) competent cells and plated on solid medium containing 50 mg / mL kanamycin resistance (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder). The medium was incubated at 37°C for 12 h. The next day, single colonies were picked for PCR verification. The correctly verified positive transformant was identified as the 6′-sialyltransferase mutant M1 (see Appendix). Figure 5 ).

[0044] Using recombinant pET-28a-Pd2,6ST as a template, and with oligonucleotide sequences containing mutation sites as primer pairs (444-F, 444-R; 445-F, 445-R; 447-F, 447-R), the above steps were repeated to construct circular recombinant plasmids pET-28a-Pd2,6ST-A444E, pET-28a-Pd2,6ST-S445G, and pET-28a-Pd2,6ST-L447A. Figure 2-4 Finally, 6′-sialyltransferase mutants M2, M3, and M4 were obtained (see Appendix). Figure 5 ).

[0045] The primers used for the aforementioned point mutations are shown in Table 1.

[0046] PCR reaction program: pre-denaturation 95℃ / 5min, (denaturation 95℃ / 5min, annealing 60℃ / 30s, annealing 72℃ / 7min) 30 cycles, and final extension 72℃ / 10min.

[0047] PCR system: ddH2O 32.5ul, 5×Buffer 10ul, dNTP 4ul, template 1ul, XXX-F 1ul (XXX is the mutation site), XXX-R 1ul, Primerstar 1ul.

[0048] Table 1 Primers used for point mutations

[0049]

[0050]

[0051] Example 2: Expression and enzyme activity assay of *E. coli* containing a 6′-sialyltransferase mutant expression vector.

[0052] (1) Expression of Escherichia coli containing the 6′-sialyltransferase mutant expression vector: Escherichia coli containing the mutant expression vector were cultured in TB medium (1.2% tryptone, 2.4% yeast extract, 0.5% sodium chloride) until OD 600 When the concentration of the bacterial cell count is 0.6-0.8, 100 mM IPTG is added to induce protein expression. After culturing for 18-20 hours, the bacterial cells are collected for use in the catalytic reaction.

[0053] (2) Enzyme activity assay: A 50 mL enzyme activity assay system contained 400 mM or 800 mM 5'-cytidine triphosphate disodium salt, 400 mM sialic acid, 400 mM lactose, 400 mM MgCl2·6H2O, 100 g / L wild-type CMP-6′-sialyltransferase, and 100 g / L wild-type 6′-sialyltransferase WT or 6′-sialyltransferase mutants M1-M4 cell permeate. The reaction mixture was stirred at 32 °C and 750 rpm for 1 h, and 5 mol / L alkaline solution was added to maintain the pH of the reaction at 7.5. The enzyme activity of wild-type 6′-sialyltransferase and its mutants was then detected. (Catalytic activity is defined as the amount of sialyl lactose produced per gram of enzyme-containing wet cells within 1 minute.)

[0054] Table 2. Differences in the activity of wild-type 6′-sialic acid transferase and its mutants at different concentrations of 5′-cytidine triphosphate disodium.

[0055]

[0056] The experimental results are shown in Table 2. The results indicate that, under the same concentration conditions, compared with wild-type 6′-sialyltransferase (WT), the activities of 6′-sialyltransferase mutants M1 and M2 were decreased, the activity of M3 showed no significant change, and the activity of M4 was significantly increased. Under different concentration conditions, the decrease in activity of M4 was relatively less compared with WT. Therefore, the catalytic activity of the 6′-sialyltransferase mutant M4 is better.

[0057] (3) Sialyllactose inversion reaction: The permeation solutions of the 6′-sialyltransferase mutants M1-M4 prepared in Example 1 and the permeation solutions of the synthesized wild-type 6′-sialyltransferase WT cells were diluted to the same OD value (OD). 600 =0.6), prepare a standard reaction mixture (1 ml): 100 μL 800 mM 5'-cytidine triphosphate disodium salt, 100 μL 400 mM sialic acid, 100 μL 400 mM lactose, 12.5 μL 400 mM MgCl2·6H2O, 587.5 μL 100 mM Tris-HCl (pH = 8.0). Stir the reaction mixture and add NaOH to adjust the pH to 8.0. Then add 50 μL 100 g / L CMP-sialic acid synthase and 50 μL of the above cell permeation solution. React at 37 °C and 180 rpm for 3 h on a shaker. The difference in CTP conversion between the modified enzyme and the wild-type was evaluated under final CTP concentrations of 40 mM and 80 mM. The results are shown in Tables 2 and 3. (Conversion rate is defined as the ratio of the actual substrate concentration consumed after 3 h of reaction under the same conditions to the initial substrate concentration.)

[0058] The permeation solution of 6′-sialyltransferase cells and its mutants M1-M4 was obtained by resuspending the cells in 750 μL of pH 8.0 Tris-HCl, adding 2‰ Triton X 100, and stirring at 200 rpm at 37 °C.

[0059] Table 3. Conversion rates of WT and mutants at a final CTP concentration of 40 mM.

[0060]

[0061] Table 4. Conversion rates of WT and mutants at a final CTP concentration of 80 mM.

[0062]

[0063] The results showed that although the conversion efficiency of 6′-sialyltransferase decreased significantly as the final concentration of substrate CTP increased from 40 mM to 80 mM, the conversion rates of the 6′-sialyltransferase mutant M4 were 18.03% and 26.40% higher than those of the wild-type 6′-sialyltransferase WT, respectively, at the same substrate concentration (40 mM and 80 mM). This indicates that the modified 6′-sialyltransferase exhibits better substrate tolerance.

[0064] Example 3: CTP ex-situ regeneration system

[0065] The CTP ectopic regeneration system refers to the process where high concentrations of CMP are converted into CTP under the action of CMP kinase and acetate kinase. Then, CTP reacts with sialic acid and lactose under the action of CMP-sialic acid synthase to produce sialyllactose and CMP. CMP then enters the reaction cycle as a substrate for the previous step, thus achieving CTP regeneration. The flowchart is shown below. Figure 6 .

[0066] The 6′-sialyltransferase mutant M4 obtained in Example 2 is compatible with the ectopic regeneration system. The specific reaction system is as follows:

[0067] (1) First step reaction: The system contained 25.84 g / L 5'-cytidine triphosphate disodium, 1.01 g / L MgCl2, 33.42 g / L polyphosphate, 5 g / L CMP kinase (calculated by wet bacterial weight, the same below), and 10 g / L polyphosphate kinase. After adding water to make up to 2 L, the reaction was carried out at 37℃, stirring speed of 200 rpm, and pH 7.0 for 2 h. The reaction was then stopped, and the pH was adjusted to 8.0 to proceed to the next step reaction.

[0068] (2) Second step reaction: Add 28.8 g / L lactose, 24.72 g / L sialic acid, 5 g / L LMP-sialic acid synthase, and 5 g / L 6′-sialic acid transferase M4 to the previous step reaction system. After reacting at 37℃, stirring speed of 200 rpm, and pH 7.0 for 2 hours, the reaction is terminated.

[0069] The reaction can eventually produce about 40 g / L of sialic acid lactose in a 2 L system after 4-5 h.

[0070] This invention provides a 6′-sialyltransferase mutant and its application, along with a method and approach. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A 6'-sialyltransferase mutant, characterized in that, The mutant is derived from the wild-type 6'-sialoyltransferase with nucleotide sequence SEQ ID NO:1, wherein the mutation is to change leucine at position 447 to alanine.

2. The nucleotide encoding the 6'-sialyltransferase mutant of claim 1, characterized in that, The sequence of the nucleotides is shown in SEQ ID NO:

9.

3. A recombinant vector, characterized in that, It contains the nucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the recombinant vector of claim 3 or the nucleotide of claim 2.

5. The method for constructing host cells according to claim 4, characterized in that, Includes the following steps: (1) Construct a recombinant plasmid containing the encoding gene of the 6'-sialyl transferase, wherein the recombinant plasmid is used as a host of Escherichia coli; (2) Using the recombinant plasmid constructed in step (1) as a template, and using the oligonucleotide sequence with the mutation site as a primer pair, a PCR product containing the base sequence shown in SEQ ID NO:9 was obtained by circular PCR amplification. Then, the PCR product was digested with DpnI enzyme to obtain a circular recombinant plasmid. (3) The circular recombinant plasmid obtained in step (2) is transformed into the host cell BL21(DE3) competent cells to obtain genetically engineered bacteria containing 6'-sialic acid transferase mutant.

6. The application of the 6'-sialyltransferase mutant according to claim 1 in the production of sialyl lactose.

7. The application of the 6'-sialyltransferase mutant according to claim 1 in the CTP ectopic regeneration system.

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