A beta-ketothiolase mutant and a method for constructing the same

Abstract

The application discloses a beta-ketothiolase mutant and a construction method thereof, and belongs to the field of genetic engineering and enzyme engineering. The mutant is constructed by means of genetic engineering and enzyme engineering, the enzyme activity of the mutant is improved on the basis of not affecting the enzymatic properties of the beta-ketothiolase Tfu_0875, and the mutant Tfu_0875 L163H , Tfu_0875 E221G , Tfu_0875 N249W The ability of condensing acetyl coenzyme A is 4.13 times, 2.52 times and 3.12 times of the wild enzyme beta-ketothiolase Tfu_0875, and has important industrial application potential.

Description

Technical Field

[0001] This invention relates to a β-ketothiolase mutant and its construction method, belonging to the fields of genetic engineering and enzyme engineering. Background Technology

[0002] Adipic acid, also known as fatty acid, is an important platform compound. Currently, its most important application is in the synthesis of nylon fibers (such as nylon 6,6). Industrially, adipic acid is mainly produced by oxidizing a mixture of cyclohexanol and cyclohexanone (ketone oil, also known as KA oil) with nitric acid. However, this method suffers from problems such as a long process flow, numerous byproducts, low product yield, and severe industrial waste emissions, particularly large amounts of greenhouse gases. Therefore, there is an urgent need to find a method to replace traditional chemical synthesis of adipic acid. With the development of biotechnology, the total biosynthesis method for producing organic acids has advantages such as low production cost, simple production process, low pollution, and high product purity, making it increasingly popular.

[0003] In their previous research, the inventors' team obtained a high-yield bacterial strain capable of converting multiple carbon sources into adipic acid. Based on bioinformatics, transcriptomics, and metabolomics analysis, they discovered that the 3-oxoadiacyl-CoA pathway is the main pathway for adipic acid synthesis (e.g., ...). Figure 1 The gene sequences used in this pathway include: Tfu_0875 (β-ketothiolase), Tfu_2399 (3-hydroxyacyl-CoA dehydrogenase), Tfu_0067 (3-hydroxyadipyl-CoA dehydrogenase), Tfu_1647 (5-carboxy-2-pentenoyl-CoA reductase), and Tfu_2577 and Tfu_2576 (succinyl-CoA synthase). The introduction of these six genes into *E. coli* also resulted in the accumulation of adipic acid. Furthermore, β-ketothiolase (Tfu_0875), the initial step in the highest-yielding adipic acid synthesis pathway, can also be used in the Claisen condensation reaction. However, the catalytic ability of this enzyme is limited, so it is necessary to develop β-ketothiolases with improved enzyme activity. Summary of the Invention

[0004] This invention provides a β-ketothiolase mutant, which is based on the starting sequence by mutating leucine at position 163 to histidine, or glutamic acid at position 221 to glycine, or asparagine at position 249 to tryptophan.

[0005] In one embodiment, the starting sequence is as shown in SEQ ID NO.1.

[0006] In one embodiment, the mutant is obtained by mutating leucine at position 163 of the β-ketothiolase shown in SEQ ID NO.1 to histidine, resulting in the mutant Tfu_0875 shown in SEQ ID NO.2. L163H .

[0007] In one embodiment, the mutant is obtained by mutating the glutamic acid at position 221 of the β-ketothiolase shown in SEQ ID NO.1 to glycine, resulting in the mutant Tfu_0875 shown in SEQ ID NO.3. E221G .

[0008] In one embodiment, the mutant is obtained by mutating asparagine at position 249 of the β-ketothiolase shown in SEQ ID NO.1 to tryptophan, resulting in the mutant Tfu_0875 shown in SEQ ID NO.2. N249W .

[0009] In one embodiment, the amino acid sequence of the mutant is shown in any one of SEQ ID NO. 2 to 4.

[0010] The present invention also provides a gene encoding the mutant.

[0011] In one embodiment, the nucleotide sequence of the gene is as shown in any one of SEQ ID NO. 6 to 8.

[0012] The present invention also provides recombinant microbial cells expressing the β-ketothiolytic enzyme mutant.

[0013] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0014] The present invention also provides a method for expressing the β-ketothiolase mutant, wherein the recombinant microbial cells are cultured in a culture medium for a period of time and the β-ketothiolase mutant is collected.

[0015] In one embodiment, the culture is carried out at 35–37°C until OD. 600 When the concentration of β-D-thiogalactopyranoside is 0.6–0.8, IPTG (isopropylthio-β-D-galactopyranoside) with a final concentration of 0.03–0.05 mM is added for induction, and fermentation is continued in a shaker at 24–26 °C for 12 h.

[0016] In one embodiment, the culture medium includes, but is not limited to, LB medium.

[0017] The present invention also provides a method for improving the activity of β-ketothiolase, which involves mutating leucine at position 163 of β-ketothiolase to histidine, or mutating glutamic acid at position 221 to glycine, or mutating asparagine at position 249 to tryptophan.

[0018] The present invention also provides the application of the β-ketothiolase mutant in the catalytic synthesis of acetylated compounds.

[0019] In one embodiment, the application includes, but is not limited to, catalyzing the production of acetyl-CoA from acetyl-CoA.

[0020] Beneficial Effects: This invention utilizes genetic engineering and enzyme engineering techniques to improve the enzyme activity of β-ketothiolase Tfu_0875 without affecting its enzymatic properties. Under appropriate culture conditions, the mutant Tfu_0875... L163H Tfu_0875 E221G Tfu_0875 N249W The ability to condense acetyl-CoA is 4.13 times, 2.52 times, and 3.12 times that of the wild-type enzyme β-ketothiolysis enzyme Tfu_0875, respectively. Attached Figure Description

[0021] Figure 1 The enzyme activity of the mutant relative to the wild-type β-ketothiolase is shown. Detailed Implementation

[0022] Enzyme activity assay: Ellman reagent (5,5'-dithiobis-(2-nitrobenzoic acid)) (DTNB) binds to the -SH group of free CoA to form a yellow compound, which causes an increase in absorbance at 412 nm.

[0023] Enzyme activity assay steps:

[0024] Preheating: The reaction system contains 10 μL of 50 mM Tris-HCl buffer (pH 7.4), 5 μL of 40 mM KCl, 20 μL of 1 mg / mL acetyl-CoA and 25 μL of 10 mg / mL purified β-ketothiolytic enzyme Tfu_0875, and is preheated in a 37°C water bath for 10 min.

[0025] Reaction: Mix the reaction solution and enzyme solution and shake well. Add 0.05 mM DTNB dissolved in 50 mM disodium hydrogen phosphate (pH 7.0) to the mixture and monitor the absorbance change. Terminate the reaction after 30 min.

[0026] Measurement: The above mixture was aspirated into an ELISA plate and the absorbance was measured at a wavelength of 412 nm to calculate the enzyme activity.

[0027] Enzyme activity is defined as the amount of enzyme required per minute for β-ketothiolase Tfu_0875 to catalyze Claysen condensation to produce 1 μmol of the product (acetyl-CoA).

[0028] Example 1: Preparation of β-ketothiolytic enzyme (Tfu_0875)

[0029] (1) Construction of recombinant β-ketothiolase Tfu_0875

[0030] Codon optimization was performed based on the Tfu_0875 base sequence (GenBank ID: MH157180.1) from GenBank, and the gene sequence Tfu_0875 of β-ketothiolase, as shown in SEQ ID NO. 5, was synthesized using a total chemical synthesis method. The plasmid pET28a(+) was used to construct the *E. coli* expression vector. The pET28a(+) plasmid and the plasmid containing the Tfu_0875 gene were double-digested with NcoI and XhoI, respectively. After gel recovery, the digestion products were ligated overnight using T4 ligase. The ligation product was transformed into *E. coli* JM109 competent cells. The transformation product was plated on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C. Three single colonies were picked from the plates and inoculated into LB liquid medium. After 8 hours, plasmid extraction was performed for verification, and the results were correct, yielding the enriched Tfu_0875 / pET28a plasmid. The plasmid Tfu_0875 / pET28a was transformed into Escherichia coli BL21(DE3) competent cells. Transformants were picked and cultured overnight at 37°C in LB liquid medium (containing 50 mg / L kanamycin). The glycerol tubes were then stored and named Tfu_0875 / pET28a / BL21(DE3).

[0031] (2) Expression of β-ketothiolase Tfu_0875

[0032] Tfu_0875 / pET28a / BL21(DE3) was inoculated into LB liquid medium (containing 50 mg / L kanamycin) via glycerol tubes and grown for 8 h. Seed culture was then inoculated into LB liquid fermentation medium (containing 50 mg / L kanamycin) at a 2% inoculation rate. *E. coli* was cultured at 37°C for 2 h until OD 600 = 0.6–0.8. Induction was initiated with 0.04 mM IPTG (isopropyl thio-β-D-galactopyranoside) and fermented on a shaker at 25°C for 12 h. A certain volume of fermentation broth was then incubated at 4°C and 10,000 rpm. -1 Centrifuge for 10 min, discard the supernatant, collect the bacterial cells, and use 50 mmol·L⁻¹ solution to collect the bacterial precipitate. -1 Resuspend the bacterial suspension in pH 8.5 Na₂HPO₄-NaH₂PO₄ buffer and mix well. Disrupt the cell walls of the bacterial suspension using an ultrasonic cell disruptor (operating conditions of the ultrasonic cell disruptor: Working probe, working time 5 minutes, 3 seconds working and 3 seconds stopping, working power 20%, then 10000 r·min -1 Centrifuge for 10 min, and the supernatant after centrifugation is the crude enzyme solution for fermentation. The crude enzyme solution was measured, and the results showed that the expression level of β-ketothiolase in the fermentation broth with an OD of 1 was 4 μg / mL.

[0033] (3) Purification of β-ketothiolase Tfu_0875

[0034] After the crude enzyme solution was filtered through a 0.45 μm filter membrane, it was subjected to Ni-NAT affinity chromatography and molecular sieve to obtain a large amount of high-purity, highly homogeneous soluble protein.

[0035] Ni-NAT affinity chromatography: The Ni-NTA affinity chromatography column was pre-equilibrated with 5 column volumes of Buffer A (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 20 mM imidazole). The lysate was then passed through the column packing material at a flow rate of 1 mL / min to ensure tight binding of the target protein to the packing material. Linear elution was performed at a flow rate of 1 mL / mL, repeated until the eluent reached OD. 280 No further changes. Collect the elution fraction from Buffer B (50mM Tris-HCl, pH 7.4, 200mM NaCl, 200mM imidazole).

[0036] Molecular sieving: The protein solution containing the target band was concentrated using protein concentration tubes. After concentration, the sample was purified a second time using a gel column equilibrated with Buffer C (20mM Tris-HCl, pH 7.4, 200mM NaCl). The target peak was collected and its purity was verified using SDS-PAGE.

[0037] Example 2: Preparation and expression of the β-ketothiolase Tfu_0875 mutant

[0038] Three mutant enzymes of the β-ketothiolase Tfu_0875 from *Thermobifida fusca*, namely L163H, E221G, and N249W, were identified. Based on the gene sequence of *Thermobifida fusca* β-ketothiolase Tfu_0875, primers for introducing the L163H, E221G, and N249W mutations were designed and synthesized. Site-directed mutagenesis was performed on the *Tfu_0875* gene, and the DNA coding sequence was determined. Specifically, the Leu codon at position 163 was changed to a His codon, the Glu codon at position 221 was changed to a Gly codon, and the Asn codon at position 249 was changed to a Trp codon. The mutant gene was placed in an appropriate expression vector and expressed in *E. coli* to obtain the single-mutant β-ketothiolase Tfu_0875. Site-directed mutagenesis of single mutations L163H, E221G, and N249W: rapid PCR was used with expression vector Tfu_0875 / pET28a(+) as template.

[0039] The site-directed mutagenesis primers for introducing the L163H mutation are:

[0040] Forward primer: 5'-ACGGTGTCT CAT GGAGAGTCCACCGAGCA-3' (underlined bases are mutant bases);

[0041] Reverse primer: 5'-CTCTCC ATG AGACACCGTCCATTGTT-3' (underlined bases are mutant bases);

[0042] The site-directed mutagenesis primers for introducing the E221G mutation are:

[0043] Forward primer: 5'-ATTCGC GGT ACGTCGGCCGAGAA-3' (underlined bases are mutant bases);

[0044] Reverse primer: 5'-GACGT ACC GCGAATGCTCTCGTCACGCT-3' (underlined bases are mutant bases);

[0045] The site-directed mutagenesis primers for introducing the N249W mutation are:

[0046] Forward primer: 5'-TCTCCTCTT TGG GACGGCGCCGCAGCATTGCTGATT-3' (underlined bases are mutant bases);

[0047] Reverse primer: 5'-CGTC CCAAAGAGGAGACGCGTTGCCTGCAGTGATCGT-3' (underlined bases are mutant bases);

[0048] Table 1. PCR system for the full plasmid Tfu_0875 / pET-28a.

[0049]

[0050]

[0051] PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles (95℃ for 10 s, 55℃ for 15 s, 72℃ for 30 s); extension at 72℃ for 5 min.

[0052] PCR products were digested with DpnI and transformed into competent E. coli JM109 cells. After being cultured overnight in LB solid medium (containing 50 mg / L kanamycin), clones were selected and cultured in LB liquid medium (containing 50 mg / L kanamycin). Plasmids were then extracted and transformed into competent E. coli BL21(DE3) cells. All mutant plasmids were correctly sequenced.

[0053] The mutant enzyme was expressed using the same method as in Example 1. The results are shown in Table 2, where the specific enzyme activity of the mutant was 2.52–4.13 times higher than that of the wild-type enzyme.

[0054] Table 2. Enzyme activity of β-ketothiolase Tfu_0875 and shake-flask OD of mutant enzymes. 600nm and enzyme activity

[0055]

[0056] Comparative example:

[0057] Following the strategy of Example 1, the difference lies in constructing mutants L163A, E221A, N249A, M120A, G147V, T222D, and F318H with different mutation sites. The recombinant bacteria expressing the mutants were cultured according to the method of Example 2, and the OD and specific enzyme activity of the fermentation broth were detected. The results showed that the specific enzyme activities of the mutants were 1.32 U / mg, 0.93 U / mg, 1.31 U / mg, 2.72 U / mg, 2.27 U / mg, 2.58 U / mg, and 2.25 U / mg, respectively.

[0058] Table 3. Enzyme activity of β-ketothiolase Tfu_0875 and shake-flask OD of mutant enzymes. 600nm and enzyme activity

[0059]

[0060]

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A β-ketothiolase mutant, characterized in that, Based on the starting sequence, leucine at position 163 is mutated to histidine, or glutamic acid at position 221 is mutated to glycine, or asparagine at position 249 is mutated to tryptophan; the starting sequence is shown in SEQ ID NO.

1.

2. The gene encoding the mutant of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence is shown in any of SEQ ID NO. 6 to 8.

4. Carrying the gene recombinant plasmid as described in claim 2 or 3.

5. Recombinant microbial cells expressing the β-ketothiolytic enzyme mutant of claim 1.

6. A recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET series plasmids as vectors, the β-ketothiolase mutant described in claim 1 was expressed.

7. A method for expressing the β-ketothiolase mutant of claim 1, characterized in that, The recombinant Escherichia coli according to claim 6 was cultured in a culture medium for a period of time, and the β-ketothiolase mutant was collected.

8. The method according to claim 7, characterized in that, The culture was carried out at 35-37℃ until OD... 600 When the concentration of the sample reaches 0.6~0.8, add IPTG at a final concentration of 0.03~0.05 mM for induction, and continue fermentation in a shaker at 24~26℃ for at least 12 hours.

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