An acyltransferase mutant with improved enzyme activity

Abstract

The present application relates to an acyltransferase mutant with improved enzyme activity, and belongs to the technical field of enzyme engineering. The present application mutates one or more of the 97th and 154th positions of the acyltransferase, effectively improves the conversion rate of the acyltransferase on trans-4-hydroxyproline, and the conversion rate is increased by more than 24.4% after mutation. Using the genetically engineered strain expressing the acyltransferase mutant of the present application, trans-4-hydroxyproline is used as a substrate to catalyze the synthesis of N-acetyl-trans-4-hydroxyproline, and the yield reaches more than 312.5mg / L. Among them, the genetically engineered strain expressing the mutant K97A / F154A can synthesize N-acetyl-trans-4-hydroxyproline with a yield of 433mg / L, which is increased by 72.4% compared with the yield before mutation. The present application has strong practicability, can be used for large-scale production of N-acetyl-trans-4-hydroxyproline, and has very high economic value.

Description

Technical Field

[0001] This invention relates to an acyltransferase mutant with enhanced enzyme activity, belonging to the field of enzyme engineering technology. Background Technology

[0002] N-acetyl-trans-4-hydroxyproline is an important amino acid derivative with a wide range of applications. It plays a vital role in the pharmaceutical industry, often used as an intermediate in the synthesis of drugs and antibiotics. This compound not only has important functions in drug synthesis but has also attracted considerable attention in biomedical research. Its remarkable antioxidant and anti-inflammatory activities make it a key component in disease treatment and health maintenance.

[0003] N-acetyl-trans-4-hydroxyproline and its precursor trans-4-hydroxyproline can be obtained through the chemical synthesis of collagen. In this process, trans-4-hydroxyproline is used as a substrate and acetylated under acidic conditions. However, this chemical synthesis method typically requires the use of certain toxic reagents, raising safety concerns.

[0004] In recent years, the biocatalytic synthesis of N-acetyl-trans-4-hydroxyproline has attracted widespread attention, especially through the action of enzymes, particularly acyltransferases. Acyltransferases are a class of enzymes with acyl transfer catalytic activity, capable of transferring acyl groups from substrate molecules. In the synthesis of N-acetyl-trans-4-hydroxyproline, acyltransferases play a crucial role, transferring the acetyl group to 4-hydroxyproline, thereby forming N-acetyl-trans-4-hydroxyproline.

[0005] Although research on the biocatalytic synthesis of N-acetyl-trans-4-hydroxyproline is still in its early stages, this method has great potential. It can not only improve synthesis efficiency and reduce the need for harmful reagents, but also improve the sustainability of the synthesis process, providing a promising direction for future pharmaceutical industry and medical research.

[0006] In existing technologies, the synthesis of N-acetyl-trans-4-hydroxyproline using acyltransferases still faces a series of challenges and problems. A bioenzymatic synthesis method for acetylhydroxyproline (CN113403350A) discloses a method for synthesizing N-acetyl-trans-4-hydroxyproline using acyltransferases. Although this technology utilizes known acyltransferases, it is only a starting point. Further development and optimization of the enzyme remain to be explored, including improving catalytic efficiency, enhancing specificity, increasing enzyme stability, or adapting to different reaction conditions to improve yield and reduce cost. Many opportunities and potential exist for in-depth research in the fields of enzymology and biocatalysis to further promote the production and application of N-acetyl-trans-4-hydroxyproline. Therefore, further exploring methods to improve acyltransferase activity to enhance the synthesis efficiency and quality of N-acetyl-trans-4-hydroxyproline has extremely high economic and practical value. Summary of the Invention

[0007] To further explore the potential of acyltransferases and improve their enzyme activity, this invention provides an acyltransferase mutant. Compared with the original mutant, the mutant has an increased conversion rate of trans-4-hydroxyproline, which further improves the yield of N-acetyl-trans-4-hydroxyproline.

[0008] The first objective of this invention is to provide an acyltransferase mutant obtained by mutation at one or more sites, such as position 97 and position 154, based on the acyltransferase shown in SEQ ID NO.2.

[0009] In one embodiment, the lysine at position 97 is mutated to alanine (K97A for short);

[0010] In one embodiment, the phenylalanine at position 154 is mutated to alanine (abbreviated as F154A);

[0011] In one embodiment, based on the mutation of lysine at position 97 to alanine, phenylalanine at position 154 is further mutated to alanine (abbreviated as K97A / F154A).

[0012] A second objective of this invention is to provide a gene encoding the aforementioned acyltransferase mutant.

[0013] The present invention also provides recombinant plasmids carrying the above-mentioned genes.

[0014] In one embodiment, the vector for the recombinant plasmid is a pET vector.

[0015] In one embodiment, the vector for the recombinant plasmid is pET-22b.

[0016] The present invention also provides a host cell carrying the above-mentioned gene or recombinant plasmid.

[0017] In one embodiment, the host cell is a bacterium or a fungus.

[0018] In one embodiment, the host cell is *Escherichia coli*.

[0019] In one implementation method, the host cell is Escherichia coli BL21(DE3).

[0020] A third objective of this invention is to provide the use of the above-mentioned acyltransferase mutant or the above-mentioned host cell in the synthesis of N-acetyl-trans-4-hydroxyproline.

[0021] The present invention also provides the use of the above-mentioned gene or the above-mentioned recombinant plasmid in the synthesis of N-acetyl-trans-4-hydroxyproline.

[0022] The fourth objective of this invention is to provide a method for synthesizing N-acetyl-trans-4-hydroxyproline, wherein the above-mentioned acyltransferase mutant or the above-mentioned host cell is added to the N-acetyl-trans-4-hydroxyproline synthesis system, and the system contains trans-4-hydroxyproline and an acyl donor.

[0023] In one embodiment, the acyl donor may be one or more of the following: ethyl acetate vinyl acetate, isopropyl acetate, isopropyl acetate, isopropyl acetate, isoamyl acetate, and p-nitrobenzene acetate.

[0024] In one embodiment, a surfactant is also present.

[0025] In one embodiment, the surfactant may be one or more of the following: DMSO, isopropanol, glycerol, PEG 4000, Triton X-100, Tween 80.

[0026] In one embodiment, the genetically engineered bacteria are cultured in ZYBM9 medium at 37°C and 220 rpm until OD reaches zero. 600 =0.8-1.0, add lactose to a final concentration of 400 μM, and induce culture at 20℃ and 220 rpm for 16 h. Centrifuge to obtain whole-cell catalyst.

[0027] In one embodiment, the N-acetyl-trans-4-hydroxyproline synthesis system initially contains 5–200 g / L of trans-4-hydroxyproline, 0.5–20% v / v of acyl donor, 0.5–5% v / v of surfactant, and a final wet cell concentration of 0.2–3 × 10⁻⁶ g / L. 10 CFU / mL.

[0028] In one implementation method, the whole-cell catalysis involves suspending wet bacterial cells in phosphate buffer. The reaction system contains 40 g / L trans-4-hydroxyproline, 10% (v / v) ethyl acetate, and 0.5-2% (v / v) DMSO. The final concentration of wet bacterial cells added to the reaction system is 0.2–3 × 10⁻⁶. 10 CFU / mL.

[0029] In one embodiment, the reaction is carried out at 25-35°C and 180-220 rpm.

[0030] Beneficial effects:

[0031] This invention improves the conversion rate by more than 24.4% by point mutation at different sites of acyltransferase, and increases the yield of N-acetyl-trans-4-hydroxyproline synthesized by genetically engineered strains to more than 0.3125 g / L.

[0032] In particular, the mutant K97A / F154A showed the best results. Using Escherichia coli BL21 as the host, the yield of N-acetyl-trans-4-hydroxyproline reached 0.4332 g / L in a substrate of 40 g / L trans-4-hydroxyproline, which was 72.4% higher than before the mutation. Attached Figure Description

[0033] Figure 1 Results of N-acetyl-trans-4-hydroxyproline synthesis catalyzed by acyltransferase mutant. Detailed Implementation

[0034] The technical solutions described below will be clearly and completely described with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] 1. ZYBM9 culture medium formula

[0036] Table 1 ZYBM9 culture medium

[0037]

[0038]

[0039] 2. Catalytic sample pretreatment:

[0040] After centrifuging the whole-cell catalytic sample at 12000 rpm for 10 min, the supernatant was collected. 50 μL of the supernatant was used for pre-column derivatization. The derivatization steps were as follows: 50 μL of supernatant was mixed with 50 μL of 50 mM 2-NPH and 100 μL of 25 mM EDC·HCl, and incubated in a metal bath at 80 °C for 5 min. Then, 200 μL of 1 M NaOH was added, and the mixture was incubated again in a metal bath at 80 °C for 5 min to obtain the derivatized solution. The derivatized solution was filtered through a 0.22 μm filter membrane and then used for HPLC detection.

[0041] 3. HPLC detection conditions for N-acetyl-trans-4-hydroxyproline:

[0042] Liquid chromatography was performed using an Agilent Diamond C-18 column (4.6 × 250 mm) with a flow rate of 0.5 mL / min. Mobile phase A consisted of 90% water and 10% acetonitrile (containing 25 mM pH 6 sodium phosphate buffer), and mobile phase B consisted of 40% water and 60% acetonitrile (containing 25 mM pH 6 sodium phosphate buffer). A gradient elution strategy was employed, increasing the elution from 10% B to 100% B over 20 min and decreasing it back to 10% B over 20–25 min. The column temperature was 45 °C, and the detection wavelength was 420 nm. Each sample was run for 25 min.

[0043] 4. Calculation of N-acetyl-trans-4-hydroxyproline conversion rate:

[0044] / (Actual molar amount of N-acetyl-trans-4-hydroxyproline in the system) * 100%

[0045] Example 1: Construction of acyltransferase mutants and engineered bacteria for acyltransferase expression

[0046] Based on the acyltransferase with the initial amino acid sequence shown in SEQ ID NO.2 (nucleotide sequence shown in SEQ ID NO.1), the lysine at position 97 was mutated to alanine, resulting in the acyltransferase mutant with the sequence shown in SEQ ID NO.4. According to the formula "amino acid before mutation + mutation site + amino acid after mutation," it was named K97A, and a genetically engineered strain was constructed and named BL21(DE3) / pET22b-MsAcT(K97A), as detailed below:

[0047] (1) Construct pET22b-MsAcT

[0048] Table 2 Primers

[0049]

[0050] Using the primers described above, the initial acyltransferase MsAcT sequence was ligated into the pUC19 vector (named pUC19-MsAcT plasmid) as its target gene template. Linearization amplification was performed using PCR technology. After linearization amplification, the target gene MsAcT was ligated into the pET22b vector by incubation at 50°C for 30 min using OneStep Seamless Cloning Mix homologous recombinase to obtain pET22b-MsAcT.

[0051] Table 3 pET22b amplification system

[0052]

[0053] Table 4 Reaction conditions for pET22b

[0054]

[0055] Table 5 pUC19-MsAcT amplification system

[0056]

[0057] Table 6 Reaction conditions for pUC19-MsAcT

[0058]

[0059]

[0060] Table 7 Connection System

[0061]

[0062] (2) The lysine at position 97 was mutated to alanine.

[0063] Table 8 K97A primers

[0064]

[0065] Using the primers described above, and pET22b-MsAcT as a template, site-directed mutagenesis was performed using PCR to obtain the mutated recombinant vector pET22b-MsAcT(K97A). The reaction system and conditions are as follows:

[0066] Table 9 Reaction System

[0067]

[0068] Table 10 Reaction conditions

[0069]

[0070] After site-directed mutagenesis, the template was digested by incubation with DpnI rapid digestion enzyme at 37°C for 1 hour. The enzyme digestion system is as follows:

[0071] Table 11 Enzyme digestion system

[0072]

[0073] (3) Construction of engineered strain (BL21(DE3) / pET22b-MsAcT(K97A))

[0074] BL21(DE3) competent cells that have been prepared and frozen at -80℃ were transformed with recombinant vectors.

[0075] Add 10 μL of pET22b-MsAcT(K97A) recombinant plasmid to thawed BL21(DE3) competent cells on ice, mix gently, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, followed by ice incubation for 5 min. Add 500 μL of LB medium, mix gently, and incubate at 37°C and 200 rpm for 40 min.

[0076] Centrifuge the bacterial culture, discard 500 μL of supernatant, mix well by pipetting and aspiration, and spread it on LB solid plates with ampicillin (Amp) resistance. Incubate overnight at 37°C with the plates inverted to obtain the genetically engineered strain BL21(DE3) / pET22b-MsAcT(K97A).

[0077] Based on the amino acid sequence SEQ ID NO.2, mutants N94A (amino acid sequence as shown in SEQ ID NO.3), F154A (amino acid sequence as shown in SEQ ID NO.5), and K97A / F154A (amino acid sequence as shown in SEQ ID NO.6) were obtained in the same manner. K97A / F154A involved mutations at both sites; N94A involved a mutation of asparagine at position 94 to alanine. Genetically engineered strains expressing the corresponding mutant enzymes and unmutated genetically engineered strains were constructed and named BL21(DE3) / pET22b-MsAcT(N94A), BL21(DE3) / pET22b-MsAcT(F154A), BL21(DE3) / pET22b-MsAcT(K97A / F154A), and BL21(DE3) / pET22b-MsAcT, respectively.

[0078] The introduction of mutation sites N94A and F154A is as follows:

[0079] Table 12 Primer sequences of N94A and F154A

[0080]

[0081] Example 2: Application of acyltransferase mutants in the synthesis of N-acetyl-trans-4-hydroxyproline

[0082] (1) Preparation of wet bacterial cells

[0083] The genetically engineered strain constructed in Example 1 was inoculated into 5 mL of LB medium and cultured overnight at 37°C and 220 rpm. Then, it was transferred at a 1% inoculum to 50 mL of ZYBM9 medium and cultured at 37°C and 220 rpm until OD (dose retardation). 600 When the concentration of lactose was 0.8–1.0, lactose was added to a final concentration of 400 μM, and the mixture was induced at 20 °C and 220 rpm for 16 h. After induction, all wet cells were collected by centrifugation at 4 °C and 8000 rpm for the catalytic synthesis of N-acetyl-trans-4-hydroxyproline.

[0084] (2) Catalytic synthesis of N-acetyl-trans-4-hydroxyproline

[0085] The wet bacterial cells obtained in step (1) were resuspended in 100 mM potassium phosphate buffer (pH = 7) for catalytic reaction. The reaction system included 40 g / L trans-4-hydroxyproline, 10% ethyl acetate (v / v), 1% DMSO (v / v), and 2*10 wet bacterial cells. 10 CFU / mL. The reaction was carried out at 35℃ and 220 rpm. After catalysis for 6 h, 1 mL of sample was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. 50 μL of the supernatant was used for pre-column derivatization and then for HPLC analysis.

[0086] The results are as follows Figure 1 As shown, the genetically engineered bacterium BL21(DE3) / pET22b-MsAcT(K97A / F154A) exhibited the highest conversion rate for trans-4-hydroxyproline, achieving a conversion rate of 1% within 6 hours, which was 72.4% higher than the unmutated strain, with a yield of 0.433 g / L for N-acetyl-trans-4-hydroxyproline. The genetically engineered bacterium BL21(DE3) / pET22b-MsAcT(K97A) achieved a conversion rate of 0.8% and a yield of 0.3481 g / L. The genetically engineered bacterium BL21(DE3) / pET22b-MsAcT(F154A) achieved a conversion rate of 0.72% and a yield of 0.3125 g / L.

[0087] Example 3: Synthesis of N-acetyl-trans-4-hydroxyproline using different acyl donors

[0088] (1) Culture of engineered strains

[0089] The detection was performed using the genetically engineered strain BL21(DE3) / pET22b-MsAcT with non-mutated acyltransferase, following the same procedure as step (1) in Example 2.

[0090] (2) Whole-cell catalysis in the synthesis of N-acetyl-trans-4-hydroxyproline

[0091] The effects of different acyl donors on the synthesis of N-acetyl-trans-4-hydroxyproline were investigated. 10% (v / v) of ethyl acetate, vinyl acetate, isopropyl acetate, isopropyl acetate, isoamyl acetate, and p-nitrobenzene acetate were added, with the remaining steps consistent with step (2) of Example 2. The yield of N-acetyl-trans-4-hydroxyproline was then determined.

[0092] Table 13 Effect of different acyl donors on catalysis

[0093]

[0094] As shown in Table 13, different acyl donors can be used for whole-cell catalysis to synthesize N-acetyl-trans-4-hydroxyproline.

[0095] The present invention relates to the following sequences:

[0096] Initial nucleotide sequence of acyltransferase MsAcT SEQ ID NO.1:

[0097] ATGGCCAAACGCATCTTATGTTTCGGAGACTGCCTGACTTGGGGATGGGTTCCCGTGGAAGACGGTGCTCCTACTGAACGCTTTGCGCCAGATGTACGCTGGACCGGCGTATTAGCTCAGCAGTTAGGCGCTGACTTTGAAGTCATTGAGGAAGGTTTGTCTGCCCGTACGACAAACATCGATGATCCCACCGACCCGCGTTTGAATGGAGCGAGCTATCTTCCCTCGTGCCTGGCAACTCACTTACCGCTGGATCTTGTTATTATCATGTTGGGCACAAACGACACCAAGGCGTATTTCCGCCGCACACCCCTTGATATTGCACTTGGGATGTCGGTGCTTGTCACACAGGTCTTGACATCCGCGGGGGGAGTAGGCACGACATATCCCGCACCGAAAGTATTAGTCGTCTCGCCTCCTCCCTTGGCACCCATGCCTCACCCCTGGTTTCAACTGATTTTTGAGGGCGGAGAACAAAAGACAACGGAACTTGCCCGTGTGTATTCAGCGCTGGCTTCGTTTATGAAAGTCCCGTTCTTTGATGCCGGTAGCGTGATCAGTACAGACGGCGTAGATGGTATCCACTTTACGGAGGCCAATAACCGTGATCTGGGGGTGGCACTGGCGGAGCAAGTTCGCTCACTTTTA

[0098] Initial amino acid sequence of acyltransferase MsAcT SEQ ID NO.2:

[0099] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGT N DTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0100] Acyltransferase mutant N94A amino acid sequence SEQ ID NO.3:

[0101] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGT A DTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0102] Acyltransferase mutant K97A amino acid sequence SEQ ID NO.4:

[0103] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDT A AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0104] Acyltransferase mutant F154A amino acid sequence SEQ ID NO. 5:

[0105] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLI A EGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0106] The amino acid sequence of the acyltransferase mutant K97A / F154A is SEQ ID NO. 6:

[0107] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDT A AYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLI A EGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0108] 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. Use of an acyltransferase mutant in synthesis of N-acetyl-trans-4-hydroxyproline. The acyltransferase mutant is based on an acyltransferase with an amino acid sequence as shown in SEQ ID NO. 2, wherein the lysine at position 97 is mutated to alanine; the phenylalanine at position 154 is mutated to alanine; or the lysine at position 97 is mutated to alanine and the phenylalanine at position 154 is mutated to alanine.

2. Use of a gene of an acyltransferase mutant or a recombinant plasmid of an acyltransferase mutant in the synthesis of N-acetyl-trans-4-hydroxyproline; wherein, The acyltransferase mutant is based on an acyltransferase with an amino acid sequence as shown in SEQ ID NO. 2, wherein the lysine at position 97 is mutated to alanine; the phenylalanine at position 154 is mutated to alanine; or the lysine at position 97 is mutated to alanine and the phenylalanine at position 154 is mutated to alanine.

3. A method of synthesizing N-acetyl-trans-4-hydroxyproline, characterized by, The acyltransferase mutant is added to a system for synthesizing N-acetyl-trans-4-hydroxyproline, which contains trans-4-hydroxyproline and an acyl donor; wherein the acyltransferase mutant is based on an acyltransferase with an amino acid sequence as shown in SEQ ID NO. 2, wherein the lysine at position 97 is mutated to alanine; the phenylalanine at position 154 is mutated to alanine; or the lysine at position 97 is mutated to alanine and the phenylalanine at position 154 is mutated to alanine.

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