Artificial transferase and use thereof

Abstract

This invention belongs to the field of biotechnology and relates to an artificial transferase and its applications, specifically an artificial carnitine acetyltransferase (CAT) with high expression and high solubility. Using the deep learning model LigandMPNN, 10-30% of amino acid sites were selected for optimization, resulting in a series of artificial sequences. After sequencing and wet experimental verification, multiple artificial enzymes with catalytic activity (2-30 U / mg) were finally confirmed, with significantly improved solubility and yield (30 mg / L vs 3 mg / L). The provided artificial carnitine acetyltransferase variant (SEQ ID NO:2) can achieve highly efficient soluble expression in *E. coli*, with a pure enzyme yield exceeding 30 mg / L, more than 10 times higher than the trace-soluble HCAT, and a specific activity of 30 U / mg, more than 15 times higher.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an artificial transferase and its application, specifically to a highly expressed and highly soluble artificial carnitine acetyltransferase (CAT). Background Technology

[0002] In eukaryotes and mammals, CAT maintains mitochondrial energy balance by regulating the acetyl-CoA / CoA ratio and participates in processes such as acetyl transmembrane transport and the excretion of excess acyl groups. Due to its central role in cellular metabolism, CAT has become an important drug target in the treatment of many diseases, especially those related to metabolism. Another important application of CAT is as a detection enzyme; its active site specifically binds to L-carnitine through multiple residues, allowing for the determination of L-carnitine content in various samples through specific catalytic reactions. In the national food safety standard GB5009.300—2025, implemented in September 2025, carnitine acetyltransferase was designated for the determination of L-carnitine content in infant formula and dairy products, further highlighting its significant value.

[0003] In this invention, Escherichia coli is used to express carnitine acetyltransferase (CAT) of eukaryotic origin. Compared with the traditional method of extraction from pigeon pectoral muscle tissue, the E. coli system has significant advantages: it does not rely on animal-derived materials such as pigeon pectoral muscle, thus overcoming supply limitations; the production process is significantly less expensive than animal extraction; and expression through E. coli can also reduce the use of experimental animals, which is in line with the 3R principles of animal welfare.

[0004] Human or pigeon-derived CAT expression in E. coli is difficult to achieve sufficient levels of active protein, with the main bottleneck being either no expression or predominantly inclusion body expression. Despite screening of expression vectors and hosts, the soluble expression ratio has not significantly improved. Summary of the Invention

[0005] To address the problems in recombinant expression, this invention provides a highly soluble and highly catalytically active artificial carnitine acetyltransferase (CAT) variant, overcoming the issues of eukaryotic CAT being mainly expressed as inclusion bodies in Escherichia coli, having low soluble yield, and insufficient activity.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] The first aspect of this invention discloses an artificial carnitine acetyltransferase (CAT) variant, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0008] (SEQ ID NO: 2).

[0009] A second aspect of the present invention discloses a fusion protein comprising the above-described artificial carnitine acetyltransferase variant and a protein or tag fused thereto.

[0010] A third aspect of the present invention discloses a nucleic acid molecule encoding the above-described artificial carnitine acetyltransferase variant or fusion protein.

[0011] The nucleic acid molecule has a codon-optimized nucleotide sequence to be suitable for recombinant expression in Escherichia coli or other host cells.

[0012] A fourth aspect of the present invention discloses a recombinant expression vector comprising the above-described nucleic acid molecules.

[0013] The vector includes pCold series vectors, pET series vectors, or pH6EX3 vectors. The prokaryotic expression vector is preferably the pCold II vector.

[0014] A fifth aspect of the present invention discloses a host cell comprising the above-described recombinant expression vector, or having the above-described nucleic acid molecules integrated into its genome.

[0015] The host cell is Escherichia coli (E. coli) BL21, BL21(DE3) or Rosetta strain.

[0016] The sixth aspect of this invention discloses the application of the above-mentioned artificial carnitine acetyltransferase variant or fusion protein in a high-efficiency carnitine acetyltransferase expression system, which is used to efficiently prepare highly soluble carnitine acetyltransferase. The prepared carnitine acetyltransferase can be used for the detection of L-carnitine content.

[0017] Compared with the prior art, the beneficial effects of this invention are as follows:

[0018] This invention utilizes the deep learning model LigandMPNN to optimize 10-30% of amino acid sites, obtaining a series of artificial sequences. After sequencing and wet experimental verification, multiple artificial enzymes with catalytic activity (2-30 U / mg) were finally confirmed, with significantly improved solubility and yield (30 mg / L vs 3 mg / L). The provided artificial carnitine acetyltransferase variant (SEQ ID NO:2) can achieve highly efficient soluble expression in E. coli, with a pure enzyme yield exceeding 30 mg / L, which is more than 10 times higher than the human CAT (approximately 3 mg / L) that can only be expressed in small amounts solublely, and its specific activity is more than 15 times higher. Attached Figure Description

[0019] Figure 1 The image shows the SDS-PAGE expression analysis of HCAT; lane 1 is the supernatant of hCAT after 0.1 mM IPTG, 15℃, 24 h; lane 2 is the precipitate after lysis and centrifugation; lanes 3-4 are the affinity chromatography binding and elution steps for other proteins; lane 5 is the HCAT purification result.

[0020] Figure 2 Display the WB expression analysis graph of HCAT.

[0021] Figure 3 The image shows the soluble expression ratio of HCAT before and after optimization. ID168 yielded a large amount of soluble protein compared to wild-type HCAT, with a specific activity of 8 U / mg.

[0022] Figure 4The text shows the soluble expression ratios of different PCAT (pigeon-derived) variants before and after optimization in different cell supernatants. Lane 1: Wild-type PCAT, no soluble expression; Lane 2: (PCAT optimized variant, increased soluble expression but inactivated); Lane 3: CAT1 / SEQ ID NO:2 (pigeon-derived optimized variant, specific activity 30 U / mg); Lane 4: PCAT optimized variant, increased soluble expression, specific activity 5 U / mg; Lane 5: CAT2 / SEQ ID NO:3 (PCAT optimized variant, specific activity 15 U / mg). Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods. Experimental methods without specific experimental conditions are generally performed according to conventional experimental conditions or the experimental conditions quarantined by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0024] Example 1: CAT Sequence Redesign and Candidate Variant Screening Based on LigandMPNN

[0025] To improve the soluble expression level of CAT, the protein sequence of CAT was redesigned based on the deep learning model LigandMPNN; this involves a bioinformatics method for analyzing conserved residues in enzymes, which is particularly suitable for identifying key functional sites through multiple sequence alignment and format processing.

[0026] Based on the known amino acid sequence of CAT, as shown in SEQ ID NO: 1:

[0027] (SEQ ID NO: 1).

[0028] Based on the above methods, amino acid sites with a conservation rate of at least 70% (amino acid numbers are as follows) were analyzed and fixed, especially key residues located in the active site, substrate binding site, or core functional domain, to ensure the conservation of protein catalytic function and structural framework. With these key conserved sites fixed, the protein sequence was intelligently redesigned to generate 200 new sequences with potential optimization properties.

[0029] All generated new sequences were evaluated and screened. Based on sequence representativeness, differential expression, and solubility, several candidate sequences with both diversity and optimization potential were selected. These candidate sequences were cloned into the expression vector pCold II and transformed into the expression host BL21(DE3) for induced expression. Protein expression and purification were assessed using SDS-PAGE analysis of the proportion of soluble protein in total soluble protein, enzyme yield, and activity assays. These techniques systematically validated the enhancement effects of the calculated, designed, and screened protein sequences on soluble expression levels and catalytic activity.

[0030] Results: Two representative candidate sequences with significant sequence differences were selected and named CAT1 and CAT2, with their amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The two representative candidate sequences, CAT1 and CAT2, were constructed into the pCold II vector and induced with 0.25 mM IPTG at 20 °C for 24 h. The culture medium was then used to prepare a crude enzyme solution, which was analyzed by SDS-PAGE.

[0031] CAT1:

[0032] (SEQ ID NO:2).

[0033] CAT2:

[0034] (SEQ ID NO:3).

[0035] Example 2: Construction of recombinant vectors for artificial carnitine acetyltransferase genes CAT1 and CAT2

[0036] 1. Using CAT1 and CAT2 plasmids as templates, the target gene fragment was amplified by PCR, recovered by agarose gel electrophoresis, and then double-digested with EcoR and Nde I. The digested products were then ligated into the double-digested pCold II linearized vector to construct a recombinant expression vector.

[0037] The ligation product was then transformed into E. coil DH5α competent cells, and positive clones were screened by colony PCR and sequenced to verify the correctness of the sequence. Finally, the verified recombinant plasmid was transformed into E. coil BL21(DE3) competent cells for subsequent protein expression and purification experiments.

[0038] 2. Preparation, purification, and SDS-PAGE of crude enzyme solution

[0039] Preparation of crude enzyme solution: Centrifuge the culture medium after induction at 20℃ to collect the bacterial cells, and wash twice with 0.85% physiological saline. After washing, remove excess water, resuspend approximately 0.35g of bacterial cells in 10mL of 0.1M pH 8.0 Tris-HCl buffer, sonicate (20W power, 20min, probe diameter φ6mm), centrifuge at 4℃, and collect the supernatant as crude enzyme solution. Add 2.5mL of glycerol to the crude enzyme solution, mix well, and store at -20℃ for later use.

[0040] Purification of the target protein: Affinity chromatography using Ni-NTA resin was performed. The crude enzyme solution was added to a nickel column pre-equilibrated with 10 mM imidazole solution. The target protein was captured by the specific binding affinity between its histone tag and nickel ions on the nickel column. During purification, a gradient elution with imidazole solution was used to separate contaminating proteins from the target protein: contaminating proteins were eluted with 20 mM imidazole solution, while the target protein was specifically eluted with 200 mM imidazole solution, thus achieving efficient separation and purification. After purification, the nickel column was thoroughly eluted with 400 mM imidazole solution to remove residual protein. The Ni-NTA packing material was then stored in 20% ethanol to maintain its performance.

[0041] Desalting and Concentration of the Target Protein: The purified enzyme solution was desalted and concentrated using a 30kD ultrafiltration tube. Ultrafiltration removes small molecule impurities such as imidazole and increases protein concentration, ultimately yielding a high-purity target protein solution. The enzyme solution treated with affinity chromatography and a 30kD ultrafiltration tube will be referred to as pure enzyme.

[0042] Example 3: Enzyme Activity Assay and Calculation

[0043] Specific activity assay: In a 1.5 mL detection system, 1420 μL of Tris-HCl buffer (pH 8.0), 30 μL of 2.5 M L-carnitine, 30 μL of 10 mM DTNB, and 15 μL of 50 mM Acetyl-CoA were added sequentially to a quartz cuvette. After reacting at 25 °C for 5 min, the cuvette was placed in a UV spectrophotometer and zeroed. Then, 5 μL of CAT1 and CAT2 were added, and the kinetics were quickly measured at 412 nm. The specific activity was calculated based on the change in absorbance within 2 min. The enzyme activity assay system is shown in Table 3.

[0044] Table 3 Enzyme Activity Assay System

[0045] The formulas for calculating enzyme activity and specific activity are as follows:

[0046]

[0047]

[0048] in The change in absorbance over 2 minutes is represented by V; V is the total volume of the reaction system, i.e., 1.5 mL; 14150 is the extinction coefficient of DTNB at 412 nm (M). -1 cm -1 ); 1 is the optical path distance (cm); 2 is the reaction time (min); 5 is the volume of protein added (μL); 1000 is the conversion of μL to mL; U is μmol / min / mg; mg is the unit of protein content in the reaction system; mg / mL is the unit of protein concentration in the reaction system.

[0049] Example 4: Determination of soluble expression yield

[0050] Human CAT (HCAT) showed only a small amount of soluble expression before optimization, as shown in the following results. Figure 1 As shown, Western blot (WB) expression analysis was performed. At this point, the pure enzyme yield was 3 mg / L, and the specific activity was 2.2 U / mg. The results are as follows. Figure 2 As shown. Using the sequence optimization method of ligandmpnn, solubility optimization was performed based on the HCAT sequence, which significantly improved the soluble expression ratio of ID168 and increased the specific activity of the enzyme to 8 U / mg.

[0051] The same method was used to optimize the pigeon CAT (PCAT) sequence, and the resulting CAT1 and CAT2 were determined by SDS-PAGE. The results are as follows: Figure 4 As shown, the solubility of the five proteins expressed was significantly improved compared with the wild-type PCAT expressed by recombinant expression, and the pure enzyme yield was >30 mg / L. The catalytic activities of lanes 3 and 5 were 30 U / mg and 15 U / mg, respectively, corresponding to CAT1 (SEQ ID NO: 2) and CAT2 (SEQ ID NO: 3).

[0052] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. A variant of artificial carnitine acetyltransferase (CAT), characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. A fusion protein, characterized in that, It comprises the artificial carnitine acetyltransferase variant of claim 1 and a protein or tag fused thereto.

3. A nucleic acid molecule encoding the artificial carnitine acetyltransferase variant of claim 1 or the fusion protein of claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that, Its nucleotide sequence has been codon-optimized to be suitable for recombinant expression in Escherichia coli or other host cells.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that, The vectors include pCold series vectors, pET series vectors, or pH6EX3 vectors.

7. The recombinant expression vector according to claim 6, characterized in that, The prokaryotic expression vector is the pCold II vector.

8. A host cell, characterized in that, It comprises the recombinant expression vector of claim 6 or 7, or the genome thereof having the nucleic acid molecule of claim 3 or 4 integrated therein.

9. The host cell according to claim 8, characterized in that, The host cell is Escherichia coli (E. coli) BL21, BL21(DE3) or Rosetta strain.

10. The use of the artificial carnitine acetyltransferase variant of claim 1 or the fusion protein of claim 2 in a carnitine acetyltransferase expression system.

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