Acyl-coenzyme A transferase mutant and application thereof in synthesis of 3-aminopropanol

Abstract

The invention provides an acyl coenzyme A transferase mutant and an application of the acyl coenzyme A transferase mutant in synthesis of 3-aminopropanol. The acyl coenzyme A transferase mutant comprises: 1) a protein having an amino acid sequence E249 mutated at the following sites of SEQ ID NO: 1 and having acyl coenzyme A transferase activity; and 2) protein which has an amino acid sequence with homology of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% with the protein in 1) and has acyl coenzyme A transferase activity. The acyl-coenzyme A transferase mutant can be used for synthesizing 3-aminopropanol by taking beta-alanine as a substrate, so that the vacancy that the current market is lack of a biosynthesis method for synthesizing 3-aminopropanol is made up, and the acyl-coenzyme A transferase mutant has the advantages of greenness, environmental protection, simplicity, convenience and the like, and is wide in application prospect.

Description

Technical Field

[0001] This invention relates to the field of 3-aminopropanol production, and more specifically, to an acyl-CoA transferase mutant and its application in the synthesis of 3-aminopropanol. Background Technology

[0002] Panthenol, commonly known as provitamin B5, is a biologically active D-panthenol that is a precursor to the synthesis of D-pantothenic acid-coenzyme A in organisms. It promotes the metabolism of proteins, fats, and carbohydrates in the body, protects the mucous membranes and luster of the skin surface, and prevents the occurrence of diseases. It is widely used in the pharmaceutical, food, and cosmetic industries.

[0003] The traditional method for preparing panthenol involves adding purified D-pantothenic acid lactone and 3-aminopropanol to a reactor to obtain the D-panthenol product. 3-Aminopropanol is a crucial raw material for panthenol preparation, and its preparation process is vital for its industrial production. Patent CN111196761B discloses a chemical synthesis process for preparing 3-aminopropanol. Under ammonia-containing conditions, a raw material containing 3-hydroxypropionitrile is catalytically hydrogenated. The resulting product is then separated and purified to obtain 3-aminopropanol, as well as recycled liquid ammonia and / or 3-hydroxypropionitrile. This method allows for the recycling of the reaction medium, ammonia, but the process is complex and the raw materials are expensive. Patent application CN113754548A discloses a substitution reaction of 1,3-propanediol and 35% hydrochloric acid under N,N-dimethylformamide catalysis, followed by extraction and distillation to obtain 3-chloropropanol from the resulting reaction solution. Then, in a closed system, the 3-chloropropanol is reacted with ammonia, and the resulting reaction solution is extracted and distilled to obtain 3-aminopropanol. However, this method requires harsh reaction conditions and is costly. Other methods, such as the acrylonitrile method, the 1,4-butyrolactone method, and the hydroxypropionitrile method, all employ chemical methods to prepare 3-aminopropanol. These processes generally suffer from a series of drawbacks, including hazardous raw materials, low yield, harsh reaction conditions, high cost, high toxicity, significant environmental harm, expensive catalysts, and complex processes. Furthermore, the produced 3-aminopropanol has a strong inherent odor, which cannot fully meet consumer demand in the cosmetics and pharmaceutical industries.

[0004] Generally, organic alcohols can be derived from their corresponding organic acids via biotransformation. Fillet et al. (World J Microbiol Biotechnol (2016) 32:152) reviewed the research progress in microbial production of fatty alcohols, concluding that the synthesis of fatty alcohols in organisms originates from the fatty acid synthesis pathway. The production of fatty alcohols from fatty acids can be summarized into two routes: 1) Coenzyme A reduction route: Fatty acids are activated by specific coenzyme A ligases to obtain the corresponding fatty acyl-CoA, which is then catalyzed by fatty acyl-CoA reductase to aldehydes or alcohols. The resulting aldehyde is further converted to the corresponding fatty alcohol by aldehyde reductase; 2) Carboxylic acid reduction route: Fatty acids are reduced to the corresponding fatty aldehydes by specific carboxylic acid reductases, which are then catalyzed by aldehyde reductase to produce the corresponding fatty alcohol. In these transformation routes, the supply of coenzyme A, cofactors, and ATP is also involved. In conclusion, developing a green and sustainable production process based on enzymatic methods that can improve the quality of 3-aminopropanol is key to upgrading the green biomanufacturing of panthenol and is also a pressing technical problem to be solved. Summary of the Invention

[0005] The main objective of this invention is to provide an acyl-CoA transferase mutant and its application in the synthesis of 3-aminopropanol, in order to solve the problem that there is no existing technology that uses biosynthesis to prepare 3-aminopropanol.

[0006] The synthetic route and mechanism of this invention are as follows: This invention uses β-alanine as a substrate, converting β-alanine into a structure containing a coenzyme A molecule via acyl-CoA transferase (ACT), and finally reducing it to 3-aminopropanol using alcohol dehydrogenase. Acyl-CoA transferase can attach a coenzyme A molecule to a carbon chain carboxylic acid in a one-step reaction without consuming ATP.

[0007] To achieve the above objectives, according to a first aspect of the present invention, an acyl-CoA transferase mutant is provided, comprising: 1) a protein having an amino acid sequence mutated at the following site in SEQ ID NO: 1: E249, and having acyl-CoA transferase activity; or 2) a protein having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the protein in 1), and having acyl-CoA transferase activity.

[0008] Furthermore, in 1) above, the mutation sites include E249D.

[0009] To achieve the above objectives, according to a second aspect of the present invention, a gene is provided that encodes the aforementioned acyl-CoA transferase mutant.

[0010] Furthermore, the above-mentioned gene has the nucleotide sequence shown in SEQ ID NO: 11.

[0011] To achieve the above objectives, according to a third aspect of the present invention, a plasmid is provided that contains the aforementioned gene.

[0012] Furthermore, the plasmid further includes the alcohol dehydrogenase gene adhE; preferably, the gene encoding the acyl-CoA transferase mutant on the plasmid has the same inducible promoter as the alcohol dehydrogenase gene adhE; preferably, the nucleotide sequence of the alcohol dehydrogenase gene adhE is SEQ ID NO: 3.

[0013] Furthermore, the plasmid mentioned above is pETduet.

[0014] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, the host cell comprising the above-described gene or the above-described plasmid.

[0015] Furthermore, the host cells mentioned above are selected from any of the following: Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

[0016] Furthermore, the host cell is selected from Escherichia coli; preferably, the Escherichia coli is BL21 cell; preferably, the Escherichia coli is BL21 cell with the endogenous alcohol dehydrogenase gene adhE knocked out.

[0017] To achieve the above objectives, according to five aspects of the present invention, a method for synthesizing 3-aminopropanol is provided, the method comprising: using a host cell capable of simultaneously expressing acyl-CoA transferase and alcohol dehydrogenase, and using β-alanine as a substrate, to perform a whole-cell catalytic reaction to synthesize the above-mentioned 3-aminopropanol; wherein the acyl-CoA transferase is selected from the above-mentioned acyl-CoA transferase mutant.

[0018] Furthermore, the catalytic system for carrying out the above-mentioned whole-cell catalytic reaction includes: a host cell capable of expressing acyl-CoA transferase and the above-mentioned alcohol dehydrogenase, the above-mentioned substrate β-alanine, acetyl-CoA and NADH, and Mg... 2+ The reaction buffer solution; preferably, the above-mentioned solution containing Mg 2+ The reaction buffer consists of Tris-HCl buffer at pH 8.0 and MgCl2.

[0019] Further, the host cell is the aforementioned host cell; preferably, the gene encoding the acyl-CoA transferase and the gene encoding the alcohol dehydrogenase are located on the same plasmid of the host cell; more preferably, the gene encoding the acyl-CoA transferase and the gene encoding the alcohol dehydrogenase have the same inducible promoter; preferably, the alcohol dehydrogenase gene is adhE, and the nucleotide sequence of the alcohol dehydrogenase gene is SEQ ID NO: 3.

[0020] Furthermore, the host cell is the host cell described above. In the above in vitro whole-cell catalytic reaction system, the final concentration of the substrate β-alanine is 1 g / L, the final concentration of the acetyl-CoA is 3-7 mM, the final concentration of NADH is 3-7 mM, the final concentration of the host cell is 1.0-1.5%, and the final concentration of MgCl2 is 15-25 mM.

[0021] The present invention comprises: 1) an amino acid sequence having a mutation at at least one of the following sites in SEQ ID NO: 1, and possessing acyl-CoA transferase activity, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology with the protein in 1), and which, under the action of an acyl-CoA transferase mutant with acyl-CoA transferase activity and an alcohol dehydrogenase, can catalyze the synthesis of 3-aminopropanol from β-alanine. This acyl-CoA transferase mutant can catalyze the synthesis of 3-aminopropanol using β-alanine as a substrate. This biosynthetic method is not only simple and economical, requiring only two enzymes and yielding the target product 3-aminopropanol in a single step, but also green and environmentally friendly, with no toxic or harmful chemicals involved in the production process. It overcomes the problems of low synthesis efficiency, poor safety performance, high pollution, and high cost of existing methods, laying the foundation for large-scale production of 3-aminopropanol and providing new ideas for further enzymatic synthesis of other chemical substances.

[0022] In some preferred embodiments, suitable mutation sites for acyl-CoA transferase are predicted and screened using bioinformatics methods, and acyl-CoA transferase mutants are constructed using homologous recombination. In other embodiments, this mutant is used to catalyze a reaction using β-alanine as a substrate to produce 3-aminopropanol. Attached Figure Description

[0023] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0024] Figure 1 The HPLC detection results of the ACT04_E249D mutant strain of Example 6 of the present invention are shown. The horizontal axis represents the retention time. A represents the ACT04_E249D mutant strain; B represents the wild-type ACT04; C represents the ACT04_H90D strain; D represents the ACT01_E244L mutant strain; E represents the ACT06_E239D mutant strain; and F represents the ACT08_E290D mutant strain. Detailed Implementation

[0025] The present invention is further illustrated by the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope or embodiments of the invention. The scope of the invention is limited by the appended claims. Based on this specification and general knowledge in the art, those skilled in the art can clearly understand the scope of the claims. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications and alterations to the technical solutions of the invention, and such modifications and alterations are also included within the scope of the invention.

[0026] As mentioned in the background section, existing technologies suffer from problems such as hazardous raw materials, low yield, harsh reaction conditions, high cost, high toxicity, significant environmental harm, expensive catalysts, and complex processes. To address these issues, this invention analyzes and studies the process from the perspective of green and environmentally friendly bio-enzyme catalysis. The research approach is as follows: β-alanine is catalytically reduced to a corresponding compound containing coenzyme A molecules under the action of acyl-CoA transferase, and then further reduced to the corresponding alcohol, namely 3-aminopropanol, under the action of alcohol dehydrogenase. The research results show that 3-aminopropanol can be efficiently biosynthesized using only two enzymes—an acyl-CoA transferase mutant and an alcohol dehydrogenase—using β-alanine as a substrate.

[0027] Previous research by Hyojung Park et al. (Biotechnology and Bioprocess Engineering 25:599-606 (2020)) revealed that acyl-CoA transferases exhibit good affinity for the natural substrate butyrate. Among them, acyl-CoA transferases ACT01, ACT04, and ACT06 showed the best affinity for butyrate, but their affinity for β-alanine was unknown. In this invention, Autodock was used to molecularly dock these three enzymes with β-alanine, and the experimental results showed that all three enzymes had good affinity for β-alanine.

[0028] Furthermore, this invention performed domain analysis on eight enzymes (ACT01-08) of the ACT series reported in the current literature. Experimental results showed that the RMSD of ACT01-08 was <2, indicating relatively conserved core domains. To obtain better docking results, this invention used Alphafold2 and Discovery Studio to further perform molecular docking of the eight ACT enzymes (ACT01-08) with β-alanine and butyrate, respectively. Combining the domain analysis results with the docking results from Alphafold2 and Discovery Studio, a comprehensive evaluation was conducted, ultimately identifying ACT01, ACT04, ACT06, and ACT08 as the targets for modification. Specifically, gene ACT01 has the nucleotide sequence shown in SEQ ID NO: 8, and the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 4; gene ACT04 has the nucleotide sequence shown in SEQ ID NO: 7, and the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 1; gene ACT06 has the nucleotide sequence shown in SEQ ID NO: 9, and the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 5; gene ACT08 has the nucleotide sequence shown in SEQ ID NO: 10, and the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 6.

[0029] Based on the above docking results, this invention performs further calculations on ACT01, ACT04, ACT06, and ACT08. First, the structures of the monomers of ACT01, ACT04, ACT06, and ACT08, as well as their complexes with β-alanine, are optimized using the RELAX operation. Then, monomer stability prediction and single-point mutation free energy calculation of the complex are performed to confirm the substrate pocket. Next, the substitution bases at the mutation sites are determined using the saturation mutagenesis method. Finally, acyl-CoA transferase mutants (i.e., SEQ ID NOs: 11-15) are constructed using homologous recombination. Specifically, the gene ACT04_E249D has the nucleotide sequence shown in SEQ ID NO: 11; the gene ACT04_H90D has the nucleotide sequence shown in SEQ ID NO: 12; the gene ACT01_E244L has the nucleotide sequence shown in SEQ ID NO: 13; the gene ACT06_E239D has the nucleotide sequence shown in SEQ ID NO: 14; and the gene ACT08_E290D has the nucleotide sequence shown in SEQ ID NO: 15.

[0030] Based on the above results, the applicant has proposed a series of technical solutions for this invention.

[0031] In a first typical embodiment of the present invention, an acyl-CoA transferase mutant is provided. This mutant comprises: 1) a protein having an amino acid sequence E249 mutated at the following site in SEQ ID NO: 1, and possessing acyl-CoA transferase activity; or 2) a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the protein in 1), and possessing acyl-CoA transferase activity. The acyl-CoA transferase mutant of SEQ ID NO: 1 mutated at site E249 can synergistically cooperate with alcohol dehydrogenase to achieve the biotransformation of β-alanine, thereby generating 3-aminopropanol.

[0032] Based on this application, by retaining the above-mentioned site mutations and then mutating the remaining sites, a protein with certain homology to the protein in 1) and the same acyl-CoA transferase activity can be obtained.

[0033] It should be noted that homology in this invention refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, it can be determined using the BLAST program in the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991.

[0034] The proteins described above that share 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) homology with the mutants shown in SEQ ID NO: 1 and possess acyl-CoA transferase activity, have active sites, active pockets, mechanisms of activity, and protein structures that are highly likely to be identical to the proteins provided in a) of the corresponding proteins.

[0035] Amino acid residues can be represented by three-letter or one-letter amino acid codes according to standards known and agreed upon in the art. In this document, the abbreviations for amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0036] Conservative amino acid substitutions or replacements are well known in the art. For example, a conserved amino acid substitution is preferably one amino acid residue from the following group (1)-(5) replaced by another amino acid from the same group: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) positively charged polar residues: His, Arg and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0037] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0038] In some preferred embodiments, the mutation site of the above-mentioned acyl-CoA transferase mutant includes one of the following: E249D. When the mutation site is E249D, the catalytic activity of the acyl-CoA transferase mutant constructed is improved compared with the activity of the wild-type acyl-CoA transferase shown in SEQ ID NO: 1. Accordingly, when it is applied to the synthesis of 3-aminopropanol, the yield of 3-aminopropanol is also increased.

[0039] In a second typical embodiment of the present invention, a gene is provided that encodes the aforementioned acyl-CoA transferase mutant. The gene encoding the improved acyl-CoA transferase mutant expresses an acyl-CoA transferase with relatively higher catalytic activity towards β-alanine, resulting in a relatively higher yield when applied to the synthesis of 3-aminopropanol.

[0040] Based on the amino acid sequence of the acyl-CoA transferase mutant described above, and in accordance with common knowledge in the art, the nucleotide sequence of its encoding gene can be determined. In a preferred embodiment of the present invention, the gene has the nucleotide sequence shown in SEQ ID NO: 11.

[0041] In a third typical embodiment of the present invention, a plasmid is provided containing the gene encoding the acyl-CoA transferase mutant described above. When the plasmid containing the gene is transferred into a specific host cell, it can express an acyl-CoA transferase with enhanced catalytic activity for β-alanine compared to the wild type, thereby resulting in a relatively higher synthesis yield when applied to the synthesis of 3-aminopropanol.

[0042] To facilitate the efficient synthesis of 3-aminopropanol, the plasmid described above contains not only the gene encoding an acyl-CoA transferase mutant but also the alcohol dehydrogenase gene adhE. Constructing both genes simultaneously on the same plasmid allows the plasmid to exhibit dual activity of acyl-CoA transferase and alcohol dehydrogenase upon induced expression, thereby improving the efficiency and yield of the catalytic synthesis of 3-aminopropanol.

[0043] The aforementioned plasmid is a plasmid for co-expression of the acyl-CoA transferase gene ACT and the alcohol dehydrogenase gene adhE. Existing plasmids capable of co-expressing both genes are applicable to this invention. In a preferred embodiment of this invention, the plasmid is pETduet.

[0044] The gene encoding the acyl-CoA transferase mutant in the above plasmids shares the same inducible promoter as the alcohol dehydrogenase gene adhE. In a preferred embodiment of the present invention, the inducible promoters of both the gene encoding the acyl-CoA transferase mutant and the alcohol dehydrogenase gene adhE are the T7 promoters.

[0045] The gene encoding the acyl-CoA transferase mutant and the alcohol dehydrogenase gene adhE can share the same promoter or use their own promoters.

[0046] The alcohol dehydrogenase gene adhE mentioned above can be from various sources. In a preferred embodiment of the present invention, it is selected from Clostridium acetobutylicum, as the alcohol dehydrogenase gene adhE from this source has the beneficial effect of synthesizing alcohols from carboxylic acids. The nucleotide sequence of the alcohol dehydrogenase gene adhE from Clostridium acetobutylicum is shown in SEQ ID NO: 3, and the protein encoded by this gene has the amino acid sequence shown in SEQ ID NO: 2.

[0047] In a fourth typical embodiment of the present invention, a host cell is provided. This host cell includes the aforementioned gene or plasmid. After induction, the host cell can directly participate in a whole-cell in vitro catalytic system using β-alanine as a substrate, thereby generating 3-aminopropanol. This host cell exhibits high synthesis yield of 3-aminopropanol.

[0048] The host cells mentioned above are selected from Escherichia coli. In a preferred embodiment of the present invention, the host cells are BL21 cells, preferably BL21 cells with the alcohol dehydrogenase gene adhE knocked out from Escherichia coli.

[0049] The alcohol dehydrogenase gene adhE mentioned above can be from various sources. In a preferred embodiment of the present invention, it is selected from Clostridium acetobutylicum, as the alcohol dehydrogenase gene adhE from this source has the beneficial effect of synthesizing alcohols from carboxylic acids. The nucleotide sequence of the alcohol dehydrogenase gene adhE from Clostridium acetobutylicum is shown in SEQ ID NO: 3. There are no particular limitations on the specific gene knockout method; existing gene knockout methods are all applicable to the present invention.

[0050] In a fifth typical embodiment of the present invention, a method for synthesizing 3-aminopropanol is provided. The method includes: using a host cell capable of simultaneously expressing acyl-CoA transferase and alcohol dehydrogenase, and using β-alanine as a substrate, to perform a whole-cell catalytic reaction to synthesize 3-aminopropanol; wherein the acyl-CoA transferase is selected from the above-mentioned acyl-CoA transferase mutant.

[0051] The above-described method for synthesizing 3-aminopropanol utilizes host cells capable of simultaneously expressing acyl-CoA transferase and alcohol dehydrogenase (which can be endogenous or exogenously introduced) to catalyze the in vitro whole-cell synthesis of 3-aminopropanol from β-alanine. This method is not only simple and economical, requiring only two enzymes, independent of ATP, and yielding the target product 3-aminopropanol in a single step, but also economical and environmentally friendly, with no toxic or harmful chemicals involved in the production process. It overcomes the problems of low synthesis efficiency, poor safety, high pollution, and high cost associated with existing methods, laying the foundation for large-scale production of 3-aminopropanol. In particular, the acyl-CoA transferase mutant constructed with the E249D mutation site exhibits higher catalytic activity and a correspondingly higher yield of 3-aminopropanol.

[0052] The catalytic system for the above-mentioned in vitro whole-cell catalytic reaction includes: host cells capable of expressing acyl-CoA transferase and alcohol dehydrogenase, β-alanine, acetyl-CoA, NADH, Tris-HCl buffer at pH 8.0, and MgCl2; preferably, in the in vitro whole-cell catalytic synthesis system, the final concentration of β-alanine is 1 g / L, the final concentration of acetyl-CoA is 5 mM, the final concentration of NADH is 5 mM, the final concentration of host cells is 1.2%, and the final concentration of MgCl2 is 20 mM.

[0053] The catalytic system containing the above components provides a suitable reaction environment, including substrates and coenzymes, for in vitro whole-cell catalytic reactions, laying the foundation for increasing the yield of 3-aminopropanol. Optimizing the proportions of each component in the catalytic system helps to synthesize the target product in higher yields with greater efficiency.

[0054] To further improve the catalytic activity and efficiency of the catalytic reaction, the host cells participating in the catalytic reaction preferably express both of the aforementioned related genes simultaneously. Therefore, in some preferred embodiments of the present invention, before the catalytic reaction, the method further includes the following pretreatment of the host cells: inoculating the host cells into a seed culture medium with shaking, adding an inducer for overnight induction, and collecting the cells by centrifugation; preferably, the inoculation amount of host cells is 1%; preferably, the inducer is added when the OD600 of the seed culture medium reaches 0.7–0.9; more preferably, the inducer is IPTG, and further, the final concentration of IPTG is 0.3 mM; preferably, the overnight induction temperature is 25°C, and the overnight induction rotation speed is 220 r / min.

[0055] The beneficial effects of the present invention will be explained in more detail below with reference to specific embodiments.

[0056] Example 1: ACT Substrate Affinity Analysis

[0057] Based on the previous research results of Hyojung Park et al. (Biotechnology and Bioprocess Engineering 25:599-606(2020)), it was found that ACT01, ACT04, and ACT06 have good affinity for the natural substrate butyrate, but whether they also have affinity for β-alanine is unknown. Therefore, we selected ACT01, ACT04, and ACT06 as enzyme modification targets. In the early stages, this invention used molecular docking and simulation prediction to dock the enzyme molecules with β-alanine. First, the PDB file was downloaded directly from the PDB database using the enzyme sequence. After obtaining the PDB file, it was processed with Autodock and saved as a PDBQT file. Subsequently, the small molecule β-alanine was also processed. After completing the preparation work, docking was performed using Autodock. Subsequently, in order to analyze the docking results more conveniently and intuitively, Autodock was combined with pymol. The results showed that all three enzymes (ACT01, ACT04, and ACT06) have a certain affinity for β-alanine.

[0058] Example 2: ACT Domain Analysis

[0059] To further investigate whether other acyl-CoA transferases also have good affinity for β-alanine, this invention performed domain analysis on eight acyl-CoA transferases (ACT01-ACT08) reported in the literature. According to the structural comparison analysis, the RMSD of ACT01-08 is <2, indicating that the core domains are relatively conserved. Specific domain analysis comparison results are shown in Table 1.

[0060] Table 1. Results of domain analysis and structural comparison

[0061]

[0062] To obtain better docking results, Alphafold2 was used to model the structure of ACT01-08, and molecular docking (docking times = 20) was performed with β-alanine (BAL) and butyric acid (BA). The results are shown in Table 2. The results show that the affinity of ACT01-08 for β-alanine and butyric acid is in the range of -4.2 to -3.2, both negative, indicating that ACT01-08 has good affinity for both β-alanine and butyric acid. Simultaneously, docking was performed again in Discovery Studio, and the results are shown in Table 3. By comparing the E and IE results of ACT01-08 and butyric acid, the results show that ACT01 and 03-08 also have good affinity for β-alanine, especially ACT08. Combining the data in Tables 2 and 3, it can be concluded that the results from both Alphafold2 and Discovery Studio indicate that ACT01-08 has good affinity for β-alanine.

[0063] Table 2 Results of molecular docking with Alphafold2

[0064]

[0065] Table 3 Discovery Studio Integration Results

[0066]

[0067]

[0068] Note: Underlined amino acid residues are key amino acid residues.

[0069] Example 3: Four ACT / adhE co-expression genes were screened and designed using a database.

[0070] Based on the analysis of the above results, four acyl-CoA transferases (ACT01, ACT04, ACT06, and ACT08) derived from *Megasphaera hexanoica* and a dehydrogenase derived from *Clostridium acetobutylicum* were selected. Database information is shown in Table 4 below.

[0071] Table 4. Database information table for enzymes

[0072] Gene name Protein ID source ACT01 WP_059076179.1 Megasphaera hexanoica ACT04 WP_059076321.1 Megasphaera hexanoica ACT06 WP_201784386.1 Megasphaera hexanoica ACT08 WP_113856284.1 Megasphaera hexanoica adhE WP_010890720.1 Clostridium acetobutylicum

[0073] Example 4: Determination of mutation sites in wild-type ACT using bioinformatics methods

[0074] Before performing calculations on the four enzymes ACT01, ACT04, ACT06, and ACT08, the structures of the monomers of ACT01, ACT04, ACT06, and ACT08, as well as their complexes with β-alanine, were optimized using the RELAX command. After configuring the environment, the prepared monomer files for the four enzymes were uploaded; then, the relevant code was executed on the cloud server to perform pre-optimization and secondary optimization of the monomers.

[0075] Next, complex optimization was performed. The pre-optimized structures of the monomers were selected, and hydrogenation, charge calculation, and atom type assignment were performed. For small molecule ligands, the following steps were required: hydrogenation (polar hydrogen), energy minimization, and generation of the small molecule topological parameter file: molfile_to_params.py -n BAL --extra_torsion_output BAL.mol2. The complex was first pre-optimized 40 times, and then a second optimization was performed, relaxing 100 times. The results of the second optimization are shown in Table 5.

[0076] Table 5 Results of secondary optimization of the complex

[0077]

[0078]

[0079] Subsequently, monomer stability prediction and single-point mutation free energy calculation of the complex were performed. Monomer stability prediction was conducted to scan for single-point mutants with stable structures. Single-point mutation free energy calculation of the complex: First, the substrate pocket was confirmed, and the prediction results are shown in Table 6. Within the enzymes, the key residues are GLU244 for ACT01, HIS90 and GLU249 for ACT04, GLU239 for ACT06, and TYR90, GLY265, and GLU290 for ACT08 (all key residues are underlined in Table 6). A β-alanine scan was performed on each enzyme, and the results are shown in Tables 7-10 (representing ACT01, ACT04, ACT06, and ACT08, respectively). The scan results show that: ACT01 becomes unstable after the key residue GLU244 is mutated to alanine; ACT04 becomes unstable after both the key residues HIS90 and GLU249 are mutated to alanine; ACT06 also becomes unstable after the key residue GLU239 is mutated to alanine; similarly, ACT08 also becomes unstable after each of the key residues TYR90, GLY265, and GLU290 is individually mutated to alanine. The instability of proteins suggests that they may bind more readily to substrates. Therefore, mutations to alanine residues at the aforementioned key residue sites all facilitate the binding of the corresponding proteins to substrates.

[0080] Table 6. Results of Substrate Pocket Confirmation

[0081]

[0082] The underlined parts represent key amino acid residues.

[0083] Table 7. ACT01 Alanine Scan Results

[0084]

[0085] The underlined part indicates the energy value of the mutation point and the stability after the key amino acid residue is mutated from glutamic acid to alanine.

[0086] Table 8. ACT04 Alanine Scan Results

[0087]

[0088]

[0089] The underlined part indicates the energy value of the mutation point and the stability after the key amino acid residue is mutated from glutamic acid to alanine.

[0090] Table 9. ACT06 Alanine Scan Results

[0091]

[0092] The underlined part indicates the energy value of the mutation point and the stability after the key amino acid residue is mutated from glutamic acid to alanine.

[0093] Table 10. ACT08 Alanine Scan Results

[0094] mutation Mutation energy Mutation effect (Mutation) (Mutation Energy) (Effect of Mutation) GLY383>ALA -0.23 NEUTRAL VAL266>ALA -0.04 NEUTRAL VAL112>ALA -0.02 NEUTRAL ASN380>ALA -0.02 NEUTRAL THR358>ALA -0.01 NEUTRAL SER385>ALA -0.01 NEUTRAL SER264>ALA 0.01 NEUTRAL GLN263>ALA 0.02 NEUTRAL ILE382>ALA 0.02 NEUTRAL PHE38>ALA 0.09 NEUTRAL THR39>ALA 0.11 NEUTRAL GLY384>ALA 0.24 NEUTRAL VAL291>ALA 0.29 NEUTRAL GLY265>ALA 0.76 DESTABILIZING TYR90>ALA 0.89 DESTABILIZING GLU290>ALA 1 DESTABILIZING

[0095] The underlined part indicates the energy value of the mutation point and the stability after the key amino acid residue is mutated from glutamic acid to alanine.

[0096] Then, we began preparing the mutfile for saturation mutagenesis of the four enzymes mentioned above. All amino acid sites within the specified range underwent saturation mutations. Saturation mutations to proline (which contains a benzene ring) were generally avoided. Finally, the free energy of single-point mutations was calculated using the cartesian_ddg script, where ΔG represents the energy difference between the mutant and the wild type, and a smaller value is better when it is less than zero. Based on the key amino acid residues screened in Tables 6-10, the free energy calculation results in Table 11, and the conserved sites of the four enzymes reported in the literature, the inventors ultimately selected the following mutants for subsequent experiments: ACT01_E244L, ACT04_H90D, ACT04_E249D, ACT06_E239D, and ACT08_E290D.

[0097] Table 11 Free Energy Calculation Results

[0098]

[0099] Example 5: Site-directed mutagenesis at mutation sites using homologous recombination.

[0100] The template sequences used in the homologous recombination method are shown in the last part of the specification. The strains and plasmids used in this invention are shown in Table 12, and the primers used in this invention are shown in Table 13.

[0101] Table 12 Strains and plasmids used in this invention

[0102]

[0103]

[0104] Table 13 Primers used in this invention

[0105]

[0106] Note: The underlined nucleotide sequence is the mutated nucleotide sequence.

[0107] Similar to conventional PCR, purified plasmids or wild-type bacterial cultures were used as templates, and high-fidelity polymerases (such as PrimeStar, Pfu) were selected. The total PCR reaction volume was 50 μL, and the amounts of each reactant added are shown in Table 14.

[0108] Table 14 Reactant Addition Table

[0109] PCR reactants Sample volume template Plasmid (5-10 ng) or bacterial culture 0.2 μL upstream primer Final concentration 0.2 μM Downstream primer Final concentration 0.2 μM PrimeSTAR Mix 25μL <![CDATA[ddH2O]]> up to 50μL

[0110] The PCR reaction was performed using the Touchdown program, with the following settings: pre-denaturation temperature 98℃, time 5 min; denaturation temperature 98℃, time 10 s; annealing temperature 63℃, time 15 s; extension temperature 72℃, time 1 min; final extension temperature 72℃, time 5 min; storage temperature 4℃.

[0111] Then, the following steps were performed: Plasmid template digestion: 1 μL of DpnI (GATC for recognizing methylation) was added to 50 μL of PCR product, and the mixture was reacted at 37°C for 1–2 hours to digest the plasmid template. Target product recovery: Loading buffer was added to the PCR product in the appropriate proportion. After 1% agarose gel electrophoresis, the target gene band was recovered from the gel, eluted into 50 μL of ddH2O, and the concentration was determined for later use. Homologous recombination reaction: Based on the principle of homologous recombination and primer design, Exnase II enzyme from Novizan was used for catalysis to efficiently recombine the target mutation site, achieving in vitro circularization of linear DNA. The specific reaction is shown in Table 15.

[0112] Table 15 Recombination Reaction System

[0113] Components Recombination reaction Rubber recycling products 7μL 5×CEⅡbuffer 2μL Exnase II 1μL

[0114] After adding the above reactants to the PCR tube, mix well and incubate at 37°C for 30 min, then cool to 4°C or immediately place on ice. Ligation and transformation: Transform 10 μL of the recombinant product into 100 μL of competent cells, plate on LB agar plates with appropriate antibiotics, select five single clones, and send for sequencing (SEQ ID NO s: 11-15). Then, induce protein expression in the above strains by inoculating 1% of the primary seed culture of the four strains. When the OD600 is about 0.8, add IPTG to a final concentration of 0.3 mM and incubate overnight at 25°C and 220 rpm. Collect the bacterial cells by centrifugation.

[0115] Example 6: Determining the in vitro whole-cell catalytic screening enzyme activity system

[0116] Using 1 g / L β-alanine as the substrate and acetyl-CoA as the coenzyme, the catalytic system is shown in Table 16.

[0117] Table 16 Catalytic Reaction System

[0118]

[0119]

[0120] The results showed that only one strain among the screened strains exhibited higher activity than the original column. Sequencing identified the mutation site (E249D) in this strain. HPLC analysis of the 3-aminopropanol content revealed that only the original strain ACT04 produced 0.02 g / L of 3-aminopropanol. Other mutant strains, including the two ACT04 mutants, produced only 0.004 g / L and 0.083 g / L of 3-aminopropanol, respectively. Compared to the original strain, the ACT04_E249D mutant strain showed a fourfold increase in catalytic activity for 3-aminopropanol synthesis. The HPLC verification chromatogram is attached. Figure 1 The remaining four mutant strains showed virtually no activity in the production of 3-aminopropanol, as shown in Table 17.

[0121] Table 17

[0122] Mutant name Wild-type active mutant activity ACT01_E244L ND ND ACT04_H90D 0.02 0.004 ACT04_E249D 0.02 0.083 ACT06_E239D ND ND ACT08_E290D ND ND

[0123] ND indicates that no activity was detected.

[0124] In summary, the above embodiments of the present invention achieve the following technical effects: The present invention proposes for the first time a method for synthesizing 3-aminopropanol via bio-fermentation using β-alanine as a substrate. This method is not only simple and economical, requiring only two enzymes and not dependent on ATP, but also yields the target product 3-aminopropanol in a single-pot reaction. Furthermore, it is green and environmentally friendly, with no toxic or harmful chemicals involved in the production process. This overcomes the problem of existing methods lacking a way to biosynthesize 3-aminopropanol, opening a new synthetic route for the biotransformation of β-alanine to 3-aminopropanol. By constructing an acyltransferase mutant with improved activity and applying it to the whole-cell catalysis of β-alanine to 3-aminopropanol in *E. coli*, a foundation is laid for the large-scale production of 3-aminopropanol, providing new ideas for further biotransformation-based synthesis of other chemical substances.

[0125] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An acyl-CoA transferase mutant, characterized in that, The acyl-CoA transferase mutants include: 1) A protein having an amino acid sequence with a mutation at position E249 of SEQ ID NO: 1 and possessing acyl-CoA transferase activity; or 2) The protein in 1) has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology and has acyl-CoA transferase activity.

2. The acyl-CoA transferase mutant according to claim 1, characterized in that, In 1), the mutation site includes E249D.

3. A gene characterized by, The gene encodes the acyl-CoA transferase mutant as described in claim 1 or 2.

4. The gene according to claim 3, characterized in that, The gene has the nucleotide sequence shown in SEQ ID NO:

11.

5. A plasmid, characterized in that, It contains the gene described in claim 3 or 4.

6. The plasmid according to claim 5, characterized in that, The plasmid also includes the alcohol dehydrogenase gene adhE; Preferably, the gene encoding the acyl-CoA transferase mutant on the plasmid has the same inducible promoter as the alcohol dehydrogenase gene adhE; Preferably, the nucleotide sequence of the alcohol dehydrogenase gene adhE is SEQ ID NO: 3; Preferably, the plasmid is pETduet.

7. A host cell, characterized in that, The host cell includes the gene as described in claim 3 or 4, or the plasmid as described in claim 5 or 6.

8. The host cell according to claim 7, characterized in that, The host cell is selected from any one of the following: Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae. Preferably, the Escherichia coli is BL21 cell; More preferably, the Escherichia coli is a BL21 cell in which the endogenous alcohol dehydrogenase gene adhE has been knocked out.

9. A method for synthesizing 3-aminopropanol, characterized in that, The synthesis method includes: Using host cells capable of simultaneously expressing acyl-CoA transferase and alcohol dehydrogenase, 3-aminopropanol was synthesized via a whole-cell catalytic reaction with β-alanine as a substrate. The acyl-CoA transferase is selected from the acyl-CoA transferase mutant described in claim 1 or 2.

10. The synthesis method according to claim 9, characterized in that, The catalytic system for carrying out the whole-cell catalytic reaction comprises: a host cell capable of expressing acyl-CoA transferase and the alcohol dehydrogenase, the substrate β-alanine, acetyl-CoA and NADH, and Mg2+. 2+ The reaction buffer solution; Preferably, the Mg-containing 2+ The reaction buffer consists of Tris-HCl buffer at pH 8.0 and MgCl2.

11. The synthesis method according to claim 10, characterized in that, The host cell is Escherichia coli; Preferably, the Escherichia coli is BL21 cell; More preferably, the Escherichia coli is a BL21 cell in which the endogenous alcohol dehydrogenase gene adhE has been knocked out; Preferably, the gene encoding the alcohol dehydrogenase is adhE, and the nucleotide sequence of the gene encoding the alcohol dehydrogenase is SEQ ID NO: 3.

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