Acylated aldehyde dehydrogenase AldDH mutant and application thereof in production of 6-aminohexanoic acid

Abstract

The application provides an acylated aldehyde dehydrogenase AldDH mutant and application thereof in production of 6-aminohexanoic acid. Specifically, the acylated aldehyde dehydrogenase AldDH mutant comprises a mutation based on a reference sequence, the reference sequence is shown as SEQ ID NO:1, and the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid mutation corresponding to at least one of positions A67, C69, N71, S75, W78, R86, I118, F238, L243, T245, W350, G399, T400, N401, A405 and T414 of SEQ ID NO:1. The 6-aminohexanoic acid production strain constructed by using the acylated aldehyde dehydrogenase AldDH mutant of the application can efficiently synthesize 6-aminohexanoic acid from glucose.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering and biofermentation technology, specifically to an acylated aldehyde dehydrogenase AldDH mutant and its application in the production of 6-aminocaproic acid. Background Technology

[0002] 6-Aminohexanoic acid is an important platform chemical, serving as a key monomer in the synthesis of high-molecular materials such as nylon-6 and polyamides, and also as a crucial intermediate in the pharmaceutical and chemical industries, possessing significant application value in biomanufacturing. In the de novo biosynthesis of 6-aminohexanoic acid using glucose as the sole carbon source, acylated aldehyde dehydrogenase (AldDH) catalyzes the reduction of adipicyl-CoA to 6-oxohexanoic acid. This process is the core node connecting the upstream reverse β-oxidation synthesis module of fatty acids with the downstream transamination module, and is also the critical rate-limiting step in the entire synthetic pathway.

[0003] Currently, naturally derived acylated aldehyde dehydrogenases generally exhibit low catalytic affinity and efficiency for the substrate adipicyl-CoA, severely hindering metabolic flux at this step. This prevents the efficient conversion of upstream adipicyl-CoA into downstream intermediates, directly resulting in insufficient throughput and low yield of 6-aminohexanoic acid synthesis. Existing technologies largely focus on the simple assembly of metabolic pathway modules without targeted modification of the core rate-limiting enzyme AldDH, making it difficult to achieve efficient conversion of glucose to 6-aminohexanoic acid. Therefore, identifying and modifying AldDH mutants with high catalytic activity towards adipicyl-CoA to overcome this metabolic bottleneck is of significant practical importance for constructing efficient 6-aminohexanoic acid cell factories and realizing green biosynthesis. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this application is to provide an acylated aldehyde dehydrogenase AldDH mutant with high catalytic activity for adipicoyl-CoA.

[0005] Specifically, this application relates to the following aspects: 1. An acylated aldehyde dehydrogenase AldDH mutant comprising a mutation based on a reference sequence as shown in SEQ ID NO: 1, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid mutation corresponding to at least one of the following sites in SEQ ID NO: 1: A67, C69, N71, S75, W78, R86, I118, F238, L243, T245, W350, G399, T400, N401, A405, T414; Preferably, the acylated aldehyde dehydrogenase AldDH mutant contains an amino acid mutation at at least one of the sites G399, T400, and A405 corresponding to SEQ ID NO: 1.

[0006] 2. The acylated aldehyde dehydrogenase AldDH mutant according to claim 1, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises any one or more of the following mutations: (1) A67G; (2) C69A or C69N; (3) N71A, N71H or N71G; (4) S75A or S75T; (5) W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78L, W78S, W78A, W78R or W78V; (6) R86V, R86C, R86I or R86L; (7) I118S; (8) F238A or F238Y; (9) L243A; (10) T245N; (11) W350A, W350P, W350Q, W350I, W350E, W350F, W350S, W350Y, W350L, W350V, W350G, W350M or W350K; (12) G399V, G399T, G399I, G399Y, G399F or G399Q; (13) T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K or T400R; (14) N401G; (15) A405K, A405G, A405S or A405H; (16) T414A or T414V.

[0007] 3. The acylated aldehyde dehydrogenase AldDH mutant according to claim 2, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises A405G.

[0008] 4. An acylated aldehyde dehydrogenase AldDH mutant according to any one of claims 1-3, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises any of the following combinations of mutations: G399V / T400K / A405G, G399C / T400I / A405G, G399C / T400R / A405G, G399R / T400R / A405G, G399C / T400L / A405G, G399Q / T400L / A405G, G399F / T400H / A405G, G399T / T400A / A405G, G399C / T400K / A405G, G399Y / T400I / A405G, G399T / T400Y / A405G, G399V / T400R / A405G, G399S / T400S / A405G, G399T / T400K / A405G, G399H / T400R / A405G, G399C / T400H / A405G, G399T / T400H / A405H, G399S / T400H / A405H, G399F / A405G, T400H / A405G, T400H / A405H.

[0009] 5. The acylated aldehyde dehydrogenase AldDH mutant according to claim 4, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises the G399C / T400R / A405G mutation combination.

[0010] 6. An acylated aldehyde dehydrogenase AldDH mutant according to any one of items 1-5, wherein the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is as shown in SEQ ID NO: 2 or 3, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0011] 7. A biomaterial comprising any one of the following materials: A1) A nucleic acid molecule encoding an acylated aldehyde dehydrogenase AldDH mutant as described in any one of items 1-6, preferably, the nucleic acid molecule having a sequence as shown in SEQ ID NO: 5 or 6, or having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 5 or 6.

[0012] A2) Expression cassette, which contains the nucleic acid molecule described in A1); A3) A recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) A recombinant microorganism comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3), preferably, the recombinant microorganism being Escherichia coli; A5) Recombinant cells comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst comprising the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

[0013] 8. A 6-aminocaproic acid producing strain, obtained by modifying a starting strain, the modification comprising: expressing an acylated aldehyde dehydrogenase AldDH mutant as described in any one of items 1-6 or introducing a nucleic acid molecule encoding an acylated aldehyde dehydrogenase AldDH mutant as described in any one of items 1-6.

[0014] 9. The 6-aminohexanoic acid producing strain according to item 8, wherein the modification further comprises: It expresses one or more of the following: 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolytic enzyme paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr.

[0015] 10. The 6-aminohexanoic acid producing strain according to item 8 or 9, wherein the starting strain is Escherichia coli.

[0016] 11. A method for constructing a 6-aminohexanoic acid producing strain, comprising: exogenously expressing an acylated aldehyde dehydrogenase AldDH mutant as described in any one of items 1-6 of the 6-aminohexanoic acid producing strain, thereby constructing a 6-aminohexanoic acid producing strain as described in any one of items 8-10.

[0017] 12. The use of the acylated aldehyde dehydrogenase AldDH mutant according to any one of items 1-6, the biological material according to item 7, and / or the 6-aminohexanoic acid producing strain according to any one of items 8-10 in any of the following: (1) Application in the production of 6-aminohexanoic acid; (2) Application in the preparation of products containing 6-aminohexanoic acid; (3) Application in increasing the production yield of 6-aminohexanoic acid; (4) Application in the genetic breeding of microorganisms for the production of 6-aminocaproic acid.

[0018] 13. A method for producing 6-aminohexanoic acid, comprising: culturing a 6-aminohexanoic acid producing strain as described in any one of items 8-10 or culturing a 6-aminohexanoic acid producing strain obtained by the method described in item 11, thereby producing 6-aminohexanoic acid; Preferably, 6-aminohexanoic acid is produced by fermentation using glucose as a carbon source.

[0019] Beneficial effects: This application provides a family of AldDH mutant acylated aldehyde dehydrogenases with significantly enhanced catalytic activity. By modifying key sites such as G399, T400, and A405, the binding and catalytic efficiency of the enzyme to the substrate adipicoyl-CoA are effectively enhanced. Introducing the mutant into recombinant strains throughout the entire pathway significantly promotes the efficient downstream flow of carbon towards 6-aminohexanoic acid, thereby effectively eliminating the flux bottleneck in the AldDH-dependent synthetic pathway. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the production of 6-aminocaproic acid from glucose as a substrate. Detailed Implementation

[0021] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0022] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0023] definition As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0024] As used herein, the term "wildtype" has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or trait that distinguishes it from mutants or variants when it exists in nature. It can be isolated from resources in nature and is not deliberately modified.

[0025] As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably and refer to artificially induced modifications. The terms “engineered strain” or “engineered strain cell” refer to bacterial cells that have been genetically modified from their natural state. For example, engineered bacterial cells may have nucleotide insertions, deletions, rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on plasmids within the bacteria or bacterial cell. The engineered bacterial cells of this disclosure may contain exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may contain exogenous nucleotide sequences stably integrated into their chromosomes.

[0026] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Therefore, a gene includes (but is not limited to) coding sequences and / or regulatory sequences required for their expression. A gene may also include unexpressed segments of DNA, such as recognition sequences that form other proteins. Genes can be obtained from a variety of sources, including cloning from sources of interest or synthesis from known or predicted sequence information, and may include sequences designed with desired parameters.

[0027] As used herein, the terms “exogenous” or “heterogeneous” are used interchangeably and refer to substances from sources other than their native origin. For example, the terms “exogenous protein” or “exogenous gene” refer to proteins or genes from non-native sources or locations that have been artificially introduced into a biological system.

[0028] As used herein, the term "expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for protein detection, such as enzyme activity assays, SDS-Page, ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.

[0029] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” “polynucleotide,” “polynucleotide sequence,” “nucleic acid molecule,” “RNA sequence,” or “DNA sequence” refer to oligonucleotides, nucleotides, or polynucleotides, and fragments or portions thereof, and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent sense or antisense strands. Sequences may be non-coding sequences, coding sequences, or mixtures of both. The nucleic acid sequences of this invention can be prepared using standard techniques well known to those skilled in the art.

[0030] As used herein, the term "coding" refers to i) a DNA sequence containing genetic information that can be transcribed into an RNA molecule, and / or ii) an RNA molecule containing genetic information that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can refer to a ribonucleotide (RNA) sequence or fragment thereof in a precursor or mature mRNA that can be translated into a protein, or it can refer to a complementary sequence or fragment thereof of a deoxyribonucleotide (DNA) sequence used as a template for transcribing the precursor or mature mRNA. Furthermore, the "coding sequence" of this application may further include polynucleotide sequences encoding proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, poly(A) tail, 5'UTR, 3'UTR, etc. Wherein, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is referred to as the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is referred to as the "coding nucleic acid" of the amino acid sequence.

[0031] As used herein, the term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0032] As used in this article, the term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0033] As used herein, the term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria mentioned may be from the genus Escherichia. (Escherichia sp. (such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium spp. Flavobacteriumsp. ), Alcaligenes ( Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp.) and Bacillus spp. ( Bacillus sp. (e.g., Bacillus). The viruses may include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus). The fungi may be derived from yeasts (e.g., Bacillus). Saccharomycessp. (such as Saccharomyces cerevisiae, Saccharomyces methylbenzene, Pichia pastoris), Fusarium spp. ( Fusariumsp. ), Rhizoctonia spp. Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicilliumsp. Aspergillus ( ) Aspergillussp. ) and Cephalosporin ( Cephalosporium sp. The actinomycetes may originate from the genus Streptomyces (…). Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanobacteria ( Cyanophyta (such as cyanobacteria), Fucus genus ( Fucus sp. ), genus *Cyclocarya* Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpus ( Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus Styracula ( Asteromonas sp. ) and the genus *Golden Color Algae* ( Boekelovia sp. )wait.

[0034] As used herein, the term “recombinant microorganism” includes microorganisms (e.g., bacteria, yeast, algae, fungi, etc.) or strains of microorganisms that have been genetically altered, modified, or engineered (e.g., genetically engineered) so that they exhibit altered, modified, or different genotypes and / or phenotypes compared to their naturally occurring or “parental” microorganisms (e.g., when genetic modifications affect the microorganism’s coding nucleic acid sequence).

[0035] When referring to, for example, cells, nucleic acids, proteins, or vectors, the term "recombinant" indicates that the cell, nucleic acid, protein, or vector has been modified by introducing a heterologous nucleic acid or protein, or that the natural nucleic acid or protein has been altered, or that the cell is derived from such a modified cell. For example, recombinant proteins are proteins produced from recombinant nucleic acid molecules. Nucleic acid molecules can include genetic material from multiple sources, thus including sequences that are not naturally occurring. Recombinant DNA can be produced using methods known in the field of molecular biology or through synthetic methods. Thus, for example, recombinant cells express genes not found in naturally occurring (non-recombinant) cell forms, or express naturally occurring genes that are abnormally expressed, underexpressed, or not expressed at all. Transgenic cells and plants are those cells and plants that express heterologous genes or coding sequences, often as a result of recombinant methods.

[0036] As used herein, the term "expression cassette" refers to a nucleotide sequence or corresponding polynucleotide or nucleic acid molecule containing a desired coding sequence and a regulatory or control sequence that is effectively linked, such that a host transformed or transfected with these sequences is able to produce the encoded protein or host cell metabolite. Exemplary expression control sequences may include any of the following: a promoter, a ribosome binding site, a transcription or translation initiation sequence and a termination sequence, or an enhancer sequence or an activator sequence.

[0037] As used herein, the term "mutation" refers to a change in the sequence of nucleotides or amino acids. In one specific implementation, the term "mutation" refers to "substitution".

[0038] As used herein, the term “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide, or protein sequence, or any fragment thereof, and refers to a naturally occurring or synthetic molecule. When “amino acid sequence” is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and similar terms are not intended to limit the amino acid sequence to the complete naturally occurring amino acid sequence associated with the described protein molecule.

[0039] In this article, "amino acid" may be referred to by its name, its commonly known three-letter symbol, or a single-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0040] As used herein, the percentage of “identity,” such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, it is the percentage of positions with identical bases or amino acid residues determined after two sequences have as many identical residues as possible by introducing vacancies, etc. The percentage of “identity” can be determined using software programs known in the art. It is preferred to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address: blast.ncbi.nlm.nih.gov / Blast.cgi.

[0041] As used herein, the term "variant" or "mutant" is interpreted as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the necessary characteristics. A typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the variant's nucleic acid sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the differences are limited, making the sequences of the reference polypeptide and the variant very similar overall and identical in many regions. The amino acid sequences of the variant and the reference polypeptide can differ by any combination of one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring (such as allelic variants) or may be variants of unknown natural origin. Non-natural variants of polynucleotides and polypeptides can be prepared by mutagenesis, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0042] As used herein, the term "recombinant cell" means any cell type that is readily transformable, transfected, transduced, etc., using nucleic acid constructs, expression cassettes, or expression vectors containing the nucleic acid molecules of this application. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0043] As used herein, the term "whole-cell catalyst" refers to a biocatalyst that directly utilizes an intact cell (such as bacteria, yeast, fungi, etc.) as a biocatalyst, catalyzing a specific chemical reaction through its internal enzyme system without the need to separate and purify individual enzymes. Exemplarily, common classifications of whole-cell catalysts include: living cell catalysts, resting cell catalysts, and immobilized whole cells. Any of the listed forms of whole-cell catalysts may be used in this application.

[0044] Acylated aldehyde dehydrogenase AldDH mutant In a first aspect, this application provides an acylated aldehyde dehydrogenase AldDH mutant containing a mutation based on a reference sequence as shown in SEQ ID NO: 1.

[0045] In this application, the terms “acylating aldehyde dehydrogenase (AldDH),” “acylating aldehyde dehydrogenase AldDH” and “AldDH” are used interchangeably.

[0046] The sequence of SEQ ID NO: 1 is as follows: MEQAVKDYLDKMVAASRIAQQEFATYPQETVDKAVRTVGKAIYDNAELLAHMAVDETKMGNYADKIAKCVNKSKSVWWRMKDKKSRGIIKRIPELGLVEVAKPIGVIGCVAPTT NPVINVMQNAMCALKCGNSMIVSPHPRAKHSSVKTVEVINEALAAALMPKNLIQVITEPSMELSAGLMSAAVDLCICTGGPGLVKAAYSSGKPAIGVGQGNVQVLVDRDADLDQVA AMVIKGRTFDNGVLCTCEQNVICPEDKKEEMIAALKKHGAYYIGNSEDAAKLRDTAFPNGGPVSKEYPGASVKKIAQLSGIQGIPEDAKVIVSCTRGYGKDEPLAKEKLFPVLAF FTYDKWEDAIHIAKTNLEMEGIGHSVVIHSNTPEHIEAVAEAIPVSRFAVNQVGGTNLGGAMDNGLNPTTTLGCGTWGNNSISENFTYYHLMNLTRVSYRVPDMYIPTDEEIWAE Among them, SEQ ID NO: 1 refers to *Aminophilus musei* (from *Aminophilus musei*). Acidaminococcus massiliensis Wild-type acylated aldehyde dehydrogenase AldDH.

[0047] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid mutation at at least one of the following sites corresponding to SEQ ID NO: 1: A67, C69, N71, S75, W78, R86, I118, F238, L243, T245, W350, G399, T400, N401, A405, and T414.

[0048] Those skilled in the art will understand that the amino acids at the aforementioned sites are determined based on the amino acid sequence shown in SEQ ID NO: 1. The positions of amino acids may vary to some extent in acylated aldehyde dehydrogenases (AldDH) from different bacterial sources. As long as the same amino acid is present at the corresponding position as at the aforementioned site, it falls within the scope of protection of this application. Specifically, this application does not limit the source of the acylated aldehyde dehydrogenase (AldDH). As long as the selected bacterial acylated aldehyde dehydrogenase (AldDH) has the same amino acid at the position corresponding to the aforementioned site shown in SEQ ID NO: 1, it meets the requirements of this application.

[0049] In this application, the above-mentioned sites are counted starting from the N-terminus. For example, the amino acid mutation at the G399 site refers to the amino acid mutation occurring at the 399th amino acid starting from the N-terminus of SEQ ID NO: 1; the amino acid mutation at the T400 site refers to the amino acid mutation occurring at the 400th amino acid starting from the N-terminus of SEQ ID NO: 1; and the amino acid mutation at the A405 site refers to the amino acid mutation occurring at the 405th amino acid starting from the N-terminus of SEQ ID NO: 1.

[0050] In this application, the term "corresponds to" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds to" means the position in one sequence that corresponds to a specified position in another sequence after homology or sequence identity comparison.

[0051] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant contains an amino acid mutation at at least one of the sites G399, T400, and A405 corresponding to SEQ ID NO: 1.

[0052] In some implementations, the mutation is a substitution.

[0053] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid substitution at at least one site corresponding to A67, C69, N71, S75, W78, R86, I118, F238, L243, T245, W350, G399, T400, N401, A405, or T414 of SEQ ID NO: 1. In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid substitution at at least one site corresponding to G399, T400, or A405 of SEQ ID NO: 1.

[0054] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant contains one or more of the following mutations: (1) A67G; (2) C69A or C69N; (3) N71A, N71H or N71G; (4) S75A or S75T; (5) W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78L, W78S, W78A, W78R or W78V; (6) R86V, R86C, R86I or R86L; (7) I118S; (8) F238A or F238Y; (9) L243A; (10) T245N; (11) W350A, W350P, W350Q, W350I, W350E, W350F, W350S, W350Y, W350L, W350V, W350G, W350M or W350K; (12) G399V, G399T, G399I, G399Y, G399F or G399Q; (13) T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K or T400R; (14) N401G; (15) A405K, A405G, A405S or A405H; (16) T414A or T414V.

[0055] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises A405G.

[0056] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant is obtained by mutation of wild-type acylated aldehyde dehydrogenase AldDH, the amino acid sequence of which is shown in SEQ ID NO: 1, and the mutation is any one or more of the following: (1) A67G; (2) C69A or C69N; (3) N71A, N71H or N71G; (4) S75A or S75T; (5) W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78L, W78S, W78A, W78R or W78V; (6) R86V, R86C, R86I or R86L; (7) I118S; (8) F238A or F238Y; (9) L243A; (10) T245N; (11) W350A, W350P, W350Q, W350I, W350E, W350F, W350S, W350Y, W350L, W350V, W350G, W350M or W350K; (12) G399V, G399T, G399I, G399Y, G399F or G399Q; (13) T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K or T400R; (14) N401G; (15) A405K, A405G, A405S or A405H; (16) T414A or T414V.

[0057] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant is obtained by mutation of wild-type acylated aldehyde dehydrogenase AldDH, the amino acid sequence of which is shown in SEQ ID NO: 1, and the mutation is as follows: (1) A67G; (2) C69A or C69N; (3) N71A, N71H or N71G; (4) S75A or S75T; (5) W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78L, W78S, W78A, W78R or W78V; (6) R86V, R86C, R86I or R86L; (7) I118S; (8) F238A or F238Y; (9) L243A; (10) T245N; (11) W350A, W350P, W350Q, W350I, W350E, W350F, W350S, W350Y, W350L, W350V, W350G, W350M or W350K; (12) G399V, G399T, G399I, G399Y, G399F or G399Q; (13) T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K or T400R; (14) N401G; (15) A405K, A405G, A405S or A405H; or (16) T414A or T414V.

[0058] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant is obtained by mutation based on wild-type acylated aldehyde dehydrogenase AldDH, the amino acid sequence of which is shown in SEQ ID NO: 1, and the mutation is A405G.

[0059] When alanine A at position 405 is mutated to glycine G, the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is shown in SEQ ID NO: 2.

[0060] The sequence of SEQ ID NO: 2 is as follows: MEQAVKDYLDKMVAASRIAQQEFATYPQETVDKAVRTVGKAIYDNAELLAHMAVDETKMGNYADKIAKCVNKSKSVWWRMKDKKSRGIIKRIPELGLVEVAKPIGVIGCVAPTTN PVINVMQNAMCALKCGNSMIVSPHPRAKHSSVKTVEVINEALAAALMPKNLIQVITEPSMELSAGLMSAAVDLCICTGGPGLVKAAYSSGKPAIGVGQGNVQVLVDRDADLDQVAA MVIKGRTFDNGVLCTCEQNVICPEDKKEEMIAALKKHGAYYIGNSEDAAKLRDTAFPNGGPVSKEYPGASVKKIAQLSGIQGIPEDAKVIVSCTRGYGKDEPLAKEKLFPVLAFF TYDKWEDAIHIAKTNLEMEGIGHSVVIHSNTPEHIEAVAEAIPVSRFAVNQVGGTNLGGGMDNGLNPTTTLGCGTWGNNSISENFTYYHLMNLTRVSYRVPDMYIPTDEEIWAE.

[0061] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises any of the following combinations of mutations: G399V / T400K / A405G, G399C / T400I / A405G, G399C / T400R / A405G, G399R / T400R / A405G, G399C / T400L / A405G, G399Q / T400L / A405G, G399F / T400H / A405G, G399T / T400A / A405G, G399C / T400K / A405G, G399Y / T400I / A405G, G399T / T400Y / A405G, G399V / T400R / A405G, G399S / T400S / A405G, G399T / T400K / A405G, G399H / T400R / A405G, G399C / T400H / A405G, G399T / T400H / A405H, G399S / T400H / A405H, G399F / A405G, T400H / A405G, T400H / A405H.

[0062] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant comprises the G399C / T400R / A405G mutant combination.

[0063] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant is obtained by mutation of wild-type acylated aldehyde dehydrogenase AldDH, the amino acid sequence of which is shown in SEQ ID NO: 1. The mutation is selected from: G399V / T400K / A405G, G399C / T400I / A405G, G399C / T400R / A405G, G399R / T400R / A405G, G399C / T400L / A405G, G399Q / T400L / A405G, G399F / T400H / A405G, G399T / T400A / A405G, G399C / T400K / A405G, G399Y / T400I / A405G, G399T / T400Y / A405G, G399V / T400R / A405G, G399S / T400S / A405G, G399T / T400K / A405G, G399H / T400R / A405G, G399C / T400H / A405G, G399T / T400H / A405H, G399S / T400H / A405H, G399F / A405G, T400H / A405G, or T400H / A405H.

[0064] In some embodiments, the acylated aldehyde dehydrogenase AldDH mutant is obtained by mutation based on wild-type acylated aldehyde dehydrogenase AldDH, the amino acid sequence of which is shown in SEQ ID NO: 1, and the mutation is G399C / T400R / A405G.

[0065] Specifically, when glycine G at position 399 is mutated to cysteine ​​C, threonine T at position 400 is mutated to arginine R, and alanine A at position 405 is mutated to glycine G, the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is shown in SEQ ID NO: 3.

[0066] The sequence of SEQ ID NO: 3 is as follows: MEQAVKDYLDKMVAASRIAQQEFATYPQETVDKAVRTVGKAIYDNAELLAHMAVDETKMGNYADKIAKCVNKSKSVWWRMKDKKSRGIIKRIPELGLVEVAKPIGVIGCVAPTTN PVINVMQNAMCALKCGNSMIVSPHPRAKHSSVKTVEVINEALAAALMPKNLIQVITEPSMELSAGLMSAAVDLCICTGGPGLVKAAYSSGKPAIGVGQGNVQVLVDRDADLDQVAA MVIKGRTFDNGVLCTCEQNVICPEDKKEEMIAALKKHGAYYIGNSEDAAKLRDTAFPNGGPVSKEYPGASVKKIAQLSGIQGIPEDAKVIVSCTRGYGKDEPLAKEKLFPVLAFF TYDKWEDAIHIAKTNLEMEGIGHSVVIHSNTPEHIEAVAEAIPVSRFAVNQVGCRNLGGGMDNGLNPTTTLGCGTWGNNSISENFTYYHLMNLTRVSYRVPDMYIPTDEEIWAE.

[0067] Those skilled in the art will understand that the acylated aldehyde dehydrogenase AldDH mutant is not limited to the specific sequences listed above. The acylated aldehyde dehydrogenase AldDH mutant should encompass sequences containing one, two, or three or more amino acid mutations compared to the sequence shown in SEQ ID NO: 2 or 3, but still substantially functionally identical, and also include sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence shown in SEQ ID NO: 2 or 3.

[0068] In some embodiments, the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is as shown in SEQ ID NO: 2 or 3, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0069] In some embodiments, the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is as shown in any one of SEQ ID NO: 28-32, 34, 36-44, 47-48, 51-58, 60-61, 63-64, 68-69, 71-76, 78-82, 84, 90-92, 95-96, 99-108, 112-114, 117, and 120, or is consistent with SEQ ID NO: The amino acid sequences shown in any one of 28-32, 34, 36-44, 47-48, 51-58, 60-61, 63-64, 68-69, 71-76, 78-82, 84, 90-92, 95-96, 99-108, 112-114, 117, and 120 have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity.

[0070] In some embodiments, the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is as shown in any one of SEQ ID NO: 121, 124, 126-135, 137, 138, 140, 141, 148, 150, and 151, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 121, 124, 126-135, 137, 138, 140, 141, 148, 150, and 151.

[0071] This application does not impose any restrictions on the method for preparing acylated aldehyde dehydrogenase AldDH mutants. Mutation can be carried out according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or construction of synthetic oligonucleotides, and then the mutated DNA sequence can be expressed in host cells to obtain mutants with amino acid sequence substitution, insertion, deletion, and / or truncation.

[0072] biomaterials Secondly, this application provides a biomaterial comprising any one of the following materials: A1) A nucleic acid molecule encoding the AldDH mutant of the acylated aldehyde dehydrogenase described in the first aspect of this application; A2) Expression cassette, which contains the nucleic acid molecule described in A1); A3) A recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) A recombinant microorganism comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst comprising the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

[0073] In some embodiments, the A1) nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 5 or 6. In some embodiments, the A1) nucleic acid molecule comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 or 6.

[0074] In some embodiments, the sequence of the nucleic acid molecule A1) is as shown in SEQ ID NO: 5 or 6. In some embodiments, the sequence of the nucleic acid molecule A1) has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 5 or 6.

[0075] In some embodiments, the A2) kit further includes any one or a combination of promoter, terminator and marker gene functional elements. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of the A1) nucleic acid molecule can be completed. There are no further restrictions on the structure and composition of the expression cassette.

[0076] In some embodiments, the A3) recombinant vector is a vector capable of delivering exogenous DNA or a target gene into a host cell for amplification and expression. The vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, can be used, as those skilled in the art can choose according to the specific circumstances, without imposing excessive limitations here.

[0077] In some embodiments, the recombinant vector is a recombinant expression vector, which is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid. In some embodiments, the vector backbone of the recombinant vector is pCDFDuet-1.

[0078] This application does not limit the types of recombinant microorganisms, as long as they can contain the nucleic acid molecules described in A1), the expression cassettes described in A2), and / or the recombinant vectors described in A3), and can achieve the expression of the acylated aldehyde dehydrogenase AldDH mutant, they meet the requirements of this application.

[0079] In some embodiments, the A4) recombinant microorganism is Escherichia coli, Corynebacterium glutamicum, or Saccharomyces cerevisiae, etc. In some embodiments, the A4) recombinant microorganism is Escherichia coli.

[0080] Those skilled in the art can use any known genetic engineering method (e.g., gene transduction, transfection, or any known gene editing method) to construct the above-mentioned biological materials.

[0081] 6-Aminohexanoic acid producing strains Thirdly, this application provides an engineered strain, namely a 6-aminocaproic acid producing strain, which is obtained by modifying a starting strain. The modification includes: expressing the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application or introducing a nucleic acid molecule encoding the acylated aldehyde dehydrogenase AldDH mutant.

[0082] In this application, the term "introduction" generally refers to the transfer of a foreign gene into recipient cells, such as eukaryotic or prokaryotic recipient cells. There are no particular limitations on the method of introduction; any known transformation method that can transfer the target gene into the recipient cell is acceptable. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria via chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electrotransformation). (2) transducing the target gene into the host bacteria via bacteriophage transduction. (3) transferring the target gene into plant recipient cells via physical or chemical methods, such as gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electroshock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method, and eddy current method, etc. (4) Using vectors to transfer the target gene into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium-mediated method), plant virus vector mediated transformation method, etc.

[0083] The methods for expressing the acylated aldehyde dehydrogenase AldDH mutant and for introducing nucleic acid molecules encoding the acylated aldehyde dehydrogenase AldDH mutant are both conventional in the art. For example, the nucleic acid molecule encoding the acylated aldehyde dehydrogenase AldDH mutant can be introduced into the starting strain via a plasmid; or it can be integrated into the chromosome of the starting strain using genetic engineering techniques.

[0084] This application does not limit the type of the starting strain, as long as it can produce 6-aminocaproic acid using the AldDH mutant of acylated aldehyde dehydrogenase described in this application, it falls within the protection scope of this application.

[0085] In some embodiments, the starting strain is a strain capable of expressing T7 RNA polymerase.

[0086] In some embodiments, the starting strain is *Escherichia coli*. In some embodiments, the starting strain is *Escherichia coli* capable of expressing T7 RNA polymerase. In some embodiments, the starting strain is *Escherichia coli* BL21Star (DE3) or a derivative thereof.

[0087] In this application, the term "BL21 Star (DE3) derivative" generally refers to a strain obtained by introducing new mutations, plasmids, or characteristics into BL21 Star (DE3) through genetic engineering. As long as it can express T7 RNA polymerase, it meets the requirements of this application.

[0088] In some embodiments, the starting strain is Escherichia coli BL21 Star (DE3), BL21(DE3)pLysS, Rosetta (DE3), or Lemo21 (DE3).

[0089] In some embodiments, the starting strain is Escherichia coli BL21 Star (DE3).

[0090] In some embodiments, the 6-aminohexanoic acid producing strain further includes the following modifications: expressing one or more of the following: 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolytic enzyme paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr.

[0091] In some embodiments, the 6-aminohexanoic acid producing strain further includes the following modifications: expression of 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolytic enzyme paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr. In some embodiments, the NCBI accession number for the 4-aminobutyric acid transaminase gabT is WP_010984019.1; the NCBI accession number for the β-ketoadysyl-CoA thiolytic enzyme paaJ is WP_001206197.1; the NCBI accession number for the 3-hydroxyadysyl-CoA dehydrogenase paaH is WP_114399327.1; the NCBI accession number for the 2,3-dehydroadysyl-CoA hydratase paaF is KPP80590.1; and the NCBI accession number for the enoyl-ACP reductase etr is EGS22849.1.

[0092] The methods for expressing 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolytic enzyme paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr are conventional in the art.

[0093] Fourthly, this application provides a method for constructing a 6-aminohexanoic acid producing strain, comprising: exogenously expressing the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application into the 6-aminohexanoic acid producing strain, thereby constructing and obtaining the 6-aminohexanoic acid producing strain described in the third aspect of this application.

[0094] The method for constructing a 6-aminohexanoic acid-producing strain is conventional in the art. For example, the nucleotide sequence encoding the acylated aldehyde dehydrogenase AldDH mutant can be cloned into a suitable expression vector (such as the pET series), and then the recombinant vector can be introduced into the host strain (i.e., the starting strain, such as Escherichia coli) using chemical transformation or electroporation. Alternatively, gene editing technologies such as CRISPR-Cas9 can be used to integrate the exogenous gene into the host chromosome to achieve stable expression.

[0095] Furthermore, those skilled in the art will understand that there are multiple metabolic pathways for the production of 6-aminohexanoic acid from the acylated aldehyde dehydrogenase AldDH. Based on the metabolic requirements of different pathways, those skilled in the art can optimize and modify the 6-aminohexanoic acid-producing strains accordingly to obtain engineered strains suitable for specific substrate utilization and efficient 6-aminohexanoic acid synthesis.

[0096] application Fifthly, this application provides the use of the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application, the biological material described in the second aspect of this application, the 6-aminohexanoic acid producing strain described in the third aspect of this application, and / or the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application in the production of 6-aminohexanoic acid.

[0097] Sixthly, this application provides the use of the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application, the biological material described in the second aspect of this application, the 6-aminohexanoic acid producing strain described in the third aspect of this application, and / or the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application in the preparation of products containing 6-aminohexanoic acid.

[0098] Seventhly, this application provides the application of the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application, the biological material described in the second aspect of this application, the 6-aminohexanoic acid producing strain described in the third aspect of this application, and / or the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application in increasing the production yield of 6-aminohexanoic acid.

[0099] Eighthly, this application provides the use of the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application, the biological material described in the second aspect of this application, the 6-aminohexanoic acid producing strain described in the third aspect of this application, and / or the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application in microbial genetic breeding for the production of 6-aminohexanoic acid.

[0100] Ninthly, this application provides the use of the acylated aldehyde dehydrogenase AldDH mutant described in the first aspect of this application, or the A1) nucleic acid molecule, A2) expression cassette, and / or A3) recombinant vector in the biological material described in the second aspect of this application, in the construction of 6-aminocaproic acid producing strains.

[0101] Methods for producing 6-aminohexanoic acid / increasing the yield of 6-aminohexanoic acid In a tenth aspect, this application provides a method for producing 6-aminohexanoic acid, comprising: culturing the 6-aminohexanoic acid producing strain described in the third aspect of this application, or culturing the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application, thereby producing 6-aminohexanoic acid.

[0102] In some embodiments, 6-aminohexanoic acid is produced by fermentation. In other embodiments, 6-aminohexanoic acid is produced by fermentation using glucose as a carbon source.

[0103] In some embodiments, when 6-aminohexanoic acid is produced by fermentation using glucose as a carbon source, a 6-aminohexanoic acid producing strain expressing acylated aldehyde dehydrogenase AldDH mutant, 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolase paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr can be inoculated into the fermentation medium, and IPTG can be added for fermentation culture to achieve the production of 6-aminohexanoic acid.

[0104] The fermentation medium can be any suitable medium, as long as it can maintain the normal growth and activity of the 6-aminohexanoic acid producing strain and support the efficient conversion of glucose to 6-aminohexanoic acid by the acylated aldehyde dehydrogenase AldDH mutant expressed by the strain. Those skilled in the art can select and appropriately optimize conventional media according to the specific strain; this application does not impose any limitations in this regard.

[0105] Eleventhly, this application also provides a method for increasing the production yield of 6-aminohexanoic acid, comprising: culturing the 6-aminohexanoic acid producing strain described in the third aspect of this application, or culturing the 6-aminohexanoic acid producing strain constructed according to the method described in the fourth aspect of this application.

[0106] In some embodiments, 6-aminohexanoic acid is produced by fermentation. In other embodiments, 6-aminohexanoic acid is produced by fermentation using glucose as a carbon source.

[0107] In this application, the inventors discovered that using the 6-aminohexanoic acid producing strain of this application for the production of 6-aminohexanoic acid results in low cost and significantly increases the yield of 6-aminohexanoic acid.

[0108] Example The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0109] Example 1: Construction of AldDH single mutant library and verification of de novo fermentation production of 6-aminohexanoic acid A schematic diagram of the production of 6-aminohexanoic acid from glucose as a substrate is shown below. Figure 1 As shown.

[0110] derived from *Aminophilus musei* (Massai) Acidaminococcus massiliensisUsing the parental acylated aldehyde dehydrogenase AldDH gene (its amino acid sequence is shown in SEQ ID NO: 1) as a template, an artificially designed and optimized gene nucleic acid sequence (its gene sequence is shown in SEQ ID NO: 4) was used. Using this gene fragment as a template, PCR was performed using primers AldDH-F (TTTTGTTTAACTTTAATAAGGAGATATACCATGGAACAGGCGG TGAAAGACTA, SEQ ID NO: 12) and AldDH-R (ATGATGGTGATGGCTGCTT CATTCCGCCCAAATCTCTTCG, SEQ ID NO: 13) to obtain an AldDH fragment of approximately 1.4 kb, which was then purified by PCR and used as the control parental fragment.

[0111] Using this optimized gene fragment as a template, overlap PCR site-directed mutagenesis was employed to perform single-point mutations on key residues in the active pocket and adjacent regions, constructing a mutant library containing the following sites: A67G, C69A, C69N, N71A, N71H, N71K, N71G, S75G, S75A, S75T, W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78H, W78I, and W78L. , W78S, W78P, W78K, W78A, W78R, W78V, R86V, R86C, R86I, R86L, I118S, I118A, F238A, F23 8Y, L243F, L243A, T245N, T245A, W350C, W350T, W350A, W350P, W350H, W350Q, W350I, W350 E. W350F, W350S, W350Y, W350R, W350L, W350V, W350G, W350M, W350K, G399H, G399V, G399 P, G399C, G399K, G399L, G399W, G399T, G399I, G399Y, G399N, G399R, G399F, G399Q, G399D , G399S, T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K, T400R, N401G, N401T , N401A, A405T, A405K, A405G, A405S, A405H, A405R, T414N, T414A, T414F, T414M, T414V.

[0112] Using A405G as an example, the mutated gene fragment was amplified segmentally using primers A405G-F (GGTAGGTGGAACCAATCTTGGTGGACATATGGATAACGGACTGAACCCAACT, SEQ ID NO: 14) and A405G-R (AGTTGGGTTCAGTCCGTTATCCATATGTCCACCAAGATTGGTTCCACCTAC, SEQ ID NO: 15). Overlap PCR extension yielded the complete AldDH_A405G gene fragment (approximately 1.4 kb), the sequence of which is shown in SEQ ID NO: 5. All other single-point mutants were constructed using the same method, resulting in a total of 94 single-point mutant gene fragments.

[0113] According to Escherichia coli ( Escherichia coli The amino acid sequence of the 4-aminobutyric acid transaminase gabT (NCBI accession number WP_010984019.1) was artificially designed and optimized (gene sequence shown in SEQ ID NO: 7). Using this gene fragment as a template, PCR was performed using primers gabT-F (ATCTTAGTATATTAGTTAAG TATAAGAAGGAGATACATATGAGCGCACTTCCGCAG, SEQ ID NO: 16) and gabT-R (GGTGGCAGCAGCCTAGGTTAATTAGATACGGGAGAACGCCTGC, SEQ ID NO: 17) to obtain a gabT fragment of approximately 1.3 kb, which was then purified by PCR.

[0114] Using the Gibson Assembly kit (NEB), the purified AldDH, its single-point mutant fragment, and the gabT fragment were ligated into the pCDFDuet-1 vector in one step and placed downstream of two T7 promoters. The resulting recombinant plasmids were named pCDF-AldDH-1-gabT, pCDF-AldDH-2-gabT to pCDF-AldDH-94-gabT, and the parental plasmid pCDF-AldDH-WT-gabT.

[0115] Using the genome of Escherichia coli MG1655 (NCBI accession number GCF_000005845.2) as a template, PCR was performed using primers paaJ-F (AGGACGCTAAAAGAAGGAGATATACATGCGTGAAGCCTTTAT TTGTGACG, SEQ ID NO: 18) and paaJ-R (TTAAGCATTATGCGGCCGCTCAA ACACGCTCCAGAATCATGG, SEQ ID NO: 19) to obtain a paaJ gene fragment of approximately 1.2 kb (NCBI accession number for amino acid sequence WP_001206197.1, nucleotide sequence as shown in SEQ ID NO: 13) and purified by PCR.

[0116] 3-hydroxyadiyl-CoA dehydrogenase paaH (NCBI accession number for amino acids WP_114399327.1, gene sequence as shown in SEQ ID NO: 14) was artificially synthesized. Using this gene fragment as a template, primers paaH-F (TTGGTGATACGTAAAAGAAGGAGATATACATGGCAGAGTC GAATGCTGC, SEQ ID NO: 20) and paaH-R (CCGATATCCAATTGAGATCTGCC ATTTAAGGTGTCGTAGTAAGAGCCGG, SEQ ID NO: 21) were used for amplification to obtain a paaH gene fragment of approximately 1.2 kb, which was then purified by PCR.

[0117] 2,3-Dehydroadipyl-CoA hydratase paaF (NCBI accession number for amino acids KPP80590.1, gene sequence shown in SEQ ID NO: 10) was artificially synthesized. Using this gene fragment as a template, primers paaF-F (GGATCGGGAGGTGGCAGCGGAGGCGGTTCGATGAGCGAACTGATCGTCAGCCG, SEQ ID NO: 22) and paaF-R (CGCATGTATATCTCCTTCTTTT AGCGTCCTTTAAAGTCGGGCG, SEQ ID NO: 23) were used for amplification, yielding a paaF gene fragment of approximately 0.8 kb, which was then purified by PCR.

[0118] An artificially synthesized enoyl ACP reductase etr (NCBI accession number EGS22849.1 for amino acids, gene sequence shown in SEQ ID NO: 11) was used as a template for amplification with primers etr-F (TAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCGAGCATTTTGTCGC, SEQ ID NO: 24) and etr-R (TGCCATGTATATCTCCTTCTTTTACGTATCACCAAAAATGAATACTCCTTTGC, SEQ ID NO: 25). An etr fragment of approximately 1.3 kb was obtained and purified by PCR.

[0119] The pETDuet-1 plasmid was ligated with the purified paaJ, paaH, paaF and etr fragments obtained above using the Gibson Assembly kit (NEB). The resulting recombinant plasmid was named pET-JHFE.

[0120] The recombinant plasmid pET-JHFE, along with the recombinant plasmid pCDF-AldDH-1-gabT containing wild-type AldDH and 94 single-point mutants respectively, and the parental plasmid pCDF-AldDH-WT-gabT, were co-transformed into Escherichia coli BL21 Star (DE3) competent cells via electroporation to obtain 58 co-expressing recombinant strains. The strains were named as follows: directly after the introduced site mutation, such as A405G, T400R, etc., and the wild-type was named WT. At the same time, negative control strains were set up, and the empty plasmids pETDuet-1 and pCDFDuet-1 were co-transformed into E. coli chassis to obtain the control strain Ec / empty.

[0121] The recombinant strain and control strain constructed above were inoculated into fermentation medium and cultured in 48-well plates at a fermentation temperature of 30℃ and a rotation speed of 990 rpm. Initial induction was achieved by adding 0.1 mM IPTG, and fermentation continued for 48 h.

[0122] The fermentation medium formula (g / L) is: glucose 10, diammonium hydrogen phosphate ((NH4)2HPO4) 4, potassium dihydrogen phosphate (KH2PO4) 6.7, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.8, yeast extract 2.5, citric acid 0.8, and 5 mL of trace metal solution; the trace metal solution composition includes (g / L): ferrous sulfate heptahydrate (FeSO4·7H2O) 10, calcium chloride dihydrate (CaCl2·2H2O) 2, zinc sulfate heptahydrate (ZnSO4·7H2O) 2.2, manganese sulfate tetrahydrate (MnSO4·4H2O) 0.5, copper sulfate pentahydrate (CuSO4·5H2O) 1, ammonium molybdate tetrahydrate ((NH4)6Mo7O) 24 ·4H2O) 0.1, Borax (Na2B4O7·10H2O) 0.02, Glutamic acid 3.

[0123] After 48 hours of fermentation, the accumulation of 6-aminohexanoic acid in the strain was detected by high-performance liquid chromatography (HPLC). The results are shown in Table 1 below. The results showed that the blank plasmid control strain Ec / empty did not synthesize 6-aminohexanoic acid. Among the mutants, the A405G mutant had the highest 6-aminohexanoic acid yield, reaching 0.78 g / L, which was 50% higher than the control (0.52 g / L). In addition, several mutants at positions G399 and T400 also showed significant activity enhancement.

[0124] Table 1. Effect of AldDH single mutant on the yield of 6-aminohexanoic acid produced by de novo fermentation.

[0125] Example 2: Prediction and Fermentation Verification of Combined Mutants Based on the single-point mutants obtained in Example 1, combined mutation predictions were performed on the three key sites G399, T400, and A405 using a protein rational design and machine learning model. This resulted in the construction of mutants including G399V / T400K / A405G, G399T / T400H / A405G, G399V / T400A / A405G, G399C / T400I / A405G, G399R / T400R / A405G, G399C / T400L / A405G, and G399Q / T400L / A405G, G399F / T400H / A405G, G399T / T400A / A405G, G399C / T400K / A405G, G399Y / T400I / A405G, G399T / T400Y / A405G, G399V / T400R / A405G, G399S / T400S / A405G, G399T / T400K / A405G, G399H / T400H / A405G, G399H / T400R / A405G, G399C / T400H / A405G, G399W / T400H / A405G, G399T / T400H / A405H, G399S / T400H / A405H, G399Q / T400H / A405H, G399N / T400H / A405H, G399P / T400H / A405H, G399F / T400H / A405H, G399H / T400H / A405H, G399R / T400H / Several combined mutants, including A405H, G399F / A405G, G399Q / A405G, T400H / A405G, and T400H / A405H, are represented by the G399C / T400R / A405G triple mutant, with the gene sequence shown in SEQ ID NO: 6. Using the recombinant plasmid pCDF-AldDH-86-gabT containing the A405G single-point mutation constructed in Example 1 as a template, site-directed mutagenesis was employed. Using primers G399C-T400R-F (GTCGGTTCGCAGTTAACCAGGTAGGTTGTGCTAATCTTGGTGGACATATGGATAAC, SEQ ID NO: 26) and G399C-T400R-R (GTTATCCATATGTCCAC CAAGATTAGCACAACCTACCTGGTTAACTGCGAACCGAC, SEQ ID NO: 27), mutations at the G399C and T400R sites were introduced directly onto the plasmid. This resulted in a recombinant plasmid carrying three mutations, which was verified to be correct by sequencing.

[0126] The above-mentioned combined mutant plasmid and pET-JHFE plasmid were co-transformed into Escherichia coli chassis BL21 Star (DE3) to obtain the corresponding recombinant strains, which were directly named according to the combined mutation sites, and the parental strain and A405G single mutant strain were used as controls.

[0127] The recombinant strain and control strain constructed above were inoculated into fermentation medium and cultured in 48-well plates at a fermentation temperature of 30℃ and a rotation speed of 990 rpm. Initial induction was achieved by adding 0.1 mM IPTG, and fermentation was continued for 48 h. The fermentation medium formulation was the same as in Example 1. The accumulation of 6-aminohexanoic acid in the strain was detected by high performance liquid chromatography (HPLC).

[0128] The results are shown in Table 2 below. The fermentation results show that the catalytic efficiency of most combined mutants is similar to that of the optimal single-point mutant A405G. Among them, the yield of the G399C / T400R / A405G triple mutant reached 0.83 g / L, which is slightly higher than that of the A405G single mutant. It is the mutant with the highest activity among all combined mutants, which proves that there is a synergistic effect between the G399, T400 and A405 sites, which can further optimize the catalytic performance of the enzyme.

[0129] Based on the screening results of single-point mutations and combined mutations, the G399C / T400R / A405G triple mutant was identified as the representative dominant mutant.

[0130] Table 2. Effects of AldDH multipoint mutation on the yield of 6-aminohexanoic acid produced from de novo fermentation.

[0131] The following sequences are involved in this application:

[0132] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. An acylated aldehyde dehydrogenase AldDH mutant comprising a mutation based on a reference sequence as shown in SEQ ID NO: 1, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises an amino acid mutation corresponding to at least one of the following sites in SEQ ID NO: 1: A67, C69, N71, S75, W78, R86, I118, F238, L243, T245, W350, G399, T400, N401, A405, T414; Preferably, the acylated aldehyde dehydrogenase AldDH mutant contains an amino acid mutation at at least one of the sites G399, T400, and A405 corresponding to SEQ ID NO:

1.

2. The acylated aldehyde dehydrogenase AldDH mutant according to claim 1, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises any one or more of the following mutations: (1) A67G; (2) C69A or C69N; (3) N71A, N71H or N71G; (4) S75A or S75T; (5) W78Y, W78D, W78C, W78T, W78E, W78M, W78G, W78L, W78S, W78A, W78R or W78V; (6) R86V, R86C, R86I or R86L; (7) I118S; (8) F238A or F238Y; (9) L243A; (10) T245N; (11) W350A, W350P, W350Q, W350I, W350E, W350F, W350S, W350Y, W350L, W350V, W350G, W350M or W350K; (12) G399V, G399T, G399I, G399Y, G399F or G399Q; (13) T400H, T400A, T400I, T400S, T400Y, T400L, T400W, T400K or T400R; (14) N401G; (15) A405K, A405G, A405S or A405H; (16) T414A or T414V.

3. The acylated aldehyde dehydrogenase AldDH mutant according to claim 2, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises A405G.

4. The acylated aldehyde dehydrogenase AldDH mutant according to any one of claims 1-3, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises any of the following combinations of mutations: G399V / T400K / A405G, G399C / T400I / A405G, G399C / T400R / A405G, G399R / T400R / A405G, G399C / T400L / A405G, G399Q / T400L / A405G, G399F / T400H / A405G, G399T / T400A / A405G, G399C / T400K / A405G, G399Y / T400I / A405G, G399T / T400Y / A405G, G399V / T400R / A405G, G399S / T400S / A405G, G399T / T400K / A405G, G399H / T400R / A405G, G399C / T400H / A405G, G399T / T400H / A405H, G399S / T400H / A405H, G399F / A405G, T400H / A405G, T400H / A405H.

5. The acylated aldehyde dehydrogenase AldDH mutant according to claim 4, wherein the acylated aldehyde dehydrogenase AldDH mutant comprises the G399C / T400R / A405G mutant combination.

6. The acylated aldehyde dehydrogenase AldDH mutant according to any one of claims 1-5, wherein the amino acid sequence of the acylated aldehyde dehydrogenase AldDH mutant is as shown in SEQ ID NO: 2 or 3, or has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 3.

7. A biomaterial comprising any one of the following materials: A1) A nucleic acid molecule encoding an acylated aldehyde dehydrogenase AldDH mutant as described in any one of claims 1-6, preferably, the nucleic acid molecule having a sequence as shown in SEQ ID NO: 5 or 6, or having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 5 or 6. A2) Expression cassette, which contains the nucleic acid molecule described in A1); A3) A recombinant vector comprising the nucleic acid molecule described in A1) and / or the expression cassette described in A2); A4) A recombinant microorganism comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3), preferably, the recombinant microorganism being Escherichia coli; A5) Recombinant cells comprising the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst comprising the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

8. A 6-aminohexanoic acid producing strain, obtained by modifying a starting strain, wherein the modification includes: Expressing the acylated aldehyde dehydrogenase AldDH mutant as described in any one of claims 1-6 or introducing a nucleic acid molecule encoding the acylated aldehyde dehydrogenase AldDH mutant as described in any one of claims 1-6.

9. The 6-aminohexanoic acid producing strain according to claim 8, wherein the modification further comprises: It expresses one or more of the following: 4-aminobutyric acid transaminase gabT, β-ketoadysyl-CoA thiolytic enzyme paaJ, 3-hydroxyadysyl-CoA dehydrogenase paaH, 2,3-dehydroadysyl-CoA hydratase paaF, and enoyl-ACP reductase etr.

10. The 6-aminohexanoic acid producing strain according to claim 8 or 9, wherein the starting strain is Escherichia coli.

11. A method for constructing a 6-aminohexanoic acid-producing strain, comprising: The 6-aminohexanoic acid producing strain is exogenously expressed with the acylated aldehyde dehydrogenase AldDH mutant as described in any one of claims 1-6, thereby constructing and obtaining the 6-aminohexanoic acid producing strain as described in any one of claims 8-10.

12. The use of the acylated aldehyde dehydrogenase AldDH mutant according to any one of claims 1-6, the biomaterial according to claim 7, and / or the 6-aminohexanoic acid producing strain according to any one of claims 8-10 in any of the following: (1) Application in the production of 6-aminohexanoic acid; (2) Application in the preparation of products containing 6-aminohexanoic acid; (3) Application in increasing the production yield of 6-aminohexanoic acid; (4) Application in the genetic breeding of microorganisms for the production of 6-aminocaproic acid.

13. A method for producing 6-aminohexanoic acid, comprising: Cultivate the 6-aminohexanoic acid producing strain as described in any one of claims 8-10 or cultivate the 6-aminohexanoic acid producing strain obtained by the method described in claim 11 to produce 6-aminohexanoic acid. Preferably, 6-aminohexanoic acid is produced by fermentation using glucose as a carbon source.

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