Buddleja officinalis acetaldehyde dehydrogenase and coding gene and application thereof

Abstract

The application provides crocin aldehyde dehydrogenase and its coding gene and application. The application provides BomALDH1, BomALDH2 and BomALDH3 aldehyde dehydrogenases, and it is proved through in-vitro enzyme activity experiment that the proteins coded by the genes all have the activity of catalyzing crocetin dialdehyde to generate crocetin. The application also provides a recombinant expression vector containing the above-mentioned genes, a recombinant engineering bacterium and its application. The application provides a new type of gene element for high-efficiency biosynthesis of crocetin and crocin, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a method derived from Buddleja officinalis (… Buddleia medicinal plant acetaldehyde dehydrogenase (Maxim.) and its encoding gene and applications. Background Technology

[0002] Crocetin is the core active ingredient of saffron, a precious traditional Chinese medicine, and a key intermediate in the biosynthesis of crocin. It belongs to the polyene-conjugated apoenoloid carotenoid class of compounds. Modern pharmacological studies have confirmed that crocetin possesses significant antioxidant, antitumor, neuroprotective, and antidepressant activities. Crocetin can also inhibit the proliferation of colon cancer cells by inducing apoptosis and effectively treat colon cancer.

[0003] Currently, commercially available saffron acid is mainly prepared from the traditional Chinese medicinal herbs saffron and gardenia through chemical hydrolysis after extraction of saffron glycosides. Saffron is the plant *Saffron crocus* (Iridaceae family). Crocus sativus The dried stigmas of *Gardenia jasminoides* (L.) yield less than 1 kg per mu (approximately 0.16 acres), and cultivation is difficult, resulting in a severe shortage of medicinal material production capacity, with nearly 80% relying on imports. Meanwhile, *Gardenia jasminoides* (L.) yields less than 1 kg per mu (approximately 0.16 acres), and cultivation is difficult, leading to a severe shortage of medicinal material production capacity Gardenia jasmine The chemical hydrolysis process for extracting crocin (J. Ellis) suffers from problems such as cumbersome procedures and high production costs. Meanwhile, the chemical synthesis of crocin also faces technical bottlenecks, including complex steps, numerous byproducts, and low yields. Resource scarcity and limitations in preparation technology have severely restricted the large-scale production and application of crocin and crocin.

[0004] As the biosynthetic pathways of crocin and crocin have been gradually elucidated, researchers have achieved de novo synthesis of both in model plants such as tobacco and tomato, as well as in microbial systems such as *Escherichia coli* and cyanobacteria. However, current yields are only at the milligram level, which still cannot meet the needs of industrialization. In the biosynthetic pathway of crocin, zeaxanthin is cleaved by carotenoid cleavage dioxygenases (CCDs, such as BomCCD4 in *Buddleja officinalis*) to produce crocin dialdehyde, which is then oxidized by aldehyde dehydrogenases (ALDHs) to produce crocin. Currently, there are reports of aldehyde dehydrogenases from saffron that can catalyze the conversion of crocin dialdehyde to crocin. In addition, existing technologies also disclose carotenoid lysing dioxygenase BomCCD4 and its encoding gene from Buddleja officinalis, which can catalyze the production of crocin from crocin dialdehyde; and acetaldehyde dehydrogenase GjALDH2C3 from Gardenia jasminoides and its encoding gene, which can catalyze the production of crocin from crocin dialdehyde. Summary of the Invention

[0005] This invention is based on screening of full-length transcriptome data from *Buddleja officinalis*. BomALDH1 , BomALDH2 , BomALDH3 The genes and their corresponding acetaldehyde dehydrogenases were identified, and in vitro enzyme activity experiments demonstrated that the proteins encoded by these genes all have the activity of catalyzing the conversion of crocin dialdehyde to crocin, providing novel gene elements for the efficient biosynthesis of crocin and crocin glycosides.

[0006] During the research process, it was discovered that Buddleja officinalis ( Buddleja officinalis Crocin and crocin glycosides were detected in the flower tissue of *Maxim.*, indicating the existence of a complete biosynthetic pathway for crocin and crocin glycosides in the plant. This invention aims to provide novel gene elements for the efficient biosynthesis of crocin and crocin glycosides by deeply exploring the key enzyme involved in the biosynthesis of crocin and crocin glycosides in *Maxim.*.

[0007] Specifically, the present invention provides the following technical solution: The first aspect of the present invention provides a Buddleja officinalis ( Buddleja officinalis (Maxim.) aldehyde dehydrogenase, wherein the aldehyde dehydrogenase is selected from any of the following: (a) Proteins with amino acid sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; (b) A derivative protein having one to three amino acids substituted, deleted or added in the amino acid sequence described in (a) and having catalytic activity in the formation of crocin from crocin dialdehyde.

[0008] Based on the full-length transcriptome data of *Buddleja officinalis*, this invention uses *Gardenia jasminoides* GjALDH2C3 (GeneBank: ARU08110) as a reference sequence. By searching the *Buddleja officinalis* transcriptome sequence library using BLASTP, three genes with homology less than 85% and greater than 70% were selected and named BomALDH1, BomALDH2, and BomALDH3, respectively. The amino acid sequences encoded by these genes are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively, and all exhibit catalytic activity in the formation of crocin from crocin dialdehyde.

[0009] Phylogenetic analysis shows that BomALDH1, BomALDH2, and BomALDH3 are in the same evolutionary branch as GjALDH2C3, belonging to the 2C subfamily of ALDH.

[0010] Tissue-specific gene expression analysis revealed that BomALDH1 and BomALDH2The highest expression levels were observed in the flower buds and leaves of *Buddleja officinalis*; while BomALDH3 The gene was expressed at the highest level in flower tissues, consistent with the saffron acid accumulation pattern, suggesting that... BomALDH3 It may be mainly involved in the synthesis of crocin and crocin in Buddleja officinalis.

[0011] In vitro enzyme activity assays showed that BomALDH1, BomALDH2, and BomALDH3 proteins all possessed catalytic activity in the conversion of crocin dialdehyde to crocin. Under the same in vitro enzyme activity reaction conditions, BomALDH3 catalyzed the highest yield of crocin, indicating that BomALDH3 has the highest catalytic efficiency in the conversion of crocin dialdehyde to crocin.

[0012] A second aspect of the invention provides a separable nucleic acid molecule that encodes the aforementioned daphne acetaldehyde dehydrogenase.

[0013] According to embodiments of the present invention, the separable nucleic acid molecule is selected from at least one of the following: (a) A nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (b) has at least 85% homology with the nucleotide sequence shown in (a), and the encoded protein has a nucleotide sequence that catalyzes the formation of crocin from crocin dialdehyde.

[0014] A third aspect of the present invention provides an expression vector containing the separable nucleic acid molecule described in the second aspect.

[0015] According to an embodiment of the present invention, the expression vector is selected from at least one of plasmids, viruses, or bacteriophages.

[0016] According to an embodiment of the present invention, the expression vector is pET-Duet- BomALDH1 pET-Duet- BomALDH2 or pET-Duet- BomALDH3 .

[0017] A fourth aspect of the present invention provides a recombinant engineered bacterium containing the gene described in the second aspect or the expression vector described in the third aspect.

[0018] According to an embodiment of the present invention, the recombinant engineered bacteria is Escherichia coli BL21(DE3).

[0019] The fifth aspect of the present invention provides the application of the above-described buddleja acetaldehyde dehydrogenase or the above-described gene in catalyzing the production of crocin from crocin dialdehyde.

[0020] The sixth aspect of the present invention provides the use of the above-described buddleja acetaldehyde dehydrogenase or the above-described gene in the preparation of crocin or crocinoside.

[0021] A seventh aspect of the present invention provides a method for producing saffron acid, comprising the following steps: The recombinant engineered bacteria described above expressed acetaldehyde dehydrogenase from Buddleja officinalis, using crocin dialdehyde as a substrate, in NAD... + In the presence of saffron, a catalytic reaction is carried out to produce crocin.

[0022] According to an embodiment of the present invention, the conditions for the catalytic reaction are: temperature 25-37°C, pH 7.0-9.0, and reaction time 1-24 hours.

[0023] The eighth aspect of the present invention provides an expression vector containing the separable nucleic acid molecule described in the second aspect and the nucleic acid molecule containing BomCCD4 (a carotenoid cleavage dioxygenase).

[0024] According to an embodiment of the present invention, the expression vector is selected from at least one of plasmids, viruses, or bacteriophages.

[0025] According to an embodiment of the present invention, the expression vector is pThio- BomCCD4-BomALDH1 pThio- BomCCD4-BomALDH2 or pThio- BomCCD4-BomALDH3 .

[0026] A ninth aspect of the present invention provides a recombinant engineered bacterium containing the genes described in the second aspect and BomCCD4 Gene or expression vector as described in the eighth aspect.

[0027] According to an embodiment of the present invention, the recombinant engineered bacteria is Escherichia coli BL21 ZEA.

[0028] The tenth aspect of the present invention provides the use of the combination of the above-described carotenoid lysin and acetaldehyde dehydrogenase or the above-described gene combination in the preparation of crocin or crocinoside.

[0029] The eleventh aspect of the present invention provides a method for producing saffron acid, comprising the following steps: The recombinant engineered bacteria described above express carotenoid lysin dioxygenase and acetaldehyde dehydrogenase, and biosynthesize saffron acid in vivo.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects: This invention marks the first identification and isolation of acetaldehyde dehydrogenases BomALDH1, BomALDH2, and BomALDH3 from *Buddleja officinalis*, which possess catalytic activity for the conversion of crocin dialdehyde to crocin, thus enriching the enzyme resource library for catalytic crocin biosynthesis. The provided BomALDH3 exhibits significantly higher catalytic efficiency than BomALDH1 and BomALDH2, and can serve as a preferred gene element for the efficient biosynthesis of crocin and crocinoside.

[0031] The amino acid sequence of acetaldehyde dehydrogenase from Buddleja officinalis and its encoding gene provided by this invention can serve as a novel gene element for the efficient heterologous synthesis of crocin and crocin in microorganisms and plants, and is expected to realize their efficient heterologous biosynthesis, which has important application value and broad market prospects. Attached Figure Description

[0032] Figure 1 The image shows the UHPLC-MS analysis results of crocin content in different tissues of Buddleja officinalis.

[0033] Figure 2 Phylogenetic tree analysis diagram of ALDH family members such as BomALDH1, BomALDH2, BomALDH3, and GjALDH2C3.

[0034] Figure 3 for BomALDH1 , BomALDH2 and BomALDH3 Graph showing the specific expression of genes in different tissues of *Buddleja officinalis*; statistical analysis was performed using Student's t-test, with a sample size of n = 4. : P <0.05; : P <0.01; : P <0.001.

[0035] Figure 4 This is a schematic diagram of the recombinant expression vectors for BomALDH1, BomALDH2, and BomALDH3.

[0036] Figure 5The image shows the SDS-PAGE electrophoresis analysis of BomALDH1 protein, where M: protein molecular weight standard; 1: uninduced cells; 2: cells after 20 hours of induction; 3: supernatant after cell disruption and centrifugation; 4: flow-through buffer after loading onto a nickel column; 5: elution product using 20 mM imidazole elution buffer; 6: elution product using 30 mM imidazole elution buffer; 7: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 8: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 9: elution product using 3 column volumes of elution buffer (containing 250 mM imidazole).

[0037] Figure 6 The image shows the SDS-PAGE electrophoresis analysis of BomALDH2 protein, where M: protein molecular weight standard; 1: uninduced cells; 2: cells after 20 hours of induction; 3: supernatant after cell disruption and centrifugation; 4: flow-through buffer after Ni-NTA loading; 5: elution product using 20 mM imidazole elution buffer; 6: elution product using 30 mM imidazole elution buffer; 7: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 8: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 9: elution product using 3 column volumes of elution buffer (containing 250 mM imidazole).

[0038] Figure 7 The image shows the SDS-PAGE electrophoresis analysis of BomALDH3 protein, where M: protein molecular weight standard; 1: uninduced cells; 2: cells after 20 hours of induction; 3: supernatant after cell disruption and centrifugation; 4: flow-through buffer after Ni-NTA loading; 5: elution product using 20 mM imidazole elution buffer; 6: elution product using 30 mM imidazole elution buffer; 7: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 8: elution product using 1 column volume of elution buffer (containing 250 mM imidazole); 9: elution product using 3 column volumes of elution buffer (containing 250 mM imidazole).

[0039] Figure 8 The above are UHPLC-MS chromatograms of the in vitro enzyme activity reaction products of BomALDH1, BomALDH2, and BomALDH3, where A is the ion extraction chromatogram of crocin in the in vitro enzymatic reaction product of BomALDH. m / z 329.1747), Control group: reaction samples without protease; BomALDH1~3: reaction samples with BomALDH1~3 proteins respectively; Standard: crocin standard; B is the MS / MS mass spectrum of crocin and its isomers in the in vitro enzymatic reaction products of BomALDH, with crocin standard as positive control.

[0040] Figure 9 This figure compares the yields of crocin and its isomers catalyzed by BomALDH1, BomALDH2, and BomALDH3 in the production of crocin dialdehyde. The control group consisted of reaction samples without protease; BomALDH1–3 consisted of reaction samples with BomALDH1–3 proteins, respectively. Statistical analysis was performed using Student's t-test with a sample size of n = 3. : P <0.05; : P <0.01.

[0041] Figure 10 pThio- BomCCD4-BomALDH1 pThio- BomCCD4-BomALDH2 and pThio- BomCCD4- BomALDH3 Schematic diagram of the recombinant expression vector.

[0042] Figure 11 For overexpression BomALDH UHPLC-MS analysis chromatogram of Escherichia coli strains, where BomCCD4: expresses only... BomCCD4 E. coli sample; BomCCD4 + BomALDH1~3: Co-expressed respectively BomCCD4 and BomALDH1 , BomALDH2 and BomALDH3 E. coli samples. Detailed Implementation

[0043] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0044] The purpose of this invention is to address the problem of insufficient catalytic efficiency of acetaldehyde dehydrogenase in the biosynthesis of crocin and crocinoside. Based on this, this invention, for the first time, utilizes full-length transcriptomics and metabolomics techniques to identify and isolate novel BomALDH1, BomALDH2, and BomALDH3 from *Buddleja officinalis*. In vitro enzyme activity experiments verified that BomALDH1, BomALDH2, and BomALDH3 all possess catalytic activity in the production of crocin from crocin dialdehyde. This invention provides *Buddleja officinalis*. BomALDH1 , BomALDH2 and BomALDH3 The gene sequence and its encoded amino acid sequence provide novel gene elements for the biosynthesis of crocin and crocin.

[0045] This invention provides an acetaldehyde dehydrogenase derived from Buddleja officinalis and a nucleic acid molecule encoding the acetaldehyde dehydrogenase.

[0046] The acetaldehyde dehydrogenase from Buddleja officinalis provided is selected from at least one of the following: (a) Proteins with amino acid sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; (b) A derivative protein having one to three amino acids substituted, deleted or added in the amino acid sequence described in (a) and having catalytic activity in the formation of crocin from crocin dialdehyde.

[0047] In this invention, the term "substituted, deleted, or added one to three amino acids" refers to a derived protein obtained by artificially introduced, limited amino acid mutations on the amino acid sequences of BomALDH1 (SEQ ID NO:2), BomALDH2 (SEQ ID NO:4), or BomALDH3 (SEQ ID NO:6) acetaldehyde dehydrogenases, which has a slightly different structure from the original sequence but retains its catalytic activity. The phrase "one to three amino acids" means that the total number of mutated amino acid residues in any of the aforementioned amino acid sequences is one, two, or three.

[0048] The separable nucleic acid molecule provided encodes the acetaldehyde dehydrogenase from *Buddleja officinalis*.

[0049] According to a specific embodiment, the separable nucleic acid molecule is selected from at least one of the following: (a) A nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (b) has at least 85% homology with the nucleotide sequence shown in (a), and the encoded protein has a nucleotide sequence that catalyzes the formation of crocin from crocin dialdehyde.

[0050] In this article, the "separable nucleic acid molecules" refers to those extracted from Buddleja officinalis (a plant) using artificial technical means. Buddleja officinalis Nucleic acid molecules are extracted from cells or tissues (Maxim.) and have had other cellular components removed from them. These nucleic acid molecules no longer exist in their naturally occurring form but are in a state of artificial separation and purification.

[0051] In this invention, the terms "homology" or "sequence identity" are used interchangeably, referring to the percentage of identical (i.e., one-to-one base matching) residues within a defined sequence length range after optimal alignment between two nucleotide sequences. Sequence alignment can be performed using various algorithms known in the art, including but not limited to BLASTN, ClustalW, Needleman-Wunsch algorithms (such as the Needle program in the EMBOSS software package), or the FASTA program, typically using the default gap penalty parameters of the aforementioned programs for calculation.

[0052] In this invention, "having at least 85% homology with the nucleotide sequence shown in (a)" means that after optimal alignment using the above sequence alignment algorithm, the nucleotide sequence to be tested and the reference sequences shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 have at least 85% identical nucleotide positions in the corresponding alignment regions (preferably more than 90%, more than 95%, more than 98% or more 99%).

[0053] Those skilled in the art will understand that, due to the degeneracy of the genetic code, multiple different nucleotide sequences can encode the same amino acid sequence. Specifically, except for methionine and tryptophan, the remaining 18 amino acids are encoded by 2 to 6 different codons; synonymous codons within the same codon family differ only in the third base (i.e., codon wobble). Therefore, without altering the amino acid sequence of the encoded protein, one or more codons in the nucleotide sequence of this invention (SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5) can be synonymously substituted. Although the resulting degenerate variant sequence differs from the original sequence in its base sequence, it still falls within the scope of "having at least 85% homology" as described in this invention, provided that the protein encoded by the variant retains the activity of catalyzing the formation of crocin from crocin dialdehyde.

[0054] In other words, the homology range described in this invention covers both non-synonymous sequence differences caused by evolution or artificial mutation, and synonymous sequence differences caused by codon degeneracy. As long as the function of the encoded protein is preserved, it falls within the protection scope of this invention.

[0055] BomALDH1 The cDNA sequence is shown in SEQ ID NO:1: The amino acid sequence of BomALDH1 is shown in SEQ ID NO: 2: MTERTLYYISDLGRRKRKIGMTAQNGSVESNIKIPTIKFTKLFINGEFVDSISGKTFETIDPRTGEVIANIAEGGKEDVDLAVKAARQAFDNGPWPRLPGSERRRILLKFADLIDENTEELAGLDAIDGGKLYSMAKILEIPGVAETIRYFAGAADKIHGATLKMSRELHGYTLREPIGVVGHIIPWNFPSQMFVMKVGPALAAGCTMLVKPAEQTPLSALYYAHLAKLAGIPDGVLNVVTGYGYSAGAAISSHMDIDKVSFTGSTEIGRLVMQAAATSNLKPVSLELGGKSPFIIFNDVDVDKVADLALQGTLFNKGEICVAGSRVFVQEGIYDEFLVKLVEKTKTWVVGDPFDPNVHQGPQVDKKQYERILSFIELGKREGATLLTGGNPCEGKGYYIEPTIFTDVADDMIIAKEEIFGPVMSVMKFKTVEEVIKRANATKYGLAAGIMTNDLNIANTVSRSIRAGVIWINCYFAFDRDLPYGGYKMSGFERDMGLESLDKYLQLKSVAMPIHNSPWL (SEQ ID NO: 2) BomALDH2 The cDNA sequence is shown in SEQ ID NO: 3: The amino acid sequence of BomALDH2 is shown in SEQ ID NO: 4: MSTSNGASILDEYSHNIKIPKIKFTKLFINGHFVDSISGNTFETIDPRTEEVIANIAEGDKDDVDLAVNAARQAFDHGPWPRLPGSERRRIMLKFADLIDENVEELAALDAIDGGKLYKMAKLADIPGAAETIRYFAGAADKIHGATLKMSRALQGYTLREPVGVVGHIIPWNFPSQMFVMKVGPALAAGCTMVVKPAEQTPLSALYYAHLAKLAGIPDGVVNVVTGYGHAAGAAISSHMDIDMVSFTGSTEVGRLVMQAAAQSNLKPVSLELGGKSPFIVFDDVDVDKVADLALQATLYNKGEICVAGTRVFIQEGIYDKVLTKLVEKAKAWVVGDPFDPNVHQGPQVNKKQYERILSFIELGKKEGATLLTGGKPCDSKKGYYIQPTIFTDVDDDMTIAKEEIFGPVMSVMKFKTVEEVIKRANASKYGLATGIMTNNLNIANTVSRSIRAGVIWINCYFAFDRDLPYGGYKMSGFGRDLGMESLSKYLQVKSIATPIYDSPWL(SEQ ID NO: 4) BomALDH3 The cDNA sequence is shown in SEQ ID NO: 5: The amino acid sequence of the BomALDH3 protein is shown in SEQ ID NO:6: MAGESNGNVETSMMKMIPKIKFTKLFINGEFIDSTSGKTFETIDPRTEEVIANVSEGDKDDVDLAVTAARQAFDHGPWPRLPGCERRRIMLKFADLMDENVEELAALDTIDAANLYSLGKGMFIHGA AELIRYYAGAADKIHGETLKMSRDLHGYTLVEPIGVVGHIIPWNFPTQMFVMKVGPALAAGCTMVVKPAEQTPLSALYYAYLAKLAGVPDGVINVVTGYGPTVGASISSHMDIDMVSFTGSTETGHRI MQAAATSNLKVVSLELGGKSPFIVFDDADVDKIAPLALTACLYNQGQICVAGTRTFVQEGIYDKFVTRLLEELKTWVVGDPFDSNVNQGPQVDKKQYEKVLSYIEHGKREGATLLSGGKPLDTKGKGK GYYIEPTIFTDVTDDMIIAKDEIFGPVMSLMKFKSVEEVIKRANGTKYGLAAGIMTNDLNIANTVSRSLRAGAVWVNCYFGFDNDLPIGGYKRSGFGKDMGMDALHKYLQHKSIAFPIYNSPWL (SEQ ID NO:6) Phylogenetic analysis of these genes showed that the three ALDH genes, BomALDH1, BomALDH2, and BomALDH3, belong to the same evolutionary branch as GjALDH2C3, and are part of the 2C subfamily of ALDH.

[0056] BomALDH1 and BomALDH2 The highest expression levels were observed in the flower buds and leaves of *Buddleja officinalis*; while BomALDH3 The gene was expressed at its highest level in the flower tissues during the full bloom stage, consistent with the saffron acid accumulation pattern, suggesting... BomALDH3 It may be mainly involved in the synthesis of crocin and crocin in Buddleja officinalis.

[0057] In vitro enzyme activity assays revealed that BomALDH1, BomALDH2, and BomALDH3 all exhibited catalytic activity in the conversion of crocin dialdehyde to crocin. Under the same in vitro enzyme activity reaction conditions, BomALDH3 produced the highest yield of crocin compared to BomALDH1 and BomALDH2. Furthermore, the yields of crocin isomers catalyzed by BomALDH3 and BomALDH2 were similar, and both yields were higher than those produced by BomALDH1. Among the three, BomALDH3 demonstrated the highest catalytic efficiency in the conversion of crocin dialdehyde to crocin.

[0058] The present invention also provides an expression vector for the aforementioned nucleic acid molecule. The expression vector includes, but is not limited to, plasmids, viruses, or bacteriophages.

[0059] According to specific implementation methods, the expression vector mentioned may also selectively include the nucleic acid molecule BomCCD4.

[0060] The BomCCD4 nucleic acid molecule mentioned is described in Chinese patent application number 202610755674.5. It is hereby incorporated in whole or in part. According to a specific embodiment, the BomCCD4 nucleic acid molecule is shown in SEQ ID NO:7: In this invention, the expression vector refers to a nucleic acid construct capable of replicating and expressing a foreign target gene in a host cell. Plasmids are circular double-stranded DNA molecules that exist outside the chromosomes of microbial cells such as bacteria and yeast and are capable of autonomous replication. Plasmid vectors that can be used in this invention include, but are not limited to: pET series vectors (such as pET-28a, pET-32a, pET-Duet, etc.), pGEX series vectors, pMAL series vectors, pUC series vectors, pBR322 and its derivative vectors, as well as pPIC series vectors suitable for yeast expression and pCAMBIA series vectors suitable for plant expression. Plasmid vectors have advantages such as simple structure, convenient operation, controllable copy number, and wide host range, and are the most commonly used type of expression vector in genetic engineering.

[0061] Viral vectors are a class of vector systems that utilize the natural ability of viruses to infect host cells to deliver exogenous genes to target cells for expression. According to specific embodiments of the present invention, the viral vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV vectors), retroviral vectors, and baculovirus vectors. Viral vectors are particularly suitable for mammalian cells, insect cells, or plant cells where it is difficult to introduce exogenous genes using conventional transformation methods, offering advantages such as high transduction efficiency and the ability to mediate stable gene integration.

[0062] Bacteriophages are a class of viruses that infect specific bacterial strains and inject their genetic material into host cells for replication and expression. The phage vectors that can be used in this invention include, but are not limited to: lambda phage and its derivative vectors (such as λEMBL3, λDashII, etc.), M13 phage and its derivative vectors (such as the M13mp series), P1 phage vectors, and Cosmid vectors. Phage vectors are commonly used to construct genomic libraries, cDNA libraries, and for the cloning and expression of large DNA fragments. Those skilled in the art can rationally select appropriate expression vectors from the above vector types based on factors such as host cell type, target gene size, and required expression level. The present invention also provides a recombinant engineered bacterium containing the nucleic acid molecules or expression vectors described above.

[0063] In this invention, the term "recombinant engineering bacterium" refers to a bacterium that, through genetic engineering, incorporates exogenous nucleic acid molecules (i.e., those of this invention) into its culture. BomALDH1 , BomALDH2 or BomALDH3 The recombinant engineered bacteria are artificially introduced into recipient host cells to obtain microbial cells with stable genetic inheritance and the ability to express exogenous genes. These recombinant engineered bacteria are not naturally occurring strains but genetically engineered bacteria constructed through artificial intervention. The nucleic acid molecules mentioned include prokaryotic cells and eukaryotic cells. The introduction of the nucleic acid molecules or expression vectors described in this invention into host cells can be performed using any transformation or transfection technique known in the art, including but not limited to: heat-shock transformation (suitable for introducing plasmid vectors into competent *E. coli* cells); electroporation (creating reversible pores in the cell membrane using a transient high-voltage electric field, suitable for various host cells); chemical transformation (treating cells with chemical reagents such as CaCl2 to increase cell membrane permeability); conjugation (transferring plasmid DNA through direct contact between bacteria); phage transduction (using bacteriophages as vectors to introduce exogenous genes into host cells); lipofection or electrotransfection (suitable for eukaryotic host cells).

[0064] The provided acetaldehyde dehydrogenase or nucleic acid molecule from Buddleja officinalis can be used to catalyze the conversion of crocin dialdehyde to crocin.

[0065] The technical solution of the present invention will be described below through specific embodiments. It should be noted that these embodiments are only used to facilitate understanding by those skilled in the art and should not be regarded as a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents used in the embodiments can be obtained commercially.

[0066] Example 1 Example 1: Saffron acid was extracted from different tissues of *Buddleja officinalis* and its content was analyzed. This included: 1. Extraction of crocin from different tissues of Buddleja officinalis Accurately weigh 20 mg of *Buddleja officinalis* tissue (leaves, buds, and flowers), add 2 mL of methanol, and extract using ultrasound-assisted extraction for 20 minutes. After centrifugation at 12,000 rpm for 10 minutes at 4°C, collect the supernatant. Extract the residue twice more using ultrasound-assisted extraction with 2 mL of methanol. Centrifuge, combine the three supernatants, and transfer to a new centrifuge tube. After vacuum centrifugation and drying, reconstitute the solution with 200 μL of methanol. Filter the reconstituted solution through a 0.22 μm filter membrane, dilute 10-fold with methanol, and use for UHPLC-MS analysis.

[0067] 2. Quantitative analysis of saffron acid Saffron acid in the reaction products was detected using an AB SCIEX 6500 ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system with a UPLC BEH C18 reversed-phase column (100 mm × 2.1 mm, 1.7 μm, Waters). The chromatographic separation conditions were as follows: mobile phase A was a water / acetonitrile / formic acid mixture (v / v) of 95:5:0.1; mobile phase B was an acetonitrile / formic acid mixture (v / v) of 100:0.1. The flow rate was 0.2 mL / min, the column temperature was 40℃, and the injection volume was 2 μL. The gradient elution program (21 minutes) was as follows: 0–12 minutes, mobile phase B increased from 5% to 100%; 12–16 minutes, maintained at 100% B; 16–17 minutes, mobile phase B decreased from 100% to 5%; 17–21 minutes, maintained at 5% B.

[0068] Mass spectrometry was performed in MRM acquisition mode and positive ion ionization mode. Ion source parameters were set as follows: Gas 1: 55 psi; Gas 2: 55 psi; Curtain gas: 40 psi; Collision gas: 9; Ion source temperature: 400℃; Spray voltage: 5500 V. Crocin MRM ion pair: m / z 329.14→293.09; 329.14→311.11.

[0069] The results are as follows Figure 1 As shown in the figure. The results showed that the crocin content was highest in the flowers of *Buddleja officinalis*, while the crocin content in the leaves and flower buds was below the detection limit. These results indicate that the biosynthesis and accumulation of crocin mainly occur in the flower tissues of *Buddleja officinalis*.

[0070] Example 2 Example 2 Based on Gardenia GjALDH2C3 ,right BomALDH Candidate genes were screened and phylogenetic analysis was performed, including: Based on the full-length transcriptome data of *Buddleja officinalis*, with *Gardenia jasminoides* as an example... GjALDH2C3 Using (GeneBank: ARU08110) as a reference sequence, a BLASTP search of the *Buddleja officinalis* transcriptome sequence library was conducted, and three candidate genes with homology between 85% and 70% were selected and named accordingly. BomALDH1 , BomALDH2 and BomALDH3 .

[0071] An adjacency-based phylogenetic tree was constructed using the p-distance model in MEGA v12, and the Bootstrap test was performed 2000 times. The phylogenetic tree morphology was then optimized using Evolview software (https: / / www.evolgenius.info / evolview-v2 / #login).

[0072] The results of the system evolution analysis are as follows Figure 2 As shown. The results indicate that the three ALDHs of Buddleja officinalis, BomALDH1, BomALDH2, and BomALDH3, are in the same evolutionary branch as GjALDH2C3, belonging to the 2C subfamily of ALDH.

[0073] Example 3 Example 3 performed tissue-specific expression analysis of the above genes, including: 1. RNA extraction Approximately 50 mg of flower tissue sample from full bloom was rapidly frozen in liquid nitrogen and thoroughly ground into powder using a tissue homogenizer (Shanghai Wanbai Biotechnology Co., Ltd.). Total RNA was then extracted from the sample according to the instructions of the TransZol Up Plus RNA Extraction Kit (Beijing TransGen). The concentration and purity of the obtained RNA were determined using a spectrophotometer, and RNA integrity was detected by agarose gel electrophoresis.

[0074] 2. qRT-PCR analysis 0.5 μg of total RNA was used as a template, and reverse transcription was performed using the Evo M-MLV Reverse Transcription Premixed Tracer Kit (Aikerui Biotechnology) to obtain cDNA. Subsequently, using the obtained cDNA as a template, real-time quantitative PCR amplification was performed using corresponding specific primers. The primer sequences are shown in Table 1. The amplification reaction was performed using the SYBR Green Pro Taq HS Premixed qPCR Kit (Aikerui Biotechnology). The qRT-PCR reaction was completed on a real-time quantitative PCR instrument (Roche Pharmaceuticals).

[0075] Table 1

[0076] The reaction procedure was as follows: pre-denaturation at 95 °C for 30 seconds; followed by 40 cycles, each cycle consisting of denaturation at 95 °C for 5 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. Using the housekeeping gene PPP2A as an internal reference, the expression levels of BomALDH1, BomALDH2, and BomALDH3 genes were normalized, and their relative expression levels were calculated using the 2-ΔΔCT method.

[0077] The results of tissue-specific gene expression analysis are as follows: Figure 3 As shown. BomALDH1 ( Figure 3 A represents BomALDH1 (expression levels in leaves, buds, and flowers) and BomALDH2 ( Figure 3 B indicates BomALDH2 The expression levels in leaves, buds, and flowers were highest in the buds and leaves of *Buddleja officinalis*. BomALDH3 Gene( Figure 3 C indicates BomALDH3 The expression level in leaves, buds, and flowers was highest in flowers, consistent with the saffron acid accumulation pattern, suggesting... BomALDH3 It may be mainly involved in the synthesis of crocin and crocin in Buddleja officinalis.

[0078] Example 4 Example 4 BomALDH The gene was cloned, and expression vectors were constructed, including: 1. Construction of gene expression vectors pET-Duet was constructed using gene amplification and seamless cloning technology. -BomALDH Recombinant expression vectors ( Figure 4 The specific steps are as follows: Using cDNA from flower tissues during the peak flowering period of Buddleja officinalis as a template, high-fidelity DNA polymerase Phanta UniFi (Novizan) and gene-specific primers (Table 1) were used to target candidate DNA molecules. BomALDH The gene fragment was amplified by PCR, and homologous arms of approximately 20 nt were introduced at both ends of the amplified fragment. The reaction program was as follows: pre-denaturation at 95℃ for 3 minutes; followed by 32 cycles, each cycle consisting of denaturation at 95℃ for 15 seconds, annealing at 58℃ for 15 seconds, and extension at 72℃ for 90 seconds; and a final extension at 72℃ for 5 minutes. After detection by agarose gel electrophoresis, the PCR product was purified using a gel extraction method to obtain the target gene fragment. For the construction of a stable expression vector for *E. coli* transformation, restriction endonucleases were first used... Bam The pET-Duet vector (Qingke) was digested with HI (NEB) to purify and obtain a linear vector fragment. Then, the candidate gene fragment and pET-Duet were ligated using a seamless cloning kit (Xinkailai, XKL0613) to obtain pET-Duet- BomALDH1 pET-Duet- BomALDH2 and pET-Duet- BomALDH3 After the recombinant expression vector is sequenced and verified to be correct, it is used for subsequent protein expression and purification.

[0079] 2. Transformation The thermal shock conversion method was used to convert correctly sequenced pET-Duet- BomALDH1pET-Duet- BomALDH2 and pET-Duet- BomALDH3 The recombinant vectors were transformed into *Escherichia coli* prokaryotic expression strain BL21(DE3) (Weidi Bio, EC1002). After antibiotic selection, positive single colonies were picked to obtain BL21 / pET-Duet- BomALDH1 BL21 / pET-Duet- BomALDH2 and BL21 / pET-Duet- BomALDH3 Recombinant engineered strains.

[0080] Example 5 Example 5 describes the expression and purification of the BomALDH protein using the following method: 1. Bacterial culture The recombinant engineered strain obtained through the above transformation was inoculated into 3 mL of 2YT medium containing 1.6% (w / v) tryptone, 1% (w / v) yeast extract, 0.5% (w / v) sodium chloride, and 0.2% (w / v) glucose, with ampicillin added to a final concentration of 100 mg / L. The culture was incubated overnight at 37°C to obtain a seed culture. The seed culture was then inoculated into 50 mL of 2YT medium containing ampicillin at a ratio of 1:50 and incubated (37°C, 180 rpm) until OD500. 600 The concentration was 0.55–0.6. 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce cell culture for 20 hours (16 °C, 160 rpm). The cells were centrifuged for 15 minutes (4 °C, 4,000 rpm) and collected. After resuspending in pure water and centrifuging again, the supernatant was removed, and the cells were collected.

[0081] 2. Protein extraction The bacterial cells were resuspended at a ratio of 9 mL of lysis buffer per 1 g of wet bacterial cells. The lysis buffer contained 50 mM NaH2PO4, 0.3 M NaCl, and 10 mM imidazole, with a pH of 8.0, and a protease inhibitor (Yisheng Biotechnology) was added. After thorough resuspending until the bacterial cells were evenly dispersed, 1 / 9 of the bacterial resuspended volume was added to ACEBiotechnology super lysis buffer. After stirring or shaking to mix, the mixture was placed on ice for 30 minutes for lysis. After lysis, the mixture was centrifuged at 4°C and 12,000 rpm for 15 minutes, and the supernatant was collected for subsequent protein purification.

[0082] 3. Protein purification The target protein was isolated and purified using Ni-NTA affinity chromatography. The specific steps were as follows: First, the Ni-NTA packing material (Tiandi Renhe Biotechnology Co., Ltd.) was rinsed with 5 column volumes of deionized water, followed by equilibration with 5 column volumes of equilibration buffer containing 50 mM NaH2PO4, 0.3 M NaCl, and 10 mM imidazole at pH 8.0. Then, the lysis supernatant was added to the Ni-NTA chromatography column, thoroughly mixed with the Ni-NTA packing material, and incubated at room temperature for 30 minutes to promote the binding of the His-tagged fusion protein to the packing material. The flow-through was then collected. Next, the chromatography column was washed with wash buffer containing 20 mM and 30 mM imidazole, respectively, and the wash buffer was collected. Finally, the target protein was eluted using an elution buffer containing 50 mM NaH2PO4, 0.3 M NaCl, and 250 mM imidazole, with a pH of 8.0. Specifically, the protein was eluted twice with 1 column volume of elution buffer, and then eluted once with 3 column volumes of elution buffer, and the elution buffer was collected separately.

[0083] 4. SDS-PAGE protein electrophoresis analysis and protein quantification The protein expression and purification results were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that BomALDH1, BomALDH2, and BomALDH3 were not significantly expressed without the addition of an inducing agent; after 20 hours of induction with 0.5 mM IPTG, clear target protein bands were observed. Figure 5-7 Further purification by Ni-NTA affinity chromatography yielded a protein band with the expected molecular weight of the target protein. Figure 5-7 This indicates that BomALDH1, BomALDH2, and BomALDH3 proteins were successfully expressed and purified.

[0084] The purified BomALDH1, BomALDH2, and BomALDH3 target proteins were concentrated and desalted using 30 kDa ultrafiltration tubes (Yisheng Biotechnology). Subsequently, the concentrations of each target protein were determined using a BCA protein assay kit (White Shark), and the proteins were diluted to 250 mg / L with 50% glycerol and sterile water. The treated protein samples were stored at -80°C for subsequent enzyme activity assays.

[0085] Example 6 Example 6 identified the in vitro enzymatic activity of the above protein using the following method: 1. In vitro enzyme activity reaction system An in vitro enzyme activity reaction system was constructed to detect the catalytic activity of BomALDH1, BomALDH2, and BomALDH3 on crocin dialdehyde. The total volume of the reaction system was 200 μL, including 100 mM Tris-HCl buffer (pH 8.0), 80 μM crocin dialdehyde, 2 mM NAD+, 10 mg / L target protein, and purified water to the final volume. After thorough mixing, the reaction system was incubated at 30 °C and 220 rpm for 2 hours. After the reaction was completed, 400 μL of acetone was added to terminate the reaction. The reaction system without the target protein was used as a negative control.

[0086] 2. Product Extraction Add 1.2 mL of a mixture of diethyl ether and petroleum ether (volume ratio 1:2) to the above reaction system, mix thoroughly, and then extract. Subsequently, centrifuge at 12,000 rpm for 10 minutes at 4°C, collect the supernatant, transfer it to a new centrifuge tube, dry it under vacuum, and then reconstitute it with 200 μL of methanol. Filter the reconstituted solution through a 0.22 μm filter membrane and analyze it using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS).

[0087] 3. Qualitative identification of saffron acid Saffron acid in the reaction products was detected using an Agilent 6560 Q-TOF ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system with a UPLC BEH C18 reversed-phase column (100 mm × 2.1 mm, 1.7 μm, Waters). The chromatographic separation conditions were as follows: mobile phase A was a water / acetonitrile / formic acid mixture (v / v) of 95:5:0.1; mobile phase B was an acetonitrile / formic acid mixture (v / v) of 100:0.1. The flow rate was 0.2 mL / min, the column temperature was 40℃, and the injection volume was 2 μL. The gradient elution program was as follows: 0–12 min, mobile phase B increased from 5% to 100%; 12–16 min, maintained at 100% B; 16–17 min, mobile phase B decreased from 100% to 5%; 17–21 min, maintained at 5% B.

[0088] Mass spectrometry detection was performed in positive ion mode. The ion source parameters were set as follows: dry gas temperature 325℃, flow rate 11.0 L / min; sheath gas temperature 400℃, flow rate 11.0 L / min; nebulizer gas pressure 45 psi; capillary voltage 3500 V.

[0089] Qualitative analysis of crocin in the reaction product was performed by comparing its retention time and secondary ion fragmentation spectrum with those of crocin chemical standards.

[0090] 4. Results and Analysis UHPLC-MS analysis showed that crocin was not detected in the control group (without ALDH protease); however, crocin was detected in all reaction systems with BomALDH1, BomALDH2, and BomALDH3 proteases, respectively, with precise mass ([M+H)). + : m / z 329.1747) and the chromatographic peak retention time are consistent with those of saffron acid standard (e.g., Figure 8 (As shown in Figure A). Further UHPLC-MS / MS analysis revealed that the catalytic product possessed the main characteristic secondary fragment ions of crocin standard, including... m / z 311.16, 293.15, 275.14, and 265.15, etc. (e.g.) Figure 8 As shown in Figure B), this confirms that the catalytic product is crocin. The above results indicate that BomALDH1, BomALDH2, and BomALDH3 all possess catalytic activity for the conversion of crocin dialdehyde to crocin.

[0091] Furthermore, in the catalytic reaction products of BomALDH1, BomALDH2, and BomALDH3, another mass-to-charge ratio [M+H] was detected at a chromatographic retention time of 9.94 minutes. + : m / z The chromatographic peak at 329.1747 (e.g.) Figure 8 (As shown in Figure A). Furthermore, the catalytic product exhibits some fragment ion characteristics of crocin, suggesting it is an isomer of crocin (e.g., ...). Figure 8 (As shown in B).

[0092] Example 7 Example 7 compared the enzyme activity efficiency of the above proteins, including: 1. In vitro enzyme activity reaction system An in vitro enzyme activity reaction system was constructed to detect the catalytic activity of BomALDH1, BomALDH2, and BomALDH3 on crocin dialdehyde. The total volume of the reaction system was 200 μL, including 100 mM Tris-HCl buffer (pH 8.0), 80 μM crocin dialdehyde, 2 mM NAD+, 10 mg / L target protein, and purified water to the final volume. After thorough mixing, the reaction system was incubated at 30 °C and 220 rpm for 2 hours. After the reaction was completed, 400 μL of acetone was added to terminate the reaction. The reaction system without the target protein was used as a negative control.

[0093] 2. Product Extraction Add 1.2 mL of a mixture of diethyl ether and petroleum ether (volume ratio 1:2) to the above reaction system, mix thoroughly, and then extract. Subsequently, centrifuge at 12,000 rpm for 10 minutes at 4°C, collect the supernatant, transfer it to a new centrifuge tube, dry it under vacuum, and then reconstitute it with 200 μL of methanol. Filter the reconstituted solution through a 0.22 μm filter membrane for UHPLC-MS analysis.

[0094] 3. Quantitative analysis of saffron acid Saffron acid in the reaction products was detected using an AB SCIEX 6500 ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system with a UPLC BEH C18 reversed-phase column (100 mm × 2.1 mm, 1.7 μm, Waters). The chromatographic separation conditions were as follows: mobile phase A was a water / acetonitrile / formic acid mixture (v / v) of 95:5:0.1; mobile phase B was an acetonitrile / formic acid mixture (v / v) of 100:0.1. The flow rate was 0.2 mL / min, the column temperature was 40℃, and the injection volume was 2 μL. The gradient elution program (21 minutes) was as follows: 0–12 minutes, mobile phase B increased from 5% to 100%; 12–16 minutes, maintained at 100% B; 16–17 minutes, mobile phase B decreased from 100% to 5%; 17–21 minutes, maintained at 5% B.

[0095] Mass spectrometry was performed in MRM acquisition mode and positive ion ionization mode. Ion source parameters were set as follows: Gas 1: 55 psi; Gas 2: 55 psi; Curtain gas: 40 psi; Collision gas: 9; Ion source temperature: 400℃; Spray voltage: 5500 V. Crocin MRM ion pair: m / z 329.14→293.09; 329.14→311.11.

[0096] 4. Results Analysis UHPLC-MS analysis (such as...) Figure 9 As shown in the figure, under the same reaction conditions, BomALDH3 catalyzed the highest yield of crocin compared to BomALDH1 and BomALDH2. Furthermore, the yields of crocin isomers obtained by the reactions catalyzed by BomALDH3 and BomALDH2 were similar, and both yields were higher than those obtained by BomALDH1.

[0097] The above results indicate that, among the three, BomALDH3 has the highest catalytic efficiency in the reaction of crocin dialdehyde to crocin.

[0098] Example 8 Example 8 describes the expression of the above protein and the synthesis of crocin in Escherichia coli using the following method: 1. Construction and transformation of gene expression vectors pThio- was constructed using gene amplification and seamless cloning technology. BomCCD4-BomALDH Recombinant expression vectors ( Figure 10 The specific steps are as follows: Using cDNA from *Buddleja officinalis* flower tissue as a template, high-fidelity DNA polymerase Phanta UniFi (Novizan) and gene-specific primers (Table 1) were used to target candidate DNA. BomALDH The gene fragment was amplified by PCR, and homologous arms of approximately 20 nt were introduced at both ends of the amplified fragment. The reaction program was as follows: pre-denaturation at 95℃ for 3 min; followed by 32 cycles, each cycle consisting of denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, and extension at 72℃ for 90 s; and a final extension at 72℃ for 5 min. After detection by agarose gel electrophoresis, the PCR product was purified using a gel extraction method to obtain the target gene fragment. For the construction of a stable expression vector for *E. coli* transformation, restriction endonucleases were first used... Sac I (NEB) digested the previously developed lab vector pTHIO-BomCCD4 with enzymes to purify and obtain a linear vector fragment. Then, a seamless cloning kit (Xinkailai, XKL0613) was used to ligate the candidate gene fragment and pTHIO- BomCCD4 To obtain pTHIO- BomCCD4-BomALDH1 pTHIO- BomCCD4-BomALDH2 and pTHIO- BomCCD4- BomALDH3 Recombinant expression vector. After the sequence was verified to be correct by sequencing, it was used for subsequent enzyme activity experiments.

[0099] 2. Transformation The vector pAC-ZEAXipi (Addgene, #53287) that specifically expresses zeaxanthin was transformed into the Escherichia coli prokaryotic expression strain BL21(DE3) (Weidi Bio, EC1002) using the heat shock transformation method to obtain the BL21 ZEA strain. The BL21ZEA strain was prepared into competent cells and stored at -80℃ for later use.

[0100] The successfully sequenced recombinant vector was transformed into the *Escherichia coli* BL21 ZEA strain, which biosynthesizes zeaxanthin, for expression. The control group consisted of strains transformed with pTHIO- BomCCD4 The recombinant vector of E. coli was used in the experimental group, which was transformed with pTHIO- BomCCD4-BomALDH1 pTHIO- BomCCD4-BomALDH2 and pTHIO- BomCCD4-BomALDH3*Escherichia coli*. Positive single colonies were picked and cultured in 3 mL of 2YT medium containing 1.6% (w / v) tryptone, 1% (w / v) yeast extract, 0.5% (w / v) sodium chloride, and 0.2% (w / v) glucose. Ampicillin (final concentration 100 mg / L) and chloramphenicol (final concentration 25 mg / L) were added, and the culture was incubated overnight at 37 °C to obtain a seed culture. The seed culture was inoculated into 50 mL of 2YT medium at a ratio of 1:50 and incubated (37 °C, 180 rpm) until OD500. 600 The concentration was 0.5. 0.2% (w / v) arabinose was added to induce cell culture for 24 hours (16 ℃, 160 rpm). The cells were centrifuged for 10 minutes (4 ℃, 4200 rpm) and collected. After resuspending in pure water and centrifuging again, the supernatant was removed, and the cells were frozen for later use.

[0101] 3. Product extraction Take an appropriate amount of *E. coli* cells and place them in a centrifuge tube. Add 5 mL of a 1:1 methanol / acetone mixture (containing 0.1% butylated hydroxytoluene), and sonicate for 5 minutes (4 seconds on, 3 seconds off, power 35%). Extract twice. After high-speed centrifugation for 10 minutes (4℃, 4200 rpm), collect the supernatant in a brown bottle. Concentrate and dry by centrifugation, redissolve in 200 μL of a 1:1 methanol / acetone mixture, filter through a 0.22 μm filter membrane, and analyze by UHPLC-MS.

[0102] 4. Saffron acid analysis Saffron acid in the reaction products was detected using an Agilent 6560 Q-TOF ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system with a UPLC BEH C18 reversed-phase column (100 mm × 2.1 mm, 1.7 μm, Waters). The chromatographic separation conditions were as follows: mobile phase A was a water / acetonitrile / formic acid mixture (v / v) of 95:5:0.1; mobile phase B was an acetonitrile / formic acid mixture (v / v) of 100:0.1. The flow rate was 0.2 mL / min, the column temperature was 40℃, and the injection volume was 2 μL. The gradient elution program was as follows: 0–12 min, mobile phase B increased from 5% to 100%; 12–16 min, maintained at 100% B; 16–17 min, mobile phase B decreased from 100% to 5%; 17–21 min, maintained at 5% B.

[0103] Mass spectrometry detection was performed in positive ion mode. The ion source parameters were set as follows: dry gas temperature 325℃, flow rate 11.0 L / min; sheath gas temperature 400℃, flow rate 11.0 L / min; nebulizer gas pressure 45 psi; capillary voltage 3500 V.

[0104] 5. Results Analysis UHPLC-MS analysis results ( Figure 11 The text appears to be a mix of Chinese characters and symbols, possibly representing a corrupted or incomplete document. A direct translation wouldn't be BomCCD4 Crocin products were not detected in E. coli samples containing the gene; however, they were not detected in samples co-expressed with the gene. BomCCD4 and BomALDH1 , BomALDH2 and BomALDH3 Crocin products were detected in all samples after gene sequencing, and their retention times were consistent with those of crocin standards (e.g., Figure 11 (As shown). The above results indicate that Buddleja officinalis BomALDH1 , BomALDH2 and BomALDH3 Genes can serve as genetic elements for the biosynthesis of crocin in Escherichia coli.

[0105] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A kind of acetaldehyde dehydrogenase from Buddleja officinalis, characterized in that, Selected from at least one of the following: (a) Proteins with amino acid sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; (b) A derivative protein having one to three amino acids substituted, deleted or added in the amino acid sequence described in (a) and having catalytic activity in the formation of crocin from crocin dialdehyde.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the acetaldehyde dehydrogenase of Buddleja officinalis as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Selected from at least one of the following: (a) A nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (b) has at least 85% homology with the nucleotide sequence shown in (a), and the encoded protein has a nucleotide sequence that catalyzes the formation of crocin from crocin dialdehyde.

4. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 2 or 3, and a nucleic acid molecule that may optionally include BomCCD4.

5. The expression vector according to claim 4, characterized in that, The expression vector is selected from at least one of plasmids, viruses, or bacteriophages.

6. A recombinant engineered bacterium, characterized in that, Contains the nucleic acid molecule as described in claim 2 or 3 or the expression vector as described in claim 4 or 5.

7. The application of the acetaldehyde dehydrogenase of Buddleja officinalis according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or the expression vector according to claim 4 or 5 in catalyzing the production of crocin from saffron dialdehyde.

8. The use of the acetaldehyde dehydrogenase of Buddleja officinalis according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or the expression vector according to claim 4 or 5, in the preparation of crocin or crocinoside.

9. A method for producing saffron acid, characterized in that, Includes the following steps: The recombinant engineered bacteria of claim 6 express acetaldehyde dehydrogenase from Buddleja officinalis, using crocin dialdehyde as a substrate, in NAD... + In the presence of saffron, a catalytic reaction is carried out to produce crocin.

10. The method according to claim 9, characterized in that, The conditions for the catalytic reaction are: temperature 25-37℃, pH 7.0-9.0, and reaction time 1-24 hours.

11. A method for producing saffron acid, characterized in that, Includes the following steps: Using the recombinant engineered bacteria described in claim 6, carotenoid lysin dioxygenase 4 and acetaldehyde dehydrogenase are expressed, and saffron acid is biosynthesized in vivo.

Images