Tryptamine hydroxylase mutant and its application

Abstract

The present invention provides a tryptamine hydroxylase mutant and its application, the tryptamine hydroxylase mutant is selected from the amino acid sequence shown in any one of the following: (a) obtained by deleting 30 to 45 amino acids from the N-terminal of the tryptamine hydroxylase shown in SEQ ID NO: 1; (b) formed by mutation of the amino acid sequence shown in (a), the mutation site being selected from the following one or more amino acid residue sites: 114th, 133rd, 247th, 309th, 401st and 464th, the position of the mutation site corresponding to the wild-type tryptamine hydroxylase having a sequence of SEQ ID NO: 1; (c) an amino acid sequence obtained by adding a tag sequence, an enzyme cleavage site sequence or a signal sequence at one or both ends of (a) or (b). The catalytic activity of the tryptamine hydroxylase mutant is greatly improved compared to the wild-type enzyme, significantly increasing the yield of 5-hydroxytryptamine.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a tryptamine hydroxylase mutant and application thereof. Background Art

[0002] First discovered in serum, 5-HT, also known as serotonin, is widely present in mammalian tissues, primarily distributed in the pineal gland and hypothalamus. It regulates a variety of physiological functions, including sleep, appetite, mood, immune regulation, and pain perception. 5-HT also plays an important role in plants, including regulating flowering cycles, morphogenesis, aging, and resistance to biotic stress.

[0003] Since serotonin was first discovered in mammals and plants, extracting it from these sources is the oldest and most established method. 5-HT is commonly extracted from rat brain for analytical and clinical applications. Audhya et al. studied the extraction of 5-HT from rat cerebrospinal fluid (CSF) using direct lumbar puncture. Furthermore, Miekus et al. mechanically homogenized brain tissue samples using formic acid or perchloric acid solutions to further extract serotonin for their research. 5-HT is also found in various plant parts, such as leaves, roots, flowers, fruits, and seeds. Several foods have also been reported to contain high concentrations of 5-HT, including bananas, tomatoes, plantains, pineapples, butter, black walnuts, and English walnuts. Furthermore, 5-HT and its precursor, 5-HTP, are found in the seeds of Moringa oleifera and the African plant Griffonia simplicifolia. However, the yield of naturally extracted serotonin is low, and the raw material supply is insufficient, resulting in a shortage of supply.

[0004] The primary challenge is how to industrially produce 5-hydroxytryptamine and its derivatives (such as melatonin) for use in the medical field for the treatment of mental illness, the development of new drugs, and in the healthcare field for mood regulation and sleep promotion. Serotonin is synthesized from tryptophan in microorganisms, plants, animals, and humans. In animals, tryptophan 5-hydroxylase hydroxylates L-tryptophan to 5-hydroxy-L-tryptophan, which is then converted to serotonin by aromatic L-amino acid decarboxylase. The first hydroxylation reaction in this pathway requires tetrahydrobiopterin (BH4) as a cofactor, involving a complex BH4 cycle and regeneration pathway. Therefore, this process is complex and costly. In plants, tryptophan is converted to tryptamine and then to serotonin after hydroxylation by tryptamine hydroxylase. Unlike the synthetic pathway in animals, this pathway is first decarboxylated and then hydroxylated. This pathway does not require BH4 and avoids the complex BH4 cycle and regeneration pathway. However, due to the long growth cycle of plants and seasonal and regional restrictions, the yield is low, making it unsuitable for industrial application. With the rapid development of synthetic biology in recent years, researchers have gradually turned their attention to microorganisms. Expressing the biosynthetic pathways of valuable secondary metabolites from plants and animals into microorganisms to synthesize the main components of some drugs is currently a hot topic in synthetic biology research. Microbial production offers advantages such as short production cycles, low costs, high yields, and ease of large-scale production. However, within the serotonin biosynthetic pathway, the synthesis of 5-hydroxytryptamine (5-HT) is limited by low tryptamine hydroxylase activity, making its large-scale biosynthesis challenging. To address this issue, it is necessary to enhance tryptamine hydroxylase activity, develop tryptamine hydroxylase mutants that produce high 5-HT yields, and optimize industrial production conditions to reduce production costs and improve synthesis efficiency. Summary of the Invention

[0005] To solve the problems existing in the background technology, the present invention provides a tryptamine hydroxylase mutant and its application. The mutant can catalyze tryptamine to produce 5-hydroxytryptamine. Compared with the wild enzyme, the mutant has significantly improved catalytic activity and increased product yield.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, the present invention provides a tryptamine hydroxylase mutant comprising an amino acid sequence shown in any one of the following:

[0008] (a) obtained by deleting 30 to 45 amino acids from the N-terminus of tryptamine hydroxylase having the amino acid sequence of SEQ ID NO: 1;

[0009] (b) formed by mutation of the amino acid sequence shown in (a), wherein the mutation site of the sequence is selected from one or more of the following amino acid residue sites: 114, 133, 247, 309, 401 and 464, and the position of the mutation site corresponds to the tryptamine hydroxylase with the sequence of SEQ ID NO: 1;

[0010] (c) An amino acid sequence obtained by adding a tag sequence, an enzyme cleavage site sequence, or a signal sequence to one or both ends of (a) or (b).

[0011] According to the above scheme, the mutation site in b) is selected from the following mutations of one or more amino acid residues:

[0012] No. 114: F114C or F114S;

[0013] No. 133: M133Q or M133N;

[0014] No. 247: C247N or C247G;

[0015] No. 309: V309I or V309W or V309P or V309L;

[0016] No. 401: F401Y or F401T;

[0017] Position 464: Q464E or Q464D.

[0018] All of the above tryptophan hydroxylase mutants have the function of catalyzing tryptamine to produce 5-hydroxytryptamine.

[0019] Preferably, the sequence mutation at the site in (b) is selected from the following site mutations of one or more amino acid residues:

[0020] No. 114: F114C;

[0021] No. 133: M133Q;

[0022] No. 247: C247N;

[0023] No. 309: V309I;

[0024] No. 401: F401Y;

[0025] No. 464: Q464E.

[0026] Further, the sequence mutation is selected from the following: F114C / M133Q, F114C / C247N, F114C / V309I, F114C / F401Y, F114C / Q464E, M133Q / C247N, M133Q / Q464E, C247N / V309I, C247N / F401Y, C247N / Q464E, F114C / M133Q / C247N, F114C / M133Q / V309I, F114C / M133Q / F401Y, M133Q / C247N / F401Y.

[0027] Furthermore, the tryptamine hydroxylase mutant in (a) is obtained by deleting 35 to 45 amino acids from the N-terminus of the tryptamine hydroxylase with the amino acid sequence of SEQ ID NO: 1, preferably, deleting 36 to 42 amino acid residues.

[0028] In a second aspect, the present invention provides a DNA molecule encoding the above-mentioned tryptamine hydroxylase mutant.

[0029] According to the above scheme, the DNA molecule encoding the tryptamine hydroxylase mutant is obtained by changing the coding sequence of the tryptamine hydroxylase encoding DNA molecule with the nucleotide sequence of SEQ ID NO: 2 according to the change of the amino acid residues of the mutant.

[0030] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned DNA molecule.

[0031] Furthermore, the recombinant vector co-expresses a redox partner Fdr-Fdx formed by ferredoxin reductase and ferredoxin.

[0032] In a fourth aspect, the present invention provides a host cell comprising the above-mentioned recombinant vector, or a host cell in which the above-mentioned DNA molecule encoding the tryptamine hydroxylase mutant is integrated into its genome.

[0033] Furthermore, the above recombinant vector co-expresses ferredoxin reductase and the redox partner Fdr-Fdx formed by ferredoxin.

[0034] The host cell may be Escherichia coli (E. coli) or Yarrowia lipolytica.

[0035] Preferably, the host cell is Escherichia coli (E. coli).

[0036] In a fifth aspect, the present invention provides the use of the above-mentioned tryptamine hydroxylase mutant, its encoding DNA molecule, recombinant vector and host cell in the production of 5-hydroxytryptamine, for catalyzing tryptamine to produce 5-hydroxytryptamine and increasing the production of 5-hydroxytryptamine.

[0037] In a sixth aspect, the present invention provides a method for producing 5-hydroxytryptamine, comprising the following steps:

[0038] 1) Producing 5-hydroxytryptamine using the above-mentioned tryptamine hydroxylase mutant or host cell;

[0039] 2) The 5-hydroxytryptamine isolated from the system of 1).

[0040] The present invention obtains multiple mutants by truncating the amino acid sequence of tryptamine hydroxylase shown in SEQ ID NO:1 and further performing specific site mutations thereon. Furthermore, the present invention further discloses DNA molecules encoding the tryptamine hydroxylase mutants, recombinant vectors comprising the DNA molecules, and host cells. The tryptamine hydroxylase mutants, recombinant vectors, or host cells can catalyze tryptamine to produce 5-hydroxytryptamine. Furthermore, co-expression of the tryptamine hydroxylase mutants with a redox chaperone Fdr-Fdx consisting of ferredoxin reductase (Fdr) and ferredoxin (Fdx) can enhance electron transfer efficiency and increase 5-hydroxytryptamine production.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1) The present invention significantly enhances the catalytic activity of the tryptamine hydroxylase mutants obtained by truncating the amino acid sequence of wild-type tryptamine hydroxylase and further mutating this sequence at specific sites. Experimental verification indicates that the catalytic activity of the tryptamine hydroxylase mutants is significantly enhanced relative to that of the wild-type tryptamine hydroxylase, significantly increasing the yield of 5-hydroxytryptamine produced from tryptamine by up to 6.5 times. This reduces the amount of catalyst used and ultimately production costs, while also providing a foundation for developing superior tryptamine hydroxylases and promoting the industrialized production of 5-hydroxytryptamine.

[0043] 2) Co-expression of the tryptamine hydroxylase mutant with the redox chaperone Fdr-Fdx, consisting of ferredoxin reductase (Fdr) and ferredoxin (Fdx), enhanced electron transfer efficiency, further improved the catalytic activity of the tryptamine hydroxylase mutant, and increased the production of 5-hydroxytryptamine. The catalytic activity of the engineered bacteria co-expressing the tryptamine hydroxylase mutant and Fdr-Fdx towards tryptamine was increased by up to 13.5 times compared to the engineered bacteria expressing the wild-type enzyme.

[0044] 3) The present invention expresses the gene encoding the above-mentioned tryptamine hydroxylase mutant in the recombinant vector by fermentation culture to obtain tryptamine hydroxylase. This process is easy to implement, the conditions are easy to control, and it is suitable for promoting industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 This is a map of the pET22b-Δ40T5H-Fdr-Fdx expression plasmid;

[0047] Figure 2 The results of the conversion of tryptamine to 5-hydroxytryptamine catalyzed by T5H, T5H-Fdr-Fdx, pET22b-Δ35T5H-Fdr-Fdx, pET22b-Δ40T5H-Fdr-Fdx, and pET22b-Δ45T5H-Fdr-Fdx are shown;

[0048] Figure 3 This is the result of the Δ40T5H-Fdr-Fdx single mutant catalyzing the conversion of tryptamine to 5-hydroxytryptamine;

[0049] Figure 4 This is a diagram showing the results of the Δ40T5H-Fdr-Fdx multiple mutant catalyzing the conversion of tryptamine to 5-hydroxytryptamine;

[0050] Figure 5 This is the HPLC chromatogram of tryptamine standard;

[0051] Figure 6 This is the HPLC chromatogram of 5-HT standard. DETAILED DESCRIPTION

[0052] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing embodiments of the present invention and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] In the present invention, the terms "tryptamine hydroxylase" and "T5H" have the same meaning and are used interchangeably herein. Both refer to the wild-type tryptamine hydroxylase having the amino acid sequence set forth in SEQ ID NO: 1, which is derived from rice (Oryza sativa Japonica Group) and has a NCBI publication number of NP_001410381.1. This enzyme catalyzes the hydroxylation of tryptamine to produce 5-hydroxytryptamine.

[0058] SEQ ID NO: 1

[0059] *

[0060] In order to improve the catalytic activity of tryptophan hydroxylase, the inventors truncated the amino acid sequence of wild-type tryptophan hydroxylase and further performed mutations at specific sites to obtain a tryptophan hydroxylase mutant with improved activity.

[0061] The inventors discovered through multiple experiments that after truncating 20 amino acids from the N-terminus of wild-type tryptamine hydroxylase, the catalytic activity of the resulting tryptamine hydroxylase mutant decreased; after truncating 30 amino acids, the catalytic activity of the resulting tryptamine hydroxylase mutant remained essentially unchanged; after truncating 35 to 45 amino acid residues, the resulting tryptamine hydroxylase mutant had significantly enhanced catalytic activity towards tryptamine, thereby increasing the production of 5-hydroxytryptamine; and after truncating 50 amino acid residues, the tryptamine hydroxylase mutant became inactivated and no longer had catalytic activity.

[0062] In some specific embodiments, after the N-terminal amino acid residues of the wild-type tryptamine hydroxylase are truncated by 30, although its catalytic activity is equivalent to that of the wild-type tryptamine hydroxylase, after the sequence is truncate, the inclusion bodies are reduced during the expression process using engineered bacteria, the soluble expression of the protein is enhanced, and it has more advantages than the wild-type enzyme in terms of enzyme expression and purification.

[0063] In some specific embodiments, after the N-terminal amino acids are truncated to 35, 40, and 45 amino acids, respectively, the production of 5-hydroxytryptamine is increased by 47%, 127%, and 82%, respectively, compared to the wild-type tryptamine hydroxylase.

[0064] The inventors further performed site-directed mutagenesis on the tryptamine hydroxylase obtained by truncation of 30 to 45 amino acid residues at the N-terminus to construct a mutant library, and ultimately obtained a tryptamine hydroxylase mutant with better activity. The amino acid sequence obtained by deleting 30 to 45 amino acids from the N-terminus of the tryptamine hydroxylase shown in SEQ ID NO: 1 was mutated at one or more of the following amino acid sites: 114, 133, 247, 309, 401 and 464. The positions of the mutation sites correspond to the wild-type tryptamine hydroxylase with the sequence of SEQ ID NO: 1.

[0065] Furthermore, the above mutation sites are selected from the following mutations of one or more amino acid residues:

[0066] No. 114: F114C or F114S;

[0067] No. 133: M133Q or M133N;

[0068] No. 247: C247N or C247G;

[0069] No. 309: V309I or V309W or V309P or V309L;

[0070] No. 401: F401Y or F401T;

[0071] Position 464: Q464E or Q464D;

[0072] The position of the above mutation site corresponds to the wild tryptamine hydroxylase with the sequence of SEQ ID NO: 1.

[0073] In some preferred embodiments, the sequence mutation at the site in (b) is selected from the following site mutations of one or more amino acid residues:

[0074] No. 114: F114C;

[0075] No. 133: M133Q;

[0076] No. 247: C247N;

[0077] No. 309: V309I;

[0078] No. 401: F401Y;

[0079] No. 464: Q464E.

[0080] Using the tryptamine hydroxylase mutant Δ40T5H obtained by truncating 40 amino acids at the N-terminus as the starting enzyme, single-point mutations F114C, M133Q, C247N, V309I, and F401Y were performed respectively. The catalytic activities of the resulting Δ40T5H-F114C, Δ40T5H-M133Q, Δ40T5H-C247N, Δ40T5H-V309I, and Δ40T5H-F401Y mutants were significantly improved compared with the wild enzyme T5H and the mutant Δ40T5H.

[0081] In some more preferred embodiments, the sequence mutation is selected from the following: F114C / M133Q, F114C / C247N, F114C / V309I, F114C / F401Y, F114C / Q464E, M133Q / C247N, M133Q / Q464E, C247N / V309I, C247N / F401Y, C247N / Q464E, F114C / M133Q / C247N, F114C / M133Q / V309I, F114C / M133Q / F401Y, M133Q / C247N / F401Y.

[0082] The tryptamine hydroxylase mutant Δ40T5H obtained by truncating 40 amino acids at the N-terminus was used as the starting enzyme, and the mutants F114C / M133Q, F114C / C247N, F114C / V309I, F114C / F401Y, F114C / Q464E, M133Q / C247N, M133Q / Q464E, C247N / V309I, F114C / F401Y, F114C / Q464E, I, C247N / F401Y, C247N / Q464E, F114C / M133Q / C247N, F114C / M133Q / V309I, F114C / M133Q / F401Y, M133Q / C247N / F401Y multi-site mutations, the resulting mutants were significantly improved compared to the wild enzyme T5H and the mutant Δ40T5H.

[0083] In the present invention, the term "tryptamine hydroxylase mutant" also includes derivatives of the above-mentioned tryptamine hydroxylase enzymes that have deletions, insertions, or substitutions of several amino acids, as well as additions or deletions of one or more amino acids at the C-terminus and / or N-terminus, but that retain the activity and function of the P450 enzyme mutants of the present invention. For example, in the art, substitution with amino acids having similar or similar properties generally does not alter protein function, and addition of one or more amino acids at the C-terminus and / or N-terminus, such as addition of a tag sequence, restriction site sequence, signal sequence, or secretion signal sequence, generally does not alter the function of the resulting protein. However, these derivatives may contain the aforementioned sequence truncations and / or mutations.

[0084] In the present invention, the term "tryptamine hydroxylase mutant" also includes derivative proteins that retain their protein activity while sharing an amino acid sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, such as 98% or more, or 99% or more, with the aforementioned tryptamine hydroxylase mutant. Similarly, these derivative proteins may contain the sequence truncations and / or mutations described above.

[0085] The tryptamine hydroxylase mutant of the present invention can be a product of chemical synthesis, or can be produced from a prokaryotic host or a eukaryotic host using recombinant technology.

[0086] The present invention also provides a DNA molecule encoding the above-mentioned tryptamine hydroxylase mutant. The DNA form includes genomic DNA, cDNA or artificially synthesized DNA. The DNA can be a coding strand or a non-coding strand.

[0087] According to the above scheme, the DNA molecule encoding the tryptamine hydroxylase mutant is obtained by changing the coding sequence of the tryptamine hydroxylase encoding DNA molecule with the nucleotide sequence of SEQ ID NO: 2 according to the change of the amino acid residues of the mutant.

[0088] SEQ ID NO:2

[0089]

[0090] The present invention also provides a recombinant vector comprising the aforementioned DNA molecule, and a host cell produced by genetic engineering using the recombinant vector or the tryptamine hydroxylase mutant gene of the present invention. The host cell comprises the aforementioned recombinant vector, or has the aforementioned DNA molecule encoding the tryptamine hydroxylase mutant integrated into its genome.

[0091] The DNA molecule sequence of the present invention can be used to express or produce a tryptamine hydroxylase mutant through conventional recombinant DNA technology. Generally, the following steps are involved: 1) transforming or transducing a suitable host cell with the tryptamine hydroxylase mutant of the present invention or a recombinant expression vector containing the DNA molecule; (2) culturing the host cell in a suitable culture medium.

[0092] In the present invention, the DNA sequence encoding the tryptamine hydroxylase mutant can be inserted into a recombinant expression vector. As long as it can stably replicate in the host, any plasmid or vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element. Methods well known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding the tryptamine hydroxylase mutant and appropriate transcription / translation control signals. The DNA sequence can be effectively linked to an appropriate promoter of the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0093] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0094] In the present invention, the host cell can be any microorganism suitable for expressing a tryptamine hydroxylase mutant or a tryptamine hydroxylase mutant fusion protein, including Escherichia coli (E. coli) and Yarrowia lipolytica (Yarrowia lipolytica), preferably Escherichia coli (E. coli). In one or more of the following embodiments, Escherichia coli BL21 is used.

[0095] It is well known that the expression results of the same nucleotide sequence in different microbial hosts often vary greatly. In order to optimally express tryptamine hydroxylase or its mutants in genetically engineered Escherichia coli, the expression genes of these enzymes can be codon optimized.

[0096] Codon optimization is a kind of technology that can be used for making protein expression in organism maximized by increasing the translation efficiency of gene of interest.Different organisms usually illustrate the special preference of one of some codons for encoding identical amino acids due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as intestinal bacteria, the composition of its genome tRNA library separately is reflected by optimizing codon.Therefore, in fast-growing microorganisms, the low frequency codon of amino acid can be used for identical amino acid but the codon displacement of high frequency.Therefore, the expression of optimized dna sequence dna is improved in fast-growing microorganisms.

[0097] In one or more embodiments of the present invention, after codon optimization, the nucleotide sequence of rice-derived tryptamine hydroxylase is as shown in SEQ ID NO: 2, and the encoding nucleotide sequences of the tryptamine hydroxylase mutant and the tryptamine hydroxylase mutant fusion protein are adjusted based on SEQ ID NO: 2 according to the changes in amino acid residues.

[0098] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers, and host cells. The recombinant cells (host cells) established by the present invention can be cultivated using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Cultivate under conditions suitable for host cell growth. After the host cells grow to a suitable cell density, induce the promoter of choice using a suitable method (such as temperature conversion or chemical induction), and the cells are further cultivated for a period of time.

[0099] On this basis, the present invention also provides a method for producing 5-hydroxytryptamine by catalyzing the hydroxylation of tryptamine using the aforementioned tryptamine hydroxylase mutant, expression vector, or host cell. For example, in a specific embodiment, the method can be performed by culturing a host cell containing the expression vector of the present invention or a host cell having the coding sequence of the tryptamine hydroxylase mutant of the present invention integrated into its genome, or by using the tryptamine hydroxylase mutant of the present invention to catalyze the production of 5-hydroxytryptamine from tryptamine, and then obtaining the produced 5-hydroxytryptamine from the catalytic system.

[0100] During expression, the tryptamine hydroxylase mutants of the present invention can be expressed intracellularly and secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, high-speed centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0101] The tryptamine hydroxylase mutant of the present invention can be used to catalyze tryptamine to produce 5-hydroxytryptamine. The enzyme includes purified enzyme, crude enzyme, etc. It should be understood that the expression product of the host cell expressing the above tryptamine hydroxylase mutant also has this use.

[0102] Tryptamine hydroxylase and its mutants catalyze the production of 5-hydroxytryptamine from tryptamine, requiring cofactors and electron transfer. Therefore, when the tryptamine hydroxylase mutants of the present invention are used to catalyze the production of 5-hydroxytryptamine from tryptamine, in addition to tryptamine and tryptamine hydroxylase, NADPH also needs to be added to the reaction system.

[0103] The host cell expressing the above-mentioned tryptamine hydroxylase mutant can catalyze tryptamine to produce 5-hydroxytryptamine because it has an electron transport system and cofactors.

[0104] Those skilled in the art know that the redox partner Fdr-Fdx composed of ferredoxin reductase (Fdr) and ferredoxin (Fdx) is an electron transport chain that can use the coenzyme NADPH as the starting electron donor to enhance electron transfer efficiency and enhance the reaction process.

[0105] In some preferred embodiments, the reaction process can be accelerated by exogenously adding Fdr-Fdx proteins or co-expressing them with the target protein in engineered bacteria. For example, co-expressing Fdr-Fdx at the C-terminus of wild-type tryptamine hydroxylase can enhance electron transfer efficiency and improve the enzyme's catalytic activity, approximately doubling product yield. However, the addition or co-expression of Fdr-Fdr is not required for wild-type tryptamine hydroxylase, further truncations, and the aforementioned tryptamine hydroxylase mutants.

[0106] In some embodiments of the present invention, sequence truncation and site-specific mutagenesis are performed using T5H-Fdr-Fdx as the starting enzyme, specifically illustrating the effects of sequence truncation and mutagenesis. Similarly, sequence truncation (N-terminal truncation of 30-50 amino acids) and site-specific mutagenesis using T5H as the starting enzyme also exhibits corresponding truncation and mutagenesis effects.

[0107] In the present invention, after the N-terminus of wild-type tryptamine hydroxylase was truncated by 35, 40, and 45 amino acids, respectively, the production of 5-hydroxytryptamine was increased by 47%, 127%, and 82%, respectively, compared with the wild-type tryptamine hydroxylase; T5H-Fdr-Fdx obtained by co-expressing wild-type tryptamine hydroxylase and Fdr-Fdx had a 94% increase in catalytic activity towards tryptamine compared with T5H; further, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, and Δ45T5H-Fdr-Fdx obtained by truncating the N-terminus of T5H-Fdr-Fdx by 35, 45, and 50 amino acids, respectively, had catalytic activities increased by 50%, 125%, and 87%, respectively, compared with T5H-Fdr-Fdx;

[0108] In some specific embodiments of the present invention, the spinach ferredoxin (Fdx) / reductase (Fdr) system is used, which is derived from eukaryotic cells and is a widely used commercial electron transport chain.

[0109] The amino acid sequences of Fdr and Fdx are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0110] SEQ ID NO:3

[0111] MLNASVAGGAATTTYGNRLFIYEVIGLRQAEGEPSDSSIRRSGSTFFKVPYSRMNQEMQRILRLGGKIVSIRPAEEAAANNGAAPLQAAAEEPAAAPTPAP AAKKHSAEDVPVNIYRPNKPFVGKVLSNEPLVQEGGIGVVQHLTFDISEGDLRYIEGQSIGIIPDGTDDKGKPHKLRLYSIASTRHGDHVDDKTVSLCVRQL QYQNEAGETINGVCSTFLCGLKPGDDVKITGPVGKEMLLPADTDANVIMMGTGTGIAPFRAYLWRMFKDNERAINSEYQFNGKAWLIFGIPTTANILYKEELEALQAQYPDNFRLTYAISREQKNEAGGRMYIQDRVAEHADEIWNLLKDEKTHVYICGLRGMEDGIDQAMTVAAAKEDVVWSDYQRTLKKAGRWHVETYGT*

[0112] SEQ ID NO:4

[0113] MATYKVTLVNAAEGLNTTIDVADDTYILDAAEEQGIDLPYSCRAGACSTCAGKVV SGTVDQSDQSFLDDDQIAAGFVLTCVAYPTSDVTIETHKEEDL*

[0114] In the following examples, the codon-optimized nucleotide sequences of Fdr and Fdx are shown in SEQ ID NO: 33 and SEQ ID NO: 34.

[0115] SEQ ID NO:33

[0116]

[0117] SEQ ID NO:34

[0118] ATGGCAACCTACAAGGTTACGCTCGTCAATGCTGCCGAAGGCTTGAACACCACGATCGACGTGGCTGACGATACCTACATCTTGGACGCCGCTGAAGAGCAAGGCATTGACCTGCCTTACTCCTGCCGTGCTGGTGCTTGCTCGACCTGT GCTGGCAAAGTCGTCTCTGGTACCGTCGACCAATCGGATCAATCCTTCTTGGATGACGACCAAATTGCAGCAGGCTTTGTCCTGACCTGCGTCGCCTATCCGACCTCCGATGTGACGATCGAAACCCACAAAGAAGAAGACCTCTACTAG.

[0119] Furthermore, the recombinant vector and host cell for co-expressing the tryptamine hydroxylase mutant and Fdr-Fdx of the present invention are also within the protection scope of the present invention.

[0120] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0121] The molecular biology experiments in the following examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, and culture medium preparation, were primarily performed with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook et al., published by Science Press. Specific experimental conditions can be determined by simple experiments, if necessary. PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions, and can be adjusted by simple experiments, if necessary.

[0122] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources:

[0123] Plasmid: pET22b, purchased from Novagen;

[0124] Wild-type tryptamine hydroxylase (T5H): derived from rice, with the amino acid sequence shown in SEQ ID NO: 1. After codon optimization, the encoding nucleotide sequence is shown in SEQ ID NO: 2. It was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0125] Detection methods for tryptamine and 5-hydroxytryptamine:

[0126] Tryptamine and 5-hydroxytryptamine were analyzed using a high-performance liquid chromatograph (HPLC). The instrument model was a Shimadzu LAT-20A high-performance liquid chromatograph (HPLC); the chromatographic column was a C18 (250 mm x 4.6 mm, 5 μm) or equivalent column. The analytical conditions were as follows: the mobile phase consisted of 12 wt% methanol and 88 wt% potassium dihydrogen phosphate aqueous solution (5 mmol / L, pH 3.0), the flow rate was 1 mL / min, the injection volume was 10 μL, the column oven temperature was 35°C, and tryptamine and 5-hydroxytryptamine were detected at a wavelength of 275 nm. The content was determined using an external standard method. Figure 5 This is the HPLC chromatogram of the tryptamine standard, with the peak time at 14.0-14.5. Figure 6 This is the HPLC chromatographic extract of 5-HT standard, with a peak time of 5.3-5.7.

[0127] Example 1 Construction of wild-type tryptamine hydroxylase (T5H) engineered bacteria

[0128] (1) Using the pET22b vector as a template, PCR amplification was performed using primers T7pro-F and pET-R (primer sequences are shown in Table 1). After the PCR product was recovered, the linearized vector pET22b-1 was obtained. The linearized vector fragment size was 5350 bp;

[0129] (2) Using the coding nucleotide sequence of wild-type tryptamine hydroxylase (T5H) (SEQ ID NO: 2) as a template, PCR amplification was performed using primers T5H-F and T5H-R (primer sequences are shown in Table 1). The amplified product was recovered to obtain the target T5H gene fragment with a fragment size of 1602 bp.

[0130] (3) The two target fragments were connected using the ClonExpress II one-step cloning kit to obtain a recombinant plasmid, which was named pET22b-T5H. The correctness was verified by sequencing, and the recombinant plasmid was successfully constructed.

[0131] (4) The pET22b-T5H plasmid was transformed into the Escherichia coli expression host strain BL21 (DE3) by electroporation and plated onto LB solid medium containing ampicillin. The LB plate was cultured at 37°C until transformants grew. Positive transformants were picked to obtain wild-type tryptamine hydroxylase engineered bacteria (T5H).

[0132] The PCR reaction system in (1) and (2) above was 1 μL of template, 2 μL of upstream and downstream primers, 25 μL of PrimeSTAR Max DNA polymerase, and 20 μL of sterilized double-distilled water; the PCR amplification program was pre-denaturation at 98°C for 5 min, denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 7 min.

[0133] Example 2 Construction of T5H Co-expressing Fdr-Fdx Engineering Bacteria

[0134] (1) Using the pET22b vector as a template, PCR amplification was performed using primers pET-F and pET-R (primer sequences are shown in Table 1). After the PCR product was recovered, the linearized vector pET22b-2 was obtained. The linearized vector fragment size was 5416 bp;

[0135] (2) Using the synthesized T5H gene (SEQ ID NO: 2) as a template, PCR amplification was performed using primers T5H-F and T5H-R (primer sequences are shown in Table 1). The amplified product was recovered to obtain the target T5H gene fragment with a fragment size of 1602 bp.

[0136] (3) Using the pET22b vector as a template, PCR amplification was performed using primers T7pro-F and T7pro-R (primer sequences are shown in Table 1). After recovery of the PCR product, the target fragment T7pro was obtained, with a fragment size of 283 bp.

[0137] (4) Using the artificially synthesized Fdr gene as a template, PCR amplification was performed using primers Fdr-F and Fdr-R. After the PCR product was recovered, the target Fdr gene fragment was obtained, with a fragment size of 1232 bp.

[0138] (5) Using the artificially synthesized Fdx gene as a template, PCR amplification was performed using primers Fdx-F and Fdx-R. After the PCR product was recovered, the target Fdx gene fragment was obtained, with a fragment size of 350 bp.

[0139] (6) The four target fragments were ligated with the personalized vector pET22b-2 using the ClonExpress II one-step cloning kit to obtain a recombinant plasmid, which was named pET22b-T5H-Fdr-Fdx. The recombinant plasmid was successfully constructed after sequencing verification.

[0140] (7) The pET22b-T5H-Fdr-Fdx plasmid was transformed into the Escherichia coli expression host strain BL21 (DE3) by electroporation and plated onto LB solid medium containing ampicillin. The LB plate was cultured at 37°C until transformants grew. Positive transformants were picked to obtain wild-type Fdr-Fdx co-expressing tryptamine hydroxylase engineered bacteria (T5H-Fdr-Fdx).

[0141] The PCR reaction system was 1 μL of template, 2 μL of upstream and downstream primers, 25 μL of PrimeSTAR Max DNA polymerase, and 20 μL of sterilized double-distilled water. The PCR amplification program was 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and 72°C extension for 7 min.

[0142] Example 3 Construction of N-terminally truncated tryptamine hydroxylase engineered bacteria

[0143] Using the engineered bacterium T5H as the starting strain, the N-terminus of tryptamine hydroxylase was truncated by 20, 30, 35, 40, 45, and 50 amino acids, respectively, to obtain engineered bacteria Δ20T5H, Δ30T5H, Δ35T5H, Δ40T5H, Δ45T5H, and Δ50T5H; using the engineered bacterium T5H-Fdr-Fdx as the starting strain, the N-terminus of tryptamine hydroxylase was truncated by 35, 40, and 45 amino acids, respectively, to obtain engineered bacteria Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, and Δ45T5H-Fdr-Fdx. The specific construction method is as follows:

[0144] (1) Using the constructed pET22b-T5H plasmid as a template, PCR amplification was performed using primers Δ40T5H-F and pET-R. The amplified product was self-ligated to obtain a recombinant plasmid with 40 amino acid residues deleted from the N-terminus of wild-type tryptamine hydroxylase. It was named pET22b-Δ40T5H and verified to be correct by sequencing. The recombinant plasmid pET22b-Δ20T5H, pET22b-Δ30T5H, pET22b-Δ35T5H, pET22b-Δ45T5H and pET22b-Δ50T5H were constructed in the same manner as above. The upstream primers were replaced with Δ20T5H-F, Δ30T5H-F, Δ35T5H-F, Δ45T5H-F and Δ50T5H-F, respectively, for reverse amplification and self-ligation.

[0145] (2) Using the constructed pET22b-T5H-Fdr-Fdx plasmid as a template, PCR amplification was performed using primers T7pro-F and pET-R. The amplified product was recovered to obtain the pET22b-Fdr-Fdx target fragment with a fragment size of 7221 bp.

[0146] (3) Using the constructed pET22b-T5H-Fdr-Fdx plasmid as a template, PCR amplification was performed using primers Δ40T5H-F and T5H-R. The amplified product was recovered to obtain the target fragment of the Δ40T5H gene, with a fragment size of 1455 bp.

[0147] (4) The two target fragments were ligated using the ClonExpress II one-step cloning kit to obtain a recombinant plasmid with 40 amino acid residues deleted from the N-terminus of the wild-type tryptamine hydroxylase. The recombinant plasmid was named pET22b-Δ40T5H-Fdr-Fdx and was successfully constructed after sequencing verification. The plasmid map is shown in FIG. Figure 1 shown.

[0148] (5) Using the constructed pET22b-Δ40T5H-Fdr-Fdx plasmid as a template, primers Δ35T5H-F, ΔT5H-R and Δ45T5H-F, ΔT5H-R were used for PCR amplification to construct the tryptamine hydroxylase engineered bacterial plasmids with different numbers of amino acids truncated at the N-terminus: pET22b-Δ35T5H-Fdr-Fdx and pET22b-Δ45T5H-Fdr-Fdx.

[0149] (6) pET22b-Δ20T5H, pET22b-Δ30T5H, pET22b-Δ35T5H, pET22b-Δ40T5H, pET22b-Δ45T5H, pET22b-Δ50T5H, pET22b-Δ35T5H-Fdr-Fdx, and pET22b-Δ40T5H-Fdr-Fdx were transformed into pET22b-Δ20T5H, pET22b-Δ30T5H, pET22b-Δ35T5H-Fdr-Fdx, and pET22b-Δ40T5H-Fdr-Fdx. The pET22b-Δ45T5H-Fdr-Fdx plasmids were transformed into the Escherichia coli expression host strain BL21 (DE3), respectively, and plated onto LB solid medium containing ampicillin. The LB plates were cultured at 37°C until transformants grew out. The positive transformants were picked to obtain engineered bacteria with N-terminal truncations of 20, 30, 35, 40, 45 and 50 amino acids of tryptamine hydroxylase (Δ20T5H, Δ30T5H, Δ35T5H, Δ40T5H, Δ45T5H, Δ50T5H, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, and Δ45T5H-Fdr-Fdx).

[0150] The PCR reaction system was 1 μL of template, 2 μL of upstream and downstream primers, 25 μL of PrimeSTAR Max DNA polymerase, and 20 μL of sterilized double-distilled water. The PCR amplification program was 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and 72°C extension for 7 min.

[0151] Table 1 Plasmid construction primer sequences

[0152]

[0153] Example 4 Construction of single mutant tryptamine hydroxylase engineered bacteria

[0154] Using the constructed pET22b-Δ40T5H-Fdr-Fdx plasmid as a template, primers were designed for plasmid amplification and mutagenesis to obtain a linearized plasmid vector with base mutations. This vector was then transformed into Escherichia coli BL21 (DE3) and repaired and circularized in vivo to obtain a plasmid with base mutations. Specifically:

[0155] (1) Using the constructed pET22b-Δ40T5H-Fdr-Fdx plasmid as a template, PCR amplification was performed using six pairs of primers. The primer sequences (F114C-F, F114C-R, M133Q-F, M133Q-R, C247N-F, C247N-R, V309I-F, V309I-R, F401Y-F, F401Y-R, Q464E- F, Q464E-R) as shown in Table 3, 6 mutant sequences were obtained, and the mutation modes were as follows: the phenylalanine at amino acid position 114 of Δ40T5H was mutated to cysteine, the methionine at position 133 was mutated to glutamine, the cysteine at position 247 was mutated to asparagine, the valine at position 309 was mutated to isoleucine, the phenylalanine at position 401 was mutated to tyrosine, and the glutamine at position 464 was mutated to glutamate. The obtained plasmids expressing mutants were named pET22b-Δ40T5H(F114C)-Fdr-Fdx, pET22b-Δ40T5H(M133Q)-Fdr-Fdx, pET22b-Δ40T5H(C247N)-Fdr-Fdx, pET22b-Δ40T5H(V309I)-Fdr-Fdx, pET22b-Δ40T5H(F401Y)-Fdr-Fdx, and pET22b-Δ40T5H(Q464E)-Fdr-Fdx.

[0156] (2) After the PCR reaction, take 8.5 μL of PCR product, add 0.5 μL of DpnI restriction enzyme, 1 μL of 10× CutSmart, and incubate in a 37°C water bath for 16 h.

[0157] (3) The six Dpn I-treated linearized plasmids were transformed into the Escherichia coli expression host strain BL21 (DE3) by electroporation and plated onto LB solid medium containing ampicillin. The LB plates were cultured at 37°C until transformants grew. The transformants were picked and sequenced to obtain Δ40T5H-Fdr-Fdx single mutant tryptamine hydroxylase engineered bacteria Δ40T5H(F114C)-Fdr-Fdx, Δ40T5H(M133Q)-Fdr-Fdx, Δ40T5H(C247N)-Fdr-Fdx, Δ40T5H(V309I)-Fdr-Fdx, Δ40T5H(F401Y)-Fdr-Fdx, and Δ40T5H(Q464E)-Fdr-Fdx.

[0158] The above PCR reaction system is 1 μL of template, 2 μL of upstream and downstream primers, 25 μL of PrimeSTAR Max DNA polymerase, and 20 μL of sterile double-distilled water; the PCR amplification program is 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min 30 s, 30 cycles, and 72°C extension for 7 min.

[0159] The catalytic activity of the tryptamine hydroxylase engineered bacteria of T5H, Δ20T5H, Δ30T5H, Δ35T5H, Δ40T5H, Δ45T5H, Δ50T5H, T5H-Fdr-Fdx, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, Δ45T5H-Fdr-Fdx and Δ40T5H-Fdr-Fdx single mutant was determined as follows:

[0160] (1) The T5H, Δ35T5H, Δ40T5H, Δ45T5H, T5H-Fdr-Fdx, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, Δ45T5H-Fdr-Fdx and single mutant tryptamine hydroxylase engineered bacteria of Δ40T5H-Fdr-Fdx were cultured overnight in a seed culture medium containing 50 μg / mL ampicillin to obtain a seed solution. The seed culture medium (mass percentage) was 1% tryptone, 1% sodium chloride and 0.5% yeast extract. The culture conditions of the seed solution were 37°C and 220 rpm.

[0161] (2) 2% of the above seed solution was inoculated into a protein expression medium containing 1.6% tryptone, 1% yeast extract, and 0.5% NaCl (mass percentage), and the culture conditions were 37° C. and 220 rpm.

[0162] (3) After culturing for 3 h, IPTG was added to a final concentration of 0.5 mM to induce expression at 25°C and 220 rpm.

[0163] (4) After 5 h of induction, shake flask fermentation was performed in a 50 mL reaction flask. The substrate tryptamine concentration was approximately 1.5 g / L. The reaction conditions were 37°C, 220 rpm, and the reaction time was 24 h to obtain a fermentation reaction solution. The fermentation reaction solution was diluted with methanol by an appropriate multiple and centrifuged at 12,000 × g for 3 min. The supernatant was filtered through a 0.22 μm organic filter membrane, and the tryptamine and 5-hydroxytryptamine contents were determined. The results are shown in Table 2 below.

[0164] Table 2 The yield of 5-hydroxytryptamine produced by the engineered bacteria prepared in Examples 1 to 4

[0165]

[0166] The production of 5-hydroxytryptamine catalyzed by some engineered bacteria can also be seen Figure 2 and Figure 3 , from Table 2 and Figure 2 、 Figure 3 The data show that compared with the T5H strain, the 5-hydroxytryptamine content catalyzed by Δ20T5H obtained by truncating 20 amino acids at the N-terminus was reduced by 51%, and the 50T5H obtained by truncating 50 amino acids at the N-terminus was inactivated and had no tryptamine hydroxylase activity; the 5-hydroxytryptamine content catalyzed by Δ30T5H obtained by truncating 30 amino acids at the N-terminus was basically unchanged, but from the perspective of the preparation process, the inclusion bodies were reduced after the amino acid sequence was truncated, the protein soluble expression was enhanced, and the preparation efficiency was improved; the 5-hydroxytryptamine content catalyzed by Δ35T5H, Δ40T5H, and Δ45T5H obtained by truncating 35, 40, and 45 amino acids at the N-terminus was significantly improved. The contents increased by 47%, 127% and 82% respectively. Truncation of 35 to 45 amino acids can significantly improve its catalytic performance and increase the production of 5-hydroxytryptamine from tryptamine. The content of 5-hydroxytryptamine catalyzed by T5H-Fdr-Fdx, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, Δ45T5H-Fdr-Fdx strains and Δ40T5H-Fdr-Fdx single mutant strain was increased. Among them, the production of 5-hydroxytryptamine by Δ40T5H(C247N)-Fdr-Fdx reached 599.8 mg / L, an increase of 349% compared with the T5H-Fdr-Fdx strain.

[0167] Example 5 Construction of multiple mutant tryptamine hydroxylase engineered bacteria

[0168] Based on the above-mentioned single mutant tryptamine hydroxylase engineered bacteria, the present invention further constructs multiple mutant tryptamine hydroxylase engineered bacteria.

[0169] (1) The multiple mutant tryptamine hydroxylase engineered bacteria were constructed by combining three single point mutations (F114C, M133Q, and C247N) with improved activity. The specific results are shown below, and the construction method is the same as that of Example 4.

[0170] Based on the existing plasmid pET22b-Δ40T5H(F114C)-Fdr-Fdx, primers M133Q-F and M133Q-R were used to construct the double combination mutant plasmid pET22b-Δ40T5H(F114C / M133Q)-Fdr-Fdx; primers C247N-F and C247N-R were used to construct the double combination mutant plasmid pET22b-Δ40T5H(F114C / C247N)-Fdr-Fdx; primers M133 The triple mutant plasmid pET22b-Δ40T5H(F114C / M133Q / C247N)-Fdr-Fdx was constructed using primers Q-F1 and C247N-R1. Based on the existing plasmid pET22b-Δ40T5H(M133Q)-Fdr-Fdx, the double mutant plasmid pET22b-Δ40T5H(M133Q / C247N)-Fdr-Fdx was constructed using primers C247N-F and C247N-R. The primer sequences are shown in Table 3.

[0171] Table 3 Primer sequences for constructing mutants in Examples 4 and 5

[0172] Primers Sequence: 5'→3' F114C-F TGAGCGGCCAGTGTCTGAGCTTTGGTT, (SEQ ID NO: 19) F114C-R TGCTTGCAACCGGTTCCAGTTCCGG, (SEQ ID NO:20) M133Q-F CGTATCATCGTCAGGCCCGCCGTGTGG, (SEQ ID NO: 21) M133Q-R TGCTTGCAACCGGTTCCAGTTCCGG, (SEQ ID NO:22) C247N-F CCGCCTGAAAAAAAATCTGGCAGATCTGCG, (SEQ ID NO:23) C247N-R CAGTTTATAATCCGGATCTTTCAGATCAATTTCACCAC, (SEQ ID NO: 24) V309I-F CTGGATATGTTTTATTGCCGGTACCGATACCACCTTTG, (SEQ ID NO:25) V309I-R CAGTTTATAATCCGGATCTTTCAGATCAATTTCACCAC, (SEQ ID NO: 26) F401Y-F CGCACCCGCGTTTATATTAATACCTTTGCAATGGG, (SEQ ID NO: 27) F401Y-R CAGTTTATAATCCGGATCTTTCAGATCAATTTCACCAC, (SEQ ID NO: 28) Q464E-F CTGGCAACCGTTGAAGTGAGCCTGGCCAGCCTGCTGT, (SEQ ID NO: 29) Q464E-R CAGCCGGATCTTACACTTCACT, (SEQ ID NO:30) M133Q-F1 CGTATCATCGTCAGGCCCGCCGTGTGG(SEQ ID NO:31) C247N-R1 AGATCTGCCAGATTTTTTTTCAGGCGGCGACG(SEQ ID NO:32)

[0173] (2) The above-mentioned multiple mutants are not limited to the above-mentioned site combinations. Multiple mutant tryptamine hydroxylase engineered bacteria constructed based on other sites can also improve enzyme activity and have a synergistic effect. For example: F114C / V309I, F114C / F401Y, F114C / Q464E, M133Q / F401Y, M133Q / V309I, M133Q / Q464E, C247N / V309I, C247N / F401Y, C247N / Q464E, F114C / M133Q / V309I, F114C / M133Q / F401Y, M133Q / C247N / F401Y, etc.

[0174] The multiple mutants constructed in this example were used to determine the catalytic activity of each strain towards the substrate tryptamine. The specific method was the same as in Example 4. The results are shown in Tables 4 and Figure 4 As shown,

[0175] Table 4

[0176]

[0177] It can be seen that compared with Δ40T5H-Fdr-Fdx, the Δ40T5H-Fdr-Fdx multiple mutant strain catalyzed a significant increase in the 5-hydroxytryptamine content, with the highest content reaching 998 mg / L. This yield can be increased by up to 6.5 times compared with the wild-type T5H-Fdr-Fdx strain and 13.5 times compared with the wild-type T5H strain.

[0178] Overall, a series of modifications to the tryptamine hydroxylase, including N-terminal truncation, single-point mutations, and combined mutations, have all resulted in a modest improvement in enzyme activity. Furthermore, co-expression of Fdr-Fdx to enhance electron transfer efficiency has been shown to further enhance the production of its product, 5-hydroxytryptamine.

[0179] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tryptamine hydroxylase mutant, characterized in that An amino acid sequence selected from any one of the following: (a) obtained by deleting 35 to 45 amino acids from the N-terminus of tryptamine hydroxylase having the amino acid sequence of SEQ ID NO: 1; (b) is formed by mutation of the amino acid sequence shown in (a), Site mutations of one or two amino acid residues selected from the group consisting of: No. 114: F114C; No. 133: M133Q; No. 247: C247N; No. 309: V309I; No. 401: F401Y; Position 464: Q464E, or Selected from any one of the following sequence mutations: F114C / M133Q / C247N, F114C / M133Q / V309I, F114C / M133Q / F401Y, M133Q / C247N / F401Y, The position of the mutation site corresponds to the tryptamine hydroxylase with the sequence of SEQ ID NO: 1; (c) The amino acid sequence obtained by adding a tag sequence, restriction site sequence, or signal sequence to one or both ends of (a) or (b).

2. A DNA molecule encoding the tryptamine hydroxylase mutant according to claim 1.

3. A recombinant vector comprising the DNA molecule according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector co-expresses a redox partner Fdr-Fdx formed by ferredoxin reductase and ferredoxin.

5. A host cell, characterized in that The recombinant vector comprises the recombinant vector according to claim 3 or 4, or the DNA molecule according to claim 2 is integrated into its genome.

6. Use of the tryptamine hydroxylase mutant according to claim 1, the DNA molecule according to claim 2, the recombinant vector according to claim 3 or 4, and the host cell according to claim 5 in producing 5-hydroxytryptamine.

7. A method for producing 5-hydroxytryptamine, characterized in that: The steps include: 1) Producing 5-hydroxytryptamine using the tryptamine hydroxylase mutant according to claim 1 or the host cell according to claim 5; 2) 5-HT isolated from the system in 1).

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