Tryptamine hydroxylase mutant and application thereof

Abstract

The invention provides a tryptamine hydroxylase mutant and application thereof. The tryptamine hydroxylase mutant is selected from any one of the following amino acid sequences: (a) the tryptamine hydroxylase mutant is obtained by deleting 30-45 amino acids from the N terminal of tryptamine hydroxylase as shown in SEQ ID NO: 1, and (b) the tryptamine hydroxylase mutant is obtained by deleting 30-45 amino acids from the N terminal of tryptamine hydroxylase as shown in SEQ ID NO: 2; (b) is formed by mutation of the amino acid sequence shown in (a), mutation sites are selected from one or more of the following amino acid residue sites: the 114 site, the 133 site, the 247 site, the 309 site, the 401 site and the 464 site, and the positions of the mutation sites correspond to the wild tryptamine hydroxylase with the sequence of SEQ ID NO: 1; (c) an amino acid sequence obtained by adding a tag sequence, a restriction enzyme cutting site sequence or a signal sequence to one end or two ends of (a) or (b). The catalytic activity of the tryptamine hydroxylase mutant is greatly improved compared with that of a wild enzyme, and the yield of 5-hydroxytryptamine is remarkably improved.

Description

Technical Field

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

[0002] 5-HT was first discovered in serum, also known as serotonin. It is widely present in mammalian tissues and is mainly distributed in the pineal gland and hypothalamus. It can regulate a variety of physiological functions, such as sleep, appetite, mood, immune regulation and pain perception. In addition, 5-HT also plays an important role in plants, including flowering cycle, morphological construction, aging, resistance to biological stress and other physiological regulation processes.

[0003] Since serotonin was first discovered in mammals and plants, extracting serotonin from these sources is the earliest and most mature method. 5-HT in rat brain is often extracted for analysis and clinical applications. Audhya et al. studied the extraction of 5-HT by collecting 30-50ul cerebrospinal fluid from rats through direct lumbar puncture. In addition, Miekus et al. used formic acid or perchloric acid solution to mechanically homogenize brain tissue samples to further extract serotonin for research. 5-HT is also found in different parts of plants, such as leaves, roots, flowers, fruits and seeds. Some foods are also reported to contain high concentrations of 5-HT, including bananas, tomatoes, plantains, pineapples, butter, black walnuts and English walnuts. In addition, 5-HT and its precursor 5-HTP are also found in the seeds of Moringa 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] How to industrially produce 5-hydroxytryptamine and its derivatives (such as melatonin) so that they can be used in the medical field for the treatment of mental illness, the development of new drugs, and in the health care field for mood regulation, sleep promotion, etc. is the primary problem to be solved. In microorganisms, plants, animals and humans, serotonin is synthesized from tryptophan. In animals, tryptophan 5-hydroxylase hydroxylates L-tryptophan to 5-hydroxy-L-tryptophan, and 5-hydroxy-L-tryptophan is converted into serotonin by aromatic L-amino acid decarboxylase. In this pathway, the first step of hydroxylation requires tetrahydrobiopterin (BH4) as a cofactor, which involves a complex BH4 cycle and regeneration pathway. Therefore, the process through this pathway is complex and costly. In plants, tryptophan is converted into tryptamine, and then converted into serotonin after being hydroxylated by tryptamine hydroxylase. Unlike the synthetic pathway in animals, in plants, decarboxylation precedes hydroxylation. This pathway does not require the participation of BH4, avoiding the complex BH4 cycle and regeneration pathway. However, due to the long growth cycle of plants, seasonal and regional restrictions, the yield is low and it is not suitable for industrial application. In recent years, with the rapid development of synthetic biology, people have gradually turned their attention to microorganisms. Expressing the biosynthetic pathways of valuable secondary metabolites in animals and plants in microorganisms to synthesize the main components of some drugs is one of the current hot spots in synthetic biology research. Microbial production has the advantages of short production cycle, low cost, high yield and easy large-scale production. However, in the biosynthetic pathway of serotonin, the synthesis of 5-hydroxytryptamine is limited by the low activity of tryptamine hydroxylase, and its large-scale biosynthesis is still challenging. Therefore, in order to solve this problem, it is necessary to increase the activity of tryptamine hydroxylase, develop tryptamine hydroxylase mutants with high yield of 5-hydroxytryptamine, and optimize its industrial production conditions to reduce production costs and improve its 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, which can catalyze tryptamine to produce 5-hydroxytryptamine. Compared with the wild enzyme, the catalytic activity of the mutant is significantly improved, thereby increasing the yield of the product.

[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 an amino acid sequence of SEQ ID NO: 1;

[0009] (b) is 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, a restriction 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] Position 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, and 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 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 the redox partner Fdr-Fdx formed by ferredoxin reductase and 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 generate 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 mutation on this basis. On this basis, the present invention further discloses a DNA molecule encoding the tryptamine hydroxylase mutant, a recombinant vector containing the above DNA molecule, and a host cell. The above tryptamine hydroxylase mutant and the recombinant vector or host cell can catalyze tryptamine to produce 5-hydroxytryptamine. Furthermore, the tryptamine hydroxylase mutant is co-expressed with a redox partner Fdr-Fdx composed of ferredoxin reductase (Fdr) and ferredoxin (Fdx), which can enhance the efficiency of electron transfer and increase the production of 5-hydroxytryptamine.

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

[0042] 1) The catalytic activity of the tryptamine hydroxylase mutant obtained by truncating the amino acid sequence of wild tryptamine hydroxylase and further performing specific site mutation on this basis has been greatly improved. Experimental verification shows that, preferably, the catalytic activity of the tryptamine hydroxylase mutant is significantly improved relative to that of the wild-type tryptamine hydroxylase, and the yield of 5-hydroxytryptamine generated by catalyzing tryptamine is significantly increased, up to 6.5 times. Therefore, the purpose of reducing the amount of catalyst used and ultimately reducing production costs can be achieved, and at the same time, it also provides a basis for developing excellent tryptamine hydroxylases and promoting the industrial production of 5-hydroxytryptamine;

[0043] 2) Co-expression of the tryptamine hydroxylase mutant with the redox chaperone Fdr-Fdx composed of ferredoxin reductase (Fdr) and ferredoxin (Fdx) can enhance the efficiency of electron transfer, further improve the activity of the tryptamine hydroxylase mutant in catalyzing tryptamine, and increase the production of 5-hydroxytryptamine. The catalytic activity of the engineered bacteria co-expressing the tryptamine hydroxylase mutant and Fdr-Fdx on tryptamine can be increased by up to 13.5 times compared with the engineered bacteria with wild enzymes.

[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. The 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0047] Figure 2 The result diagram of the conversion of tryptamine into 5-hydroxytryptamine catalyzed by T5H, T5H-Fdr-Fdx, pET22b-Δ35T5H-Fdr-Fdx, pET22b-Δ40T5H-Fdr-Fdx, and pET22b-Δ45T5H-Fdr-Fdx;

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

[0049] Figure 4 This is a graph showing the result of Δ40T5H-Fdr-Fdx multiple mutants catalyzing the conversion of tryptamine into 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 in conjunction with 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 to describe embodiments of the present invention, and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary 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 can be used interchangeably herein, and both refer to the wild-type tryptamine hydroxylase having an amino acid sequence as shown in SEQ ID NO: 1, which is derived from rice (Oryza sativa Japonica Group) and has a publication number of NP_001410381.1 in NCBI. The enzyme can catalyze the hydroxylation of tryptamine to generate 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 have found through multiple experiments that after truncating 20 amino acids at the N-terminus of wild-type tryptamine hydroxylase, the catalytic activity of the resulting tryptamine hydroxylase mutant is reduced, after truncating 30 amino acids, the catalytic activity of the resulting tryptamine hydroxylase mutant remains essentially unchanged, after truncating 35 to 45 amino acid residues, the resulting tryptamine hydroxylase mutant has significantly improved catalytic activity for tryptamine, and can increase the production of 5-hydroxytryptamine, and after truncating 50 amino acid residues, the tryptamine hydroxylase mutant is inactivated and no longer has 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 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, respectively, the production of 5-hydroxytryptamine is increased by 47%, 127%, and 82%, respectively, compared with the wild-type tryptamine hydroxylase.

[0064] The inventors further carried out site-directed mutagenesis on the tryptamine hydroxylase obtained by truncating 30 to 45 amino acid residues at the N-terminus to construct a mutant library, and finally obtained a tryptamine hydroxylase mutant with better activity, which was formed by mutating the amino acid sequence obtained by deleting 30 to 45 amino acids at the N-terminus of the tryptamine hydroxylase shown in SEQ ID NO: 1 at one or more of the following amino acid sites: 114, 133, 247, 309, 401 and 464, and the position of the mutation site corresponds to the wild tryptamine hydroxylase with the sequence of SEQ ID NO: 1.

[0065] Furthermore, the above mutation site is 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 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] Position 464: Q464E.

[0080] Using the tryptamine hydroxylase mutant Δ40T5H obtained by truncating 40 amino acids at the N-terminus as the starting enzyme, single-site 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 following were performed, including but not limited to F114C / M133Q, F114C / C247N, F114C / V309I, F114C / F401Y, F114C / Q464E, M133Q / C247N, M133Q / Q464E, C247N / V309 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" should also include: based on the above-mentioned tryptamine hydroxylase, several amino acids are deleted, inserted or substituted, and one or more amino acids are added or deleted at the C-terminus and / or N-terminus, but the derivatives formed by maintaining the activity and function of the P450 enzyme mutant of the present invention, for example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and adding one or more amino acids at the C-terminus and / or N-terminus, such as adding a tag sequence, a restriction site sequence, a signal sequence or a secretion signal sequence, usually does not change the function of the resulting protein. However, in these derivatives, the above-mentioned sequence truncations and / or mutations exist.

[0084] In the present invention, the term "tryptamine hydroxylase mutant" should also include: derivative proteins that retain their protein activity and have an amino acid sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98%, more than 99% with the above-mentioned tryptamine hydroxylase mutant. Similarly, these derivative proteins must contain the sequence truncations and / or mutations described above in the present invention.

[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 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 above 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 contains the above recombinant vector, or the DNA molecule encoding the tryptamine hydroxylase mutant is integrated into the genome.

[0091] The DNA molecule sequence of the present invention can be used to express or produce a tryptamine hydroxylase mutant by 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 tryptamine hydroxylase mutant encoding DNA sequence can be inserted into a recombinant expression vector, and any plasmid and vector can be used as long as it can stably replicate in the host. An important feature of the 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 tryptamine hydroxylase mutant encoding DNA sequence and suitable transcription / translation control signals. The DNA sequence can be effectively connected to an appropriate promoter of the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0093] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[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), 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 are often very different. In order to optimally express tryptamine hydroxylase or its mutants in genetically engineered bacteria Escherichia coli, the codons of the expression genes of these enzymes can be optimized.

[0096] Codon optimization is a kind of technology that can be used for maximizing protein expression in organism by increasing the translation efficiency of gene of interest.Different organisms usually show the special preference for one of some codons of the same amino acid of coding due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its genome tRNA library separately.Therefore, in fast-growing microorganisms, the low frequency codon of amino acid can be used for the codon displacement of the same amino acid but high frequency.Therefore, the expression of optimized DNA sequence 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 according to the changes in amino acid residues based on SEQ ID NO: 2.

[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 cultured 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 in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the promoter selected is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured 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 above-mentioned tryptamine hydroxylase mutant, expression vector or host cell. For example, in a specific embodiment, the 5-hydroxytryptamine produced can be obtained from the catalytic system 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 catalyzing the tryptamine hydroxylase mutant of the present invention to produce 5-hydroxytryptamine.

[0100] During expression, the tryptamine hydroxylase mutant of the present invention can be expressed intracellularly and secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 breaking, 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 and 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 tryptamine to produce 5-hydroxytryptamine, which requires cofactors and electron transfer. Therefore, when the tryptamine hydroxylase mutant of the present invention is used to catalyze tryptamine to produce 5-hydroxytryptamine, in addition to tryptamine and tryptamine hydroxylase, NADPH needs to be added to the reaction system.

[0103] The host cell expressing the above tryptamine hydroxylase mutant can catalyze tryptamine to produce 5-hydroxytryptamine because it has an electron transfer 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 the efficiency of electron transfer and the reaction process.

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

[0106] In some specific embodiments of the present invention, T5H-Fdr-Fdx is used as the starting enzyme to perform sequence truncation and site mutation, and the specific description of the sequence truncation effect and mutation effect is performed. However, similarly, using T5H as the starting enzyme to perform sequence truncation (N-terminal truncation of 30-50 amino acids) and site mutation also has corresponding truncation and mutation effects.

[0107] In the present invention, after the N-terminus of the wild tryptamine hydroxylase is truncated by 35, 40, and 45 amino acids, respectively, the production of 5-hydroxytryptamine is increased by 47%, 127%, and 82% respectively compared with the wild-type tryptamine hydroxylase; T5H-Fdr-Fdx obtained by co-expression of the wild tryptamine hydroxylase and Fdr-Fdx has a catalytic activity for tryptamine that is increased by 94% 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, have catalytic activities that are 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 the above-mentioned 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 nucleotide sequences of Fdr and Fdx after codon optimization 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 in conjunction with specific examples. It should be understood that these examples are only used 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 include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly with reference to "Molecular Cloning Laboratory Guide" (3rd Edition), edited by J. Sambrook et al., Science Press, and the specific experimental conditions can be determined by simple experiments when necessary. PCR amplification experiments are carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier, and can be adjusted by simple experiments when 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, the amino acid sequence is shown in SEQ ID NO: 1, after codon optimization, the encoding nucleotide sequence is shown in SEQ ID NO: 2, and it was chemically synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.;

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

[0126] High performance liquid chromatography was used to analyze tryptamine and 5-hydroxytryptamine. Instrument model: Shimadzu LAT-20A High Performance Liquid Chromatography (HPLC); chromatographic column: C18 (250mm*4.6mm, 5μm) or equivalent chromatographic column; analysis conditions were as follows: mobile phase: 12wt% methanol and 88wt% potassium dihydrogen phosphate aqueous solution (5mmol / L, pH3.0), flow rate: 1mL / min, injection volume: 10μL, column oven temperature: 35°C, tryptamine and 5-hydroxytryptamine were detected at 275 wavelength, and the content was determined by 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 the 5-HT standard, with a peak time of 5.3-5.7.

[0127] Example 1 Construction of wild-type tryptamine hydroxylase (T5H) engineering 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, a 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 fragment of the T5H gene, 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 recombinant plasmid was successfully constructed and verified to be correct by sequencing.

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

[0132] The PCR reaction system in (1) and (2) above 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 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 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, a 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 fragment of the T5H gene, 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 the PCR product was recovered, the target fragment T7pro was obtained, and the fragment size was 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 fragment of the Fdx gene was obtained, and the fragment size was 350 bp.

[0139] (6) The four target fragments were connected to 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 and verified to be correct by sequencing.

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

[0141] The 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 sterilized 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 30 s, 30 cycles, and 72°C extension for 7 min.

[0142] Example 3 Construction of an engineered bacterium producing an N-terminally truncated tryptamine hydroxylase

[0143] Using the engineered bacteria T5H as the starting bacteria, 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 bacteria T5H-Fdr-Fdx as the starting bacteria, 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, and the amplified product was self-ligated to obtain a recombinant plasmid. The N-terminal of the wild-type tryptamine hydroxylase lacked 40 amino acid residues and was named pET22b-Δ40T5H. The recombinant plasmid was successfully constructed and verified by sequencing. The recombinant plasmids pET22b-Δ20T5H, pET22b-Δ30T5H, pET22b-Δ35T5H, pET22b-Δ45T5H and pET22b-Δ50T5H were constructed in the same manner as above, and 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 connected using the ClonExpress II one-step cloning kit to obtain a recombinant plasmid, which lacked 40 amino acid residues at the N-terminus of the wild-type tryptamine hydroxylase and was named pET22b-Δ40T5H-Fdr-Fdx. The recombinant plasmid was successfully constructed and verified by sequencing. The plasmid map is shown in FIG. Figure 1 shown.

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

[0149] (6) The pET22b-Δ20T5H, pET22b-Δ30T5H, pET22b-Δ35T5H, pET22b-Δ40T5H, pET22b-Δ45T5H, pET22b-Δ50T5H, pET22b-Δ35T5H-Fdr-Fdx, pET22b-Δ40T5H-Fdr-Fdx and pET22b-Δ45T5H-Fdr-Fdx plasmids were transformed into the Escherichia coli expression host strain BL2 by electroporation. 1(DE3) and spread on LB solid medium containing ampicillin. The LB plate was cultured at 37°C until transformants grew out. The positive transformants were picked to obtain engineered bacteria with N-terminal truncated 20, 30, 35, 40, 45 and 50 amino acids of tryptamine hydroxylase (Δ20T5H, Δ30T5H, Δ35T5H, Δ40T5H, Δ45T5H, Δ50T5H, Δ35T5H-Fdr-Fdx, Δ40T5H-Fdr-Fdx, Δ45T5H-Fdr-Fdx).

[0150] The 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 sterilized 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 30 s, 30 cycles, and 72°C extension for 7 min.

[0151] Table 1 Primer sequences for plasmid construction

[0152]

[0153] Example 4 Construction of single mutant tryptamine hydroxylase engineering 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, which was then transformed into Escherichia coli BL21 (DE3) and repaired and cyclized 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 6 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 the 114th amino acid of Δ40T5H was mutated to cysteine, the methionine at the 133rd amino acid was mutated to glutamine, the cysteine ​​at the 247th amino acid was mutated to asparagine, the valine at the 309th amino acid was mutated to isoleucine, the phenylalanine at the 401th amino acid was mutated to tyrosine, and the glutamine at the 464th amino acid 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 endonuclease and 1 μL of 10×CutSmart, and incubate in a 37°C water bath for 16 h.

[0157] (3) The six linearized plasmids treated with Dpn I were transformed into the Escherichia coli expression host BL21 (DE3) by electroporation and plated on LB solid culture medium containing ampicillin. The LB plates were cultured at 37°C until transformants grew out. The transformants were picked and sequenced to obtain Δ40T5H-Fdr-Fdx single mutant tryptamine hydroxylase engineering 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 engineering 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, and the specific steps are as follows:

[0160] (1) 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, and 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) at 37° C. and 220 rpm.

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

[0163] (4) After 5 hours of induction, shake flask fermentation was carried out in a 50 mL reaction bottle, the concentration of substrate tryptamine was about 1.5 g / L, the reaction conditions were 37°C, 220 rpm, and the reaction time was 24 hours to obtain a fermentation reaction solution. The fermentation reaction solution was diluted with methanol by an appropriate multiple, centrifuged at 12000×g for 3 minutes, and the supernatant was filtered through a 0.22 μm organic filter membrane to detect the tryptamine and 5-hydroxytryptamine contents. 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-HT produced by some engineered bacteria catalyzing tryptamine 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 Δ20T5H obtained by truncating 20 amino acids at the N-terminus reduced the content of 5-hydroxytryptamine by 51%, and the Δ50T5H obtained by truncating 50 amino acids at the N-terminus was inactive and had no tryptamine hydroxylase activity; the Δ30T5H obtained by truncating 30 amino acids at the N-terminus had basically no change in the content of 5-hydroxytryptamine catalyzed, 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 Δ35T5H, Δ40T5H, and Δ45T5H obtained by truncating 35, 40, and 45 amino acids at the N-terminus catalyzed the production of 5-hydroxytryptamine The contents increased by 47%, 127% and 82% respectively. Truncating 35 to 45 amino acids can significantly improve its catalytic performance and increase the production of 5-hydroxytryptamine from tryptamine. The contents 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 were all increased, among which the output of Δ40T5H(C247N)-Fdr-Fdx reached 599.8 mg / L, which was 349% higher than that of T5H-Fdr-Fdx strain.

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

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

[0169] (1) The multiple mutant tryptamine hydroxylase engineering bacteria constructed by combining three single point mutations (F114C, M133Q, C247N) with improved activity. The specific results are shown below, and the construction method is the same as 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 mutant plasmid pET22b-Δ40T5H(F114C / M133Q)-Fdr-Fdx; primers C247N-F and C247N-R were used to construct the double 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 construction sequences of mutants in Example 4 and Example 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 engineering bacteria constructed based on other sites can also improve the 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 measure the catalytic activity of each strain on 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 5-hydroxytryptamine content catalyzed by the Δ40T5H-Fdr-Fdx multiple mutant strains was significantly increased, with the highest content reaching 998 mg / L. The yield was up to 6.5 times higher than that of the wild-type T5H-Fdr-Fdx strain and 13.5 times higher than that of the wild-type T5H strain.

[0178] In general, a series of modifications to the tryptamine hydroxylase, including N-terminal truncation, single point mutation, and combined mutation, have improved the activity of the enzyme to a certain extent. On this basis, co-expression of Fdr-Fdx to enhance its electron transfer efficiency is conducive to further improving 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tryptamine hydroxylase mutant, It is characterized in that An amino acid sequence selected from any of the following: (a) obtained by deleting 30 to 45 amino acids from the N-terminus of tryptamine hydroxylase having an amino acid sequence of SEQ ID NO: 1; (b) is 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; (c) An amino acid sequence obtained by adding a tag sequence, a restriction site sequence or a signal sequence to one or both ends of (a) or (b).

2. The tryptamine hydroxylase mutant according to claim 1, It is characterized in that The mutation site in (b) is selected from the following mutations of one or more amino acid residues: No. 114: F114C or F114S; No. 133: M133Q or M133N; No. 247: C247N or C247G; No. 309: V309I or V309W or V309P or V309L; No. 401: F401Y or F401T; Position 464: Q464E or Q464D.

3. The tryptamine hydroxylase mutant according to claim 2, It is characterized in that The sequence mutation at the site in (b) is selected from the following site mutations of one or more amino acid residues: No. 114: F114C; No. 133: M133Q; No. 247: C247N; No. 309: V309I; No. 401: F401Y; Position 464: Q464E.

4. The tryptamine hydroxylase mutant according to claim 3, It is characterized in that 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.

5. The tryptamine hydroxylase mutant according to any one of claims 1 to 4, It is characterized in that 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.

6. A DNA molecule encoding the tryptamine hydroxylase mutant according to any one of claims 1 to 5.

7. A recombinant vector comprising the DNA molecule of claim 6.

8. The recombinant vector according to claim 7, It is characterized in that The recombinant vector co-expresses a redox partner Fdr-Fdx formed by ferredoxin reductase and ferredoxin.

9. A host cell, It is characterized in that The recombinant vector of claim 7 or 8 is included, or the DNA molecule of claim 6 is integrated into its genome.

10. Use of the tryptamine hydroxylase mutant according to any one of claims 1 to 5, the DNA molecule according to claim 6, the recombinant vector according to claim 7 or 8, or the host cell according to claim 9 in producing 5-hydroxytryptamine.

11. A method for producing serotonin, It is characterized in that The steps include: 1) Producing 5-hydroxytryptamine using the tryptamine hydroxylase mutant according to any one of claims 1 to 5 or the host cell according to claim 9; 2) The 5-hydroxytryptamine isolated from the system of 1).

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