Melatonin synthetase derived from intestinal bacteria and application of melatonin synthetase

Abstract

The invention discloses an intestinal bacterium-derived melatonin synthetase and an application thereof. Specifically, the melatonin synthetase is selected from one or more of a DDC enzyme, an AANAT enzyme and an AST enzyme, the DDC enzyme comprises an amino acid sequence as shown in SEQ ID NO.2 or an active fragment of the amino acid sequence, or an analogue of which the similarity with the amino acid sequence as shown in SEQ ID NO.2 is more than 80%, and the DDC enzyme comprises an amino acid sequence as shown in SEQ ID NO.2 or an active fragment of the amino acid sequence as shown in SEQ ID NO.2 or an analogue of which the similarity with the amino acid sequence as shown in SEQ ID NO.2 is more than 80%. The AANAT enzyme comprises an amino acid sequence as shown in SEQ ID NO.34 or an active fragment thereof, or an analogue of which the similarity with the amino acid sequence as shown in SEQ ID NO.34 is more than 80%, and the AST enzyme comprises an amino acid sequence as shown in SEQ ID NO.42 or an active fragment thereof, or an analogue of which the similarity with the amino acid sequence as shown in SEQ ID NO.42 is more than 80%. On one hand, the melatonin synthetase can be used for improving the melatonin level of a host and improving related diseases; on the other hand, the method can be used for biosynthesis of melatonin and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to melatonin synthase derived from intestinal bacteria and its applications. Background Technology

[0002] Melatonin is a human hormone that regulates circadian rhythms, sleep and body temperature, and relieves intestinal inflammation.

[0003] The biosynthesis of melatonin involves the metabolic processes of multiple enzymes (such as...) Figure 11 (As shown). Currently, commercially available melatonin is mainly chemically synthesized. These chemical synthesis methods involve many steps, are costly, and cause serious environmental pollution. Direct extraction of melatonin from animals and plants is very inefficient. Designing and optimizing melatonin biosynthetic pathways in microorganisms holds promise as a cheap and environmentally friendly method for industrial production of melatonin.

[0004] A research team from the Institute of Microbiology, Chinese Academy of Sciences, introduced pterin-4α-methanolamine dehydratase from Pseudomonas, phenylalanine-4-hydroxylase from Xanthomonas, decarboxylase from Streptomyces, caffeic acid 3-O-methyltransferase from rice, and methyltransferase from Streptomyces into Escherichia coli, achieving the biosynthesis of melatonin (Zhang, Y., He, Y., Zhang, N., Gan, J., Zhang, S., & Dong, Z. Combining protein and metabolic engineering strategies for biosynthesis of melatonin in Escherichia coli. Microbial cell factories, 2021, 20(1), 170.). A research team from Zhejiang University reported tryptophan-5-hydroxylase (TPH) derived from environmental bacteria *Streptomyces griseofuscus* and *Entotheonella factor*, and heterologously expressed it along with methyltransferase and decarboxylase genes in an engineered strain lacking the tryptophan dioxygenase gene, producing melatonin via microbial fermentation [CN115992189A]. A research team from the University of California reported DDC enzyme (PMID: 25263219) derived from human intestinal bacteria *Ruminococcus gnavus* and *Clostridium sporogenes*.

[0005] Currently, most existing biosynthesis schemes utilize enzymes from eukaryotic organisms for synthesis and production. However, heterologous expression of melatonin biosynthetic enzymes from other sources in engineered microbial chassis suffers from problems such as very low expression levels, low soluble protein content, and poor catalytic activity. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a novel method for the in vitro synthesis of melatonin. This invention utilizes experiments such as heterologous protein expression, single-bacterial culture, and LC-MS metabolic detection to synthesize melatonin from human intestinal bacteria *Bifidobacterium* (…). Bifidobacterium ) and Bacteroides multifiliis ( Bacteroides dorei We screened and discovered melatonin synthases derived from gut bacteria.

[0007] Based on this, the first aspect of the present invention provides a melatonin synthase, wherein the melatonin synthase is selected from one or more of DDC enzyme, AANAT enzyme, and ASMT enzyme, wherein the DDC enzyme comprises the amino acid sequence shown in SEQ ID NO.2 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.2; the AANAT enzyme comprises the amino acid sequence shown in SEQ ID NO.34 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.34; and the ASMT enzyme comprises the amino acid sequence shown in SEQ ID NO.42 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.42.

[0008] In some embodiments, the active fragment or analogue of the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.34, or SEQ ID NO.42 includes one or more of the following: (a) Having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.34 or SEQ ID NO.42 or having an alternative amino acid sequence with the same functional group; (b) A derivative sequence which is derived from the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.34 or SEQ ID NO.42 by substituting, adding and / or deleting one or more amino acids and having the same function as the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.34 or SEQ ID NO.42.

[0009] In some embodiments, the melatonin synthase comprises an enzyme composition comprising a combination of AANAT and ASMT enzymes, a combination of DDC and AANAT enzymes, a combination of DDC and ASMT enzymes, or a combination of DDC, AANAT, and ASMT enzymes.

[0010] A second aspect of the present invention provides a nucleic acid molecule that encodes the melatonin synthase described in the first aspect of the present invention.

[0011] In some implementations, the sequence of the nucleic acid molecule encoding the DDC enzyme is shown in SEQ ID NO.60.

[0012] In some implementations, the sequence of the nucleic acid molecule encoding the AANAT enzyme is shown in SEQ ID NO.92.

[0013] In some implementations, the sequence of the nucleic acid encoding the ASMT enzyme is shown in SEQ ID NO.100.

[0014] A third aspect of the present invention provides a pharmaceutical preparation comprising the melatonin synthase described in the first aspect of the present invention.

[0015] A fourth aspect of the present invention provides a bacterial agent comprising a first bacterium expressing the AANAT enzyme described in the first aspect of the present invention, a second bacterium expressing the ASMT enzyme described in the first aspect of the present invention, and a third bacterium expressing the DDC enzyme described in the first aspect of the present invention.

[0016] In some implementations, the microbial agent includes intestinal bacteria or engineered bacteria.

[0017] In some implementations, the first and / or third bacteria are Bifidobacteria.

[0018] In this invention, the Bifidobacteria include, but are not limited to, Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium animalis.

[0019] In some implementations, the second bacterium is Bacteroides multifiliis.

[0020] The fifth aspect of this invention provides a strain of Bacteroides multifiliis, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46612.

[0021] The sixth aspect of this invention provides a method for in vitro synthesis of melatonin or melatonin synthesis-related metabolites, the method comprising: Add the melatonin synthase described in the first aspect of the present invention, the pharmaceutical preparation described in the third aspect of the present invention, the bacterial agent described in the fourth aspect of the present invention, or the Bacteroides multifiliis strain described in the fifth aspect of the present invention to a system containing a melatonin synthesis substrate. The melatonin synthesis substrates include one or more of 5-HTRP, 5-HT, and NAS; The melatonin synthesis-related metabolites include 5-HT and / or NAS.

[0022] The sixth aspect of this invention provides the use of the melatonin synthase described in the first aspect of this invention, the nucleic acid molecule described in the second aspect of this invention, the pharmaceutical preparation described in the third aspect of this invention, the bacterial agent described in the fourth aspect of this invention, or the Bacteroides dysporidis strain described in the fifth aspect of this invention in any of the following: (1) Prepare products that promote melatonin synthesis; (2) To prepare drugs for the prevention or treatment of diseases related to melatonin secretion or function; (3) Prepare foods or health products that help improve sleep; In some embodiments, the health products include probiotic supplements; the probiotic supplements described in this invention include, but are not limited to, probiotic strips and probiotic gummies.

[0023] In this invention, the diseases related to melatonin secretion or function include sleep disorders, nervous system disorders, mental health disorders, ophthalmological disorders, or inflammatory bowel disease. Specifically, sleep disorders include, but are not limited to, insomnia or delayed sleep phase syndrome; nervous system disorders include, but are not limited to, Alzheimer's disease or Parkinson's disease; mental health disorders include, but are not limited to, depression; and ophthalmological disorders include, but are not limited to, age-related macular degeneration and glaucoma.

[0024] In some implementations, the products include pharmaceutical preparations and reagents.

[0025] In some implementations, the drug can also be used to relieve intestinal inflammatory responses.

[0026] The seventh aspect of the present invention provides a method for preventing or treating diseases related to melatonin secretion or function, comprising administering to a subject the melatonin synthase described in the first aspect of the present invention, the pharmaceutical preparation described in the third aspect of the present invention, or the bacterial agent described in the fourth aspect of the present invention.

[0027] Beneficial effects: This invention discloses several melatonin synthases derived from human gut microbiota, which can be used to increase the host's melatonin levels and improve related diseases; and can also be used for the biosynthesis of melatonin.

[0028] The melatonin synthase disclosed in this invention is derived from human intestinal microorganisms. When it is biosynthesized by engineered chassis bacteria such as Escherichia coli, it can achieve more stable expression and high-efficiency enzyme activity. Attached Figure Description

[0029] Figure 1 Screening for DDC enzymes derived from gut bacteria for heterologous expression; Figure 2 Screening for AANAT enzymes derived from gut bacteria for heterologous expression; Figure 3 Screening for ASMT enzymes derived from gut bacteria for heterologous expression; Figure 4 To assess similarity to previously reported DDC enzymes derived from gut bacteria; Figure 5 To determine the activity parameters of melatonin synthase derived from intestinal bacteria; Figure 6 For the determination of enzyme activity in other Bifidobacterium species containing DDC or AANAT homologous sequences; Figure 7 Intestinal bacteria that express melatonin synthase can increase melatonin levels in germ-free mice; Figure 8 Intestinal bacteria or engineered bacteria that express melatonin synthase can increase melatonin levels in SPF mice; Figure 9 Engineered bacteria that express melatonin synthase can increase melatonin levels in disease hosts and improve disease performance. Figure 10 Gut bacteria that express melatonin synthase can improve disease performance by increasing melatonin levels in disease-affected hosts. Figure 11 Intestinal bacteria that express melatonin synthase can be used for the biosynthesis of melatonin; Figure 12 This refers to the metabolic pathway from tryptophan to melatonin. Figure 13 This invention describes a functional enzyme derived from gut bacteria that was discovered in the melatonin metabolic synthesis pathway.

[0030] Microbial preservation for patented procedures: 1. The Bacteroides strain of the present invention Bacteroides dorei DA75 Deposit date: September 15, 2025 Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Accession number: CGMCC No. 46612; Classification and nomenclature: Bacteroides multifiliis Bacteroides dorei ; Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0031] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0032] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0033] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0034] Example 1 1. Screening for melatonin synthases derived from gut bacteria Our laboratory previously discovered Bifidobacterium adolescentis (… Bifidobacterium adolescentis ) possesses DDC and AANAT enzyme activity, Bacteroides multifiliis ( Bacteroides dorei Both bacteria possess ASMT enzyme activity. Bioinformatics analysis of the genomes of the two bacteria identified 28 potential DDC enzymes, 13 AANAT enzymes, and 17 ASMT enzymes.

[0035] The amino acid sequences of the 28 DDC enzymes are shown below: c_DDC1: MTASPLAQTPNDMFNAPIAEADPEIAEILDAELSRQQNGLEMIASENFVPRAVLQAQGSVLTNKYAEGYPGRRYYGGCEQVDKIETIARERAKSLFGAEYANVQPHSGAQANAAVYQALVKPGDTVLGLALDHGGHLTHGMKINFSGRFYHAEAYGVNPETFRIDPEIIRQRALETHPAMIIGGWSAYPRIEDFKAMKEIADEVGAKFWVDMAHFAGL VAAGLHPSPVPYADVVSSTAHKTLGGPRSGFILAKQEYAKKLNSAVFPGQQGGPLMHVIAGKAVAFKVAATPEFKDRMQRTLDGAKILAERLMADDVKNNGISVLTGGTDVHLVMVDLRNSEMDGKQGEDLLAQCGITINRTNVPFDPRPASVASGLRIGTSALATRGFGPKEYEEVADIIGTALAAGQDVDALKARVDKLAEDFPLYPGLDQIH (SEQ ID NO.1) >c_DDC2: MATEQLETLGTEDAKWLGEYSQVHMNVFGTPLRVMDHGEGAHIWDVDGNEYLDFLAGIAVNALGYAHPKWVKAVSEQAAKAAHVSNYFATEPQIKLAAKLVKLAGAPEGSRVYFGNSGAEGNEALKLAKLYGRTLPGASPEIGGKPARIISMTHGFHGRTMGALSATWKPAIREKFEPLVPNIEFVEAGNVEALHDAFAETGQGKYGKGPVAAVIMELIQGEAGVMPLGADYVKAARKLCDEHKALLIIDEVQTGIGRTGAWFAFQREDLSGGVTPDIVTFAKVGGGFPMGGMISFGAELSALFTPGSHGSTFAGNPLGASAALATLGVIEEDSLVDNAEERGKQLRDGIASCGNPLFVSVRGRGLLDAIELAHPCSHAAMNWALEHGLIVNAVAPNALRLAPPLVITAQDVDQAVSILAKIPSDLPND(SEQ ID NO. 2) >c_DDC3: MTDFAAIRSQFPILDQEIHGHPLVYLDSAATSQKPQCVIDAESDFYRTINAGVHRGAHELAARSTMAFEDARAKVAKLVGADSAEGEEEIVVTAGATAGLNLLATAFGNASLGRGGEAAKRFALKPGDEIVVTKAEHHSVLLPFQELALRTGATLKWFDLDEEGRVRSDTANEVITDRTKIVAVTHVGNTTGAITDIAPIIRRAHEVGAVFVLDACQSVPHLKVDFHALDVDFAAWSAHKMYGPTGVGFLYGKREMLEALPPANFGGSMVELAWMDQEAQYMEPPARFEAGTQPVAQVVAAGVAAEWLMNIGMENLEAHERTITDELLKMGDIDGIRILGPRSNKERIGTVAFEVEGVHPHDVGQFIDAQGIAIRVGHHCAQPVHRHFGVYASNRASSGIYNSVEDAQALIEALGKVRPFFGVE(SEQ ID NO.3) >c_DDC4: MQPQLSKRAQIANPFRAMVFGAMADEMIANGTDVVKLSLGEPDFGAPPAVRDAMREQYDGRPLPYTAAMGLPELRQAISDFYKERHHVDVDPKRIAITAGGSTALLLSAALTVNDDDEVLIADPSYPCNRELVRAFGGKVVDVPASAATRFHLTPELCKEYWTDRTKAVMITSPSNPTGTTIAFDTLKAVCDLARERGAWRIVDETYLDLADREPDGSDVKSVLACDPDAIVCSSFSKFFGMTGWRLGWMVVPEYALEAMDDLATNFFLCAHTPTQHAALACFTPETLAVCEERRQELLERRRIVVDGLAEIGLPLEVEPNGAFYAYFNISGTGLDAWTFCERALKEAHIALTPGRDFGEATADTHVRLSYAASREALREGLKRLGDFVAKVRG(SEQ ID NO.4) >c_DDC5: MEKQNRIFSGFDADAIDRTTAADLAEVGSDKWTRYPGCIGAFIAEMDYGLAPCIQQAIDSACDHCKLGYIPEPWKRRVAEACAGWQKSHYGWDVDPDIIRVVPDVLEAYEIFLRELVGAGNAVVVPTPAYMPFLSVPKLYDVDVIEIEMLQGTDETTGEREWLFDFNAIERAFAAGCHAFVLCNPHNPIGKVLTLAEEQRLCELAERYDVRIFNDEIHAPFVFEGRHIPYPTISKAAARQSMTATSASKSFNIPGTKCAQVLLTNPADREMWAERAEWSEHQTATIGAIATTTAYNEGDPWFQDALSYVRRNLGLFDKQMRTRFSGVGYIRPRGTYIAWLDFGPLGIDDPAAYFLEKAKVALTDGRSCGRAGAGCVRVNMAMPYPLLVDCLNRMHDALQADGLL(SEQ ID NO.5) >c_DDC6: MSETSSNAIPAYLPLRNDLIGEEPYGAPQLDVPVCLNVNENPYAPEPAVVETIAQRVKEIAPTLNRYPDREHIELRKAFSTYLERESGVKLSVDQLWGANGSNEIMLQLFQAFGGPGRIALGCDPTYSMYPEYARDTFTTWKLAHRNADFTLDVDATIKAIEDVKPAMIVLTSPNNPTGTPLKPEDLKRVLEAARTAEVAGAAEGVHPVVVVDEAYIEFRDPGTPTALELIGEYENLAVSRTMSKAFAFAGARVGYLAANKGIIDCVRIVRMPYHLSAVTQAAALAAFEHTDEQLSRVAHLRETRNATSAWLKEQTYKGQPLEVAETQSNFILFGGHFDDRDRIFDELLKRGVLIRVVGPEGWMRVCMGTDEEMARFREALVEVLRIVEQG(SEQ ID NO.6) >c_DDC7: MTYDFDTPIDRSGTYSLKWEEAGDALPMWVADMDFQTAPVIREALRRRVEHGVFGYSIVPPEWNQAYVDWWGRRHGLAIDPDSLVFCTGVVPAISSMVRKLTTPNENVVIMTPVYNIFFNSILNNGCRVLESPLAYDGEGHYEIDWADFETKLADPQTTLMILCNPHNPIDRIWDCETLERIGELCWKHHVTVVSDEIHCDLTDPGFDYVPFASVGEHCAMNSVTCMAPTKTFNIAGLNSAAVMIPNPVLRHKVWRALNTDEVAEPNAFAMDATLAAFNEGEPWLNELRAYLAGNKATARAMFDKYNASVPADRRIIMVEGHATYLLWVDCSAITHDTDALCDYLKREHKVMFSEGSEYGGNGHDFVRINVACPRARMIEGLARFIDGLLAYRAQSFPKMD(SEQ ID NO.7) >c_DDC8: MLSFENDYSCAATPEIIARLAETAPNQYPGYGTDDVCESAKAKIREACACPDAEIFFLVGGTQTNQVVIDAITPPYAGVVAATTGHVNVHEAGAIEFTGHKVLTLPHHDGKINADELDEYCATFYADGNYTHMVFPGCVYISQATEYGTLYTLAELEAIRKVCTKYDMPLFIDGARLGYALTATGNDVTIEDIARLADVFYIGGTKVGAMFGEAVVFTHGNMPKHFVTIVKQHGALLAKGWLLGLQFDTLFTDGLYLRIARHANDAADRIRKVLVERGYTLTFDAPTNQIFVTLDNETSERLQRDVRLGFTEKADDAHTVMRICTSWATTDEQVDELVALL(SEQ ID NO.8) >c_DDC9: MADWQTLSDRINHVAPSATLAVDSKAKAMKAAGIDVIGFGAGEPNFPTPAPIVAAAAAAVQDPKNYRYTPTAGLPELRQAIADKTLRDSGYEVDPSQVIVTNGKQAVYESFQILLNEGDEVIIPTPYWTSYPEAVKLAGGVPVTVFAGADRNFEPSIAEAIEAARTEHTKAIIVNTPNNPTGAVWKPETVKAIAQWAIEHHVWVISDEIYEHLNYDGAKTTYIGAAVPEVRSQLLVLNGVAKTYAMPGWRVGWMIAPVDVAKAASKLQGHMTSNVNNISQRAAIEAVSGSLDAVYEMRAAFDKRRQTIVAALNDIEGVNCPTPTGAFYAFADVTALLNKPIGTNKTTFANTSELAALLDEGHVAAVPGEAFGAPGYLRFSYALADDDLVEGMRRMKRWIED(SEQ ID NO. 9) >c_DDC10: MINEAYKAMLGGKSVIRELSEYATARGAEIGYENVFDYSLGNPSVPCPQDYTDAVIDMYKTAEPVALHGYSPSLGISSVRKAIAESLNRRFDMDYTADYIFPTTGAAGALTHALRVVTKPGDEVITFAPYFPEYQPYVNGTGAHLTVVPADTENFQINFDAFEKALNPGTAAVLINTPNNPSGAVYSAETLTRLAEVLNAKQTEYGHDIFLIADEPYREIVFDGGEQPYPSKFYDNTLSCYSYSKSLSLPGERIGYVAVNPRATDAKLIVPMCGQISRGTGHNCPSSTAQLAVAKVVDETSDLSVYETNMNLLYDALTRLGFDVVRPGGTFYIFPKALEEDANAFCMKAKDYDLILVPSDSFGVPGYFRMAYCIDTEKVERSIAALEKFVHEVYGR(SEQ ID NO. 10) >c_DDC11: MGFHEFSSPYDWSRIASYKRTAKAAFGGMVDLSVGSPVDPVPDSVRKALAVSSNDPNAYGYPVTAGTADLRDAIHEWFAAARNVDLRAIGADAVPTVGSKEGVALMASLLHFGEGDVVVQPKVSYPTYEIGTQLAGAQTLKVDDVADADSWKDVPGVKAVWVNSPCNPTGEVLSAEQMAGIVRAARAIGAVVLSDECYALMQWEDDVAEADADTLASTPCALRADVCGGSAEGVLVLYSLSKQSNMAGYRTALIAGDQSLIGPMAAYRKQIGQIIPGPVQAAMAAGLRDFDAVKTQHSRYQERLGMLVSALRAYGYCTDMPQGALYVWVKAKSGDCWEDLRQLAEIGIVASPGEFYGAPEYLRFSATASDAAIASAAERLAR(SEQ ID NO.11) >c_DDC12: MRFSSRVDISEPNPIAKAEATAKTAGRPLGKLNDSNPTRHALAPDLVPDIYSADPRGQRYAREALAAFLDMQEIGHCSPDDLYILSSTSEAYSWLIKLLCDAGDAVLAPKPGYPLIESIARLECVDMIEYQQRFDGSWYIDVAELKTALEGPDGNRIRALVLINPNNPTGSYVKPSEREAIVRLCRDHDVAIIADEVFYDYDLEPFEGNARLAGERGALTFALDGFSKMLAAPHAKVGWIQVSGPAEDVAEAKRRLDVIADDYLPMSEIVAKQIPALLEAAPLQTARVQERVRGNLKALHAMLDADEQGLVSVLRAEGGWNVLLRVPSVLDENELVLHMIERHGVSGQPGYFFDMTSNGYLAISLLPEPDDFRHNVYVVIDTVNELIG(SEQ ID NO.12) >c_DDC13: MTRFNTQLVHGLPVNDNNTGAVNPPIYNSSTYAFESVGTMPRWDYARSGNPTREFLEKQIAQLEHGTRGFAFGSGLAAIHAVLSIFQPGDRIIVGKNIYGGTYSLFHEYFERWGVIVEEVGTTDYKALDAAVSGESAKGNAHGCPAKAVYFEVLTNPLLQVNNVTAIAGIAHRHGAIAIVDNTFVTPYLQQPLDQGADIVIHSATKY LAGHSEVNAGLVVVKDDELGKRVYFAQNRLGGVLAPNECDSVRRGIQTLALRMDRQQENARAISSYLLLHPLVKSVHYPGLPGHEYELASNGLKGGGAVLSFEVVPGDPADVLDNLHIFRLAVSLGAVESLAELPCRMTHFELPREERLKVGITDELVRLAVGIEDKQDLIEDLGQAFDIAYENYLKRHAGESLFTFAQHVYA(SEQ ID NO.13) >c_DDC14: MTNTVNIPTNMLPEDGRFSGPSKIRPEQVQALDAGARNLLGTSHRQTPVKQVVGSIREGLSNFFQIPDGYEVVLGNGGASAFWDIACASLITRKAAFGTYGSFSAKFAKSAQSAPFLEDPEIFAGKPGTYRLPELTEYVDAYCWAHNETSTGVAAPVKRVEGSKEQGALTLIDATSGAGALNVDISQTDAYYFSPQKAFGSDGGLWIAVLSPAAIDRAYGIAKSVSLPGARRWIPPFSLTSAIENSRKDQTLNTPAVATLVMLENQVRWLNDNGGLAWATARCARSASLLYQWAEKSDYAQPFVSDENARSNAVVTIDLDESINASQVVAALRENGIVDTNGYRKLGRNQLRIGVFPSVEPSDVEALTKCVDYVAEHL(SEQ ID NO. 14) >c_DDC15: MANKNYRFETLQLHVGQEQADPATDSRAVPIYQTTSYVFHNFDHAEARFGLADPGNIYGRLTNSTQGVFEDRIAALEGGTAGLAVASGAAAVEYAVRNITQSGDHIVAAKNVYGGTFNLLRHTLPRDGITTTFVSAENPQEFEDAIQENTKLVYFETFGNPNADLPDFEAITAIAHKHHLPVIVDNTFASPYLFRPLEHGADVVVESATKFIGGHGTTLGGVIVEGGKFNWAEVPGKFPTLTEPDPSYHGLNFYEALGGAAFVTRVRAILLRDTGATLSPFAAFLLLQGTETLSLRVERHVQNALKVIDYLKTVPEVESISHPSIEGRKDNDLYKKYFPNGGGSIFTFDIKGGKDAARVFIDNLHLFSLLANVADAKSLVIHPASTTHSQETLEELEDQGIHQGTIRLSIGLENIDDIIEDLESGFKALRESGLAK(SEQ IDNO.15) >c_DDC16: MSENVDTICVQGGYQPGNGESRTPPIIQATTFKYQSSEQMSRLFDLSESGYFYTRLQNPTNDTVAAKICELEGGAAAMLTSSGQAANFFALFNICNNGDHIVASSAIYGGTFNLINVTMRKMGVECTFVSPDCTEEELEAAFQPNTKAVFGESISNPALIVLDFEKFANAAHRHGVPLIVDNTFPTPINCRPFQYGVDIVTHATTKYMDGHGSCVGGAIVDSGNFDWMAHAEKFPGLTTPDDSYHGVTYAKEFGKAAYITKATAQLMRDFGSTPAPINSWIMGMHLESLGVRMERHCANALAVAKWLDADPRVSWVSFPGLETDKYHALAEKYMPNGTCGVISFGVPGGREKVSAFLDHLKMVSIATHVADARSCTLYPAGTTHRQLTEEQLEEAGVGIDLVRLSCGIENTADIIADLDQALAAVQ(SEQ ID NO.16) >c_DDC17: MTISTNTEALNATALATRAIHAGQEPDPTTGAVVTPIYMTSTFKQDGVGGLRNGYDYSRSINPTRNSFDEQLAAVEGAKYALSFSSGLAAIDVLLRSTLKPGDNILLGNDVYGGTYRLLSKVFVPWGIGLDVVDITDTAAVSAALAKKQYAYIWVETPSNPLLNITDIAATAAVAHAHGTKVVVDNTFASPVLQHPLADGADVVVYSTTKYIGGHSDVVGGAVVLNDKETRDAVAFLQNAAGAVPSPFDSWLDIRGLKTLDLRVKQHSRNAMKVAQWLETRPEVERVWYPGLESHPGHDIAARQMHGGFGGMISIQIAAGFEAAKKFAGATEVFTLAESLGGVESLIEHPGAMTHASVAGTTLEVPANLIRLSVGLEDADDLIADLEQAFRQI(SEQ ID NO.17) >c_DDC18: MARIAPLNIDWKPDRTTGEPVTEQIVGYMCEQVHSGNWPIGSRLPSQRALAEWFGVNRSTVIAAINELSDYGIVEGQHGAGTRIVSNTWSLMLPGAPDWADYVDSGFFKANNDTIQAINRYEFTPNMTRIGTGELDPRLFPKAMWRQVLGEAADEIETLGYPPPEGLPELREAIAAHMRATGVDCTASQVLVTSGALQALQMISVSLLPAGTTVYAESPSYIKSLQVFQSAGMHLEGVPMDDDGLNVQALRGLLAEGELDTGRPAPLNARNRKGNAILYTIPTNHNPTGVTMSDKRRHELVELSVDSRLPIIEDGAYRDLYFEDDAPLPSLKALDETGMVITLGSASKSLAPGLRIGWLVAAEPIVQRLADVKMQMDYGASVLSQWTFARFLTNGMYDEYVAGLKRELRHRRDRALVTLQEKMDGLAHWNVPSGGFYIWMTFDRPIRMNRLFQLAAERGVLLNPGDIYDFAADNSLRLSYSYTTPEEFADAVDVLAAVARELA(SEQ ID NO.18) >c_DDC19: MTESFSTRLNDMALRELKQIDFVHAAEKFNIKLGKSHMSQYVSGKTVPRADIAHFLAAYLRVNEDWLMGKDVPMEEHAAILPDFAGEQPDHVDDASEQPTEGRTMRTFTKSHKLDNVLYDVRGPVADEAARMEAAGTHILKLNIGNPAPFGFRTPDEVVYDMSQQLPDTEGYSPSKGLFSARKAIMQYAQLKNIPNVSIDDIYTGNGVSELINLSLSALLDNGDEVLVPSPDYPLWTACVNLAGGTAVHYVCDEDSEWYPDIDDMRSKITDKTKAIVIINPNNPTGALYPKEVLQQIVDLAREHQLMIFSDEIYDRLVMDGLEHISIASLAPDLFCVTFSGLSKSHMIAGWRVGWMVLSGNKRLAKDYIEGLNMLANMRMCSNVPAQSVVQTALGGHQSVKDYLVPPGGRIYDQRELVYNMLNDIPGITAVKPKAAFYIFPKIDVKKFNIHSDEQFALDLLHDKHILISHGGAFNWQEPDHFRVVYLPRISMLKETIGEIGDFFSTYWQA(SEQ ID NO. 19) >c_DDC20: MSDVDIFHAPRDFEFHDFDTRNVTASDGGTATLCFPRLDADAVHALAASVRRHRAEKLARYSTDGIVDVIARAVELWTDPEYPERRLAERLIPAVTGYDASMVRIELKRYMRMFRRRELLRFVDDELNCPQMLDEYRPNRSGGYTRLYGPELSFHVFSSNVPGIPVWSMTMGLLTKSAILGKSSFDEPLMPVLFARSLASVDPDMADALAIVPWRGGTVDLEDAAIGEADAVVAYGSSHTTEAIRPRVRAGKPFLSYGAKIGFSLIGREALRADLYAGTVHRMAIDVATYDQQSCLAPQTMFVERGGAMSPAQVAELLASELDGQQRKYPRSTPSEEESMAIQRLRSTAQMKALMLGAGPMAAAAGNADDAPSDDPFVVQSRSGTDWTVLYYPSIGMADSLSTPLNRTVNIVAVDHVEDALPTLRPYAQWLQTCGVALAPDRLFDVAQRVGETGIDRICPVGEMNRAKSGWHHDGGFNLLDLVHAVDIERNTDMYCDGFDMDVE(SEQ ID NO.20) >c_DDC21: MYGLEPILETMLNVSIVIPAWNEQERINDCLLNATRQTVMPHEVIVVDNRSTDSTVAVVEQFMKDHPEAPVKLLHQDDEQGLIPTRDYGLNHATGDILGRFDADCMIRPDWVEVVSGIFTEDPDAMGATGPVMYYDLPSRHFGLRGDNSTRKRIYRADDGQPLLFGSNMALRATAWREIADEVCQDKADVMHEDIDISLHLLGKDLKTVYCPRMIAGISARRMDTSPASFLNYMRRFKNTFNAHPQHYRKHKPEILITALYPVMHLLYPVWQKVLNTADINPAEAAWINEQMELAEQEGHELYDDTPTDEEWSEKHEE(SEQ ID NO.21) >c_DDC22: MSEYAETTAKEQTTDQRARELPERIVIPPIKGEMVHLRPATIDDIARMEVIEAYVGASGITGKDRVAERGAVNAWVRRSVAWSNGEKSNESGVGDPESRRTIAWSIVTETDHDGDGELDAASSDNVIGMIFLIDIDGWARSARIQVVLGKDYRGRGYSRDIMPRVMTYGFAPEPAGLGMHRIWVAVPEQNTRSVSVYQSLGFVPSGRSRDALWNASENRYQDLIVMDTLVDEFDPIRSLDAFGMHVIEDNPGVKEALSAREHSIAIQLNSVHKGDADDAAAAGQSGDTAQTSADETSAAPSQESQPNEASGTAKTDQKAGDADNNWPYSSDERKTSKDAWWRTLGRGRKRDTEGRR(SEQ ID NO.22) >c_DDC23: MPSDLLTGVLPRILQYLPATLLYWIVPSVLALFFGTLLCIVRVGRHNVLYWLCTVFISFFRGTPGIVQIYLVFFGLPKVFYLFGLDIDDWDPGVFFIIATAMNLSCFICETLRGGYLGVDKGQIETGLSIGYNRVQNFIHVIAPQTLKVAVLNLKNLEIDVLKDTSVAYTIGTVEILGSAKAIISAQYGTGQLWILGLVAIIYFVLCSLIEVGFTFASHRMERYERRYA(SEQ ID NO.23) >c_DDC24: MKALDVISRVAMPQQIAEIARPIALRHHVNELYLFGSMARGEGHADSDIDFIYQFDDTANPMIDEWALRDDLASTFGREIDLVKKRYITTELQDRLAEMQRVIFVNSITSNPMFRII(SEQ ID NO.24) >c_DDC25: MSDNPSSFHEGATPEDFPLISVIVPVYKVERYLDRCVQSIVDQTYRNLEIILVDDGSPDSCPDMCDAWIAKDYRIRVIHQENQGSGAARNNGIESSTGELIGFVDSDDWLEPTMYEALFNSLKNNDADISMTASIMEYPDGRQICHYQPNVHLVMTTSESFKYVNLPGYSTISPWSKLIKRSVLGDIRFPEGCVNGEDTRFTYEVLAASKRTTFDSVPLYHYRTTEGSLSHTYTNYNWAGYEATKQMVELVRRRFPEQLPFALYGLMRSMISLYDQALVTRSSKEQQWRRFAKDTQAFVRRNIGLVAESVDLPRGRSMQLKLMAVSPVLYSVSFLIYKRIHPERSK(SEQ ID NO.25) >c_DDC26: MTSTNNTYDDVTMVVADLDGTLLHDGETFEDRFLTQRSIDSINRMHDAGIKFVVETARPVSTGYSFVQKLPVDAVAYLNGALLDFNPVSSDYDMLTSPTLPKDGHLQKIGFSSKRACEVCKYLLEELPDMEVGIVMDDVRYTNFDVTKYWKTQTWRYTDFDDVPDGTADKLILFPNEEQWKRVGRLIPDDFDVHISEGSLWMLMNPQANKEHALSILADRMSTPLSRTVSFGDDLVDIAMLRESGRGVAVANANPDVLKIADEICPSNNDDGVAQWVENNLL(SEQ ID NO.26) >c_DDC27: MADSLASQIITAIGGPENVRSLTHCATRLRFELADASKVDQNALEHMKGVLGAVPQSGDRFQVVIGGGVATVYENIMHLPEMANAGAASASGEGQKSNADVKAEARSKARGKVAWLDSFFEYLADSFRPILGVLLGASIIIALVNLLISLNVIPNDEASAGWVFVKAIWKGVFYFLPIMVAYNASKKLKVDPWLGGAIMAILMTPQFTSLIDAKTTTCVENAALGTKSCTANIFGIPMALSDYSGNVFVPLLMAAVLALVYHGLKKIIPESVQLVFVPFFCMIIVGALTAFIIGPIGVWVGNGLGVGLAWMNTHAPFIFAIIIPLLYPFLVPLGLHWPLNALMLMNIQTLGYDFIQGPMGVWNFACFGATAGVLFIAVRDKDKDMRQTALGALAAGLLGGVSEPSLYGIHLRYKLVYKRMLVGCGLGGVVIAVLGWLFPSVTAAGQTVHGVTTTAFAFTSLLTIPVFDQMWVYAVSIAVSFLTSFFLIITFDYRTPEQKAEVLARAAADQKAAAPAVEAKEAAPAATTATATATATKTEAPAAAAAATTVVNAPVAGHVIALDETGDPVFASRALGEGVGIQPTDSEVVAPVSGVLQTVAETGHAFGIKTDDGVEVLVHVGIDTVKMNGEGFVVKVKADERVNAGDPLVSVDFAKVKDAGYSTTTLMTVLNTAALTSVTPKTGIDVKAGDEVIDIQR(SEQ ID NO.27) >c_DDC28: MATSRPLVSIIVPVYNSERYLPYCVESILNQSYTNLEIILVDDGSKDTSPRICDDYAEKDSRVIVIHQKNGGIAKAQNTGLDAAHGEYIAFADNDDILDSRNIELLLHAILNTGADMSKGRWQQFGVSQLAEIKARAAQGSAAPDKTTSFKNPLHAYQTVFCKSLRILGNLLGKNSEALYFNEANWCRLYKRELWDGLRFDLGHYAQDIRMAAPLYSRMKLVTDIDSVLYYWLQEPDSVTHSKRTPNFWHDNVTAAAKNFQATLDQGIVPCRNYFGLVASVKDEEKVLRNSSNEVTDADWQNLQDDLTLSSQLILKLTPLQRLHCYILFIIRHYENKIYDRKIHDMK (SEQ ID NO.28) The amino acid sequences of 8 AANAT enzymes are shown below: >c_AANAT1: MHMLIRHATMEDLDAIEAVEAACFPPAEAASNESLTARVAAYPDHFWLLVNTDDDDTPFPSRVEDGTLVGFVDGMATNEPNLADAMYDDADMHDEHGDWQMIFGVDVAPVYQHRGCASYLLRQVILDTAQAGRKGLVLTCKERLVDFYARLGFVDEGLSESTHGNVVWHQMRLALTHAVGTGNPTANGSAKTAERTNDADTTAMSGVDHDTETLEFPQVEPTA (SEQ ID NO.29) >c_AANAT2: MIVDHSQIPEDTAVGQIAALEKDLFGRGAWSEQSVRQEFHAPARTYLLDIEGDAVQTADPVVRGYAGYWYDGDDAEIMTIGVGRPYQRQGIAAALLETLIVSARRQGAKRMLLEVRVDNVPALALYERFGFTRMGLRKRYYQPEGIDAYTMSLDLEPRVVGFSATADTEHTSATHTTEQQ (SEQ ID NO.30) >c_AANAT3: MKYAGAGSFQRNRPLLSFAAGYAGRMADYSFRIATQDDIQAITDIYNAAVIRGGSSADTTPRTYAQRKAWVESHHDPYAVFVTTVPATDAATEGGERIIGFSALSVFYDRAGYDGVTDLAYYIAPEWQGRGAGTFTLARLLDECRNRHMRKACGIIFADNRGSIALMKHFGFTQFGLMPQAATDSTGTMRDMSYWHLDL(SEQ ID NO.31) >c_AANAT4: MSETTSERRFRRATMDDLPVMLKIYQHARELMAANGNPTQWGNTFPREEVILDDIRQQRTMLLVDEADGKERVLAQFALCTGEDPTYAHIDGAWLDDDSYVTIHRIASSGIVKGAAKDCINWCIEHYGNVRADTHPNNKAMQHVLESNGFARCGLIQLLDRPTDTTRIAYQRHEW(SEQ ID NO.32) >c_AANAT5: MEIREYDMMTDEARGIRTAVFVVEKDYRPEFDEWDEPGRATHLLAFGNGRAVATCRLYPDLDHADQPGRWVIGRLAVVADERGHGIGKTVLAEAERLIRKAGGHVAAVHSEDKNFAMYEHLGYRITSELFDNGTHGWLVKALN(SEQ ID NO.33) >c_AANAT6: MRRPGNNSFEMPEFIQSHSMRVPVSLRPLTGADYDEWNEVRWRNDAWLKPWESGDPLHGAPITYKEWMKQLRRNERAGTGAVFAIEQHGRIVGQISLGAICYGAMRSGVVGYWVDERCAGRGYAPMAVALLADWAMFDPAGPRLHRMEIDILPENKRSRAVARKVGATLEGVRRAYMYVNGQWRDHESYVLMAEDVPNGFTARLMTGNSPS(SEQ IDNO.34) >c_AANAT7: MTGPAPTIRRMKIEDYPLVYGLWTRCTGFTMRDIDDSEDAIKTFLKRNPDTCFVAEDDGGRIIGAILAGHDSRRSRIYHTAVDPDARGMGIGSLLVGRVVETLRAIGLPKVAVGVPADNDAGNDFWEHQGFAVRDDLVYRELPL(SEQ ID NO.35) >c_AANAT8: MVITYTDEKKFTKEQTQRLFLSVGWVSGQYPERLHKALMGSSTVFSAWDGERLVGLVRVLDDTEMVAYMHYVLVDPEYQGRGIAGHLVGMVKQRYHDYLYIEVMPEESKNATFYEKHGFHIMEDGVAMQICNPGERH(SEQ IDNO.36) >c_AANAT9: MTCETPTIRPLRRTDFPALEELIRLAWYDDGNDSHNTHDEVGQTSGPDKHRAELRKATRLRNMHRLAAIDMQDCLARTTKAYVAELNGQVLGVILGSLRSDITGRQRTRHMLRRHCLTLPLLASHEGRHGLLAQLAILQADEALKHDAGKEYEAEVVLFVVSPAARGMGVGRRLFNHMLGVFHDAGLREYFLFTDSTCDVGFYDHRGLIRKAERDDRNDGSSRPNPTDSSSSGLTVGTTSLLADDEPMSYFLYEGRC(SEQ ID NO.37) >c_AANAT10: MEGYAEQGARTDSTDQIGFGGARRNRKKRRGLTVFQLKVIGAVALALSAGSTTIVPLFFGSDTNNMTSLTAMVLSEVVSWFAIPIYAWLLVQGFRETHSRVAYGLQLFILAVVCEVPYDLATSHKAFDLGSQNPVFGLFVAFVVLAALDWVASRYQKAMKVLLSVGLVVIGLLWDLLLRIGLRQHMMSIGSVTLGFVLIFALMRQYENSMMFTAGLFGAVMMIAPGVGVAFVHYYNGKLGYKHQWTKWAFYAVYPVILIICAICATLA(SEQ ID NO.38) >c_AANAT11: MSDEVMQDNMQHTMQDNKNPWNADLTDPMLRLRLASERLSIVRYVFLVQVEDGIASADERASLEYADAVLIGWPETDASDVITPDAAQLDDIDEQMRVMEQHIAKFSAMERDHDIVGMTDTLIRVTERVAGIRSIYQPDFPLPTFAEIRRVVQDEWDEDMDKIDPDNASPTADSIEQETADADKEQQAEKQAEQQADGAQKSGASA (SEQ ID NO.39) >c_AANAT12: MSDTLVPEPQASIFDSVTKALGNDEDAARRVMLNPNLAKGFKDGKPVNKESKKDGQDTCGCGNHADACGCSQPQDDDIPLKAVDDIGRATAADVKEALHQVIDPELGIDVIDLGLVYGIEIDELGRAIITMTLTTPACPLTDLIEDECASTLAGLVEEFRIDWTWQPRWTMDKITPEGRDQLAALGFNFDNLPKY (SEQ ID NO.40) >c_AANAT13: MSEYAETTAKEQTTDQRARELPERIVIPPIKGEMVHLRPATIDDIARMEVIEAYVGASGITGKDRVAERGAVNAWVRRSVAWSNGEKSNESGVGDPESRRTIAWSIVTETDHDGDGELDAASSDNVIGMIFLIDIDGWARSARIQVVLGKDYRGRGYSRDIMPRVMTYGFAPEPAGLGMHRIWVAVPEQNTRSVSVYQSLGFVPSGRSRDALWNASENRYQDLIVMDTLVDEFDPIRSLDAFGMHVIEDNPGVKEALSAREHSIAIQLNSVHKGDADDAAAAGQSGDTAQTSADETSAAPSQESQPNEASGTAKTDQKAGDADNNWPYSSDERKTSKDAWWRTLGRGRKRDTEGRR (SEQ ID NO.41) The amino acid sequences of 4 ASMT enzymes are shown as follows: >c_ASMT1: MMHKRYTKEQLTAVEAQRLAQEIAFGPVVFQVSRLMLKFGIFQLLADKRKGYTLQEISVQTGLTRYAAQVLLEASLTIGTILLEEDRYVLAKAGWFLLNDKMARVNMEFNHDVNYQGLFYLEEALLNGQPEGLKVFGGWPTIYEGLSQLPEQVQKSWFGFDHFYSDNSFSQALEIVFLHSPKRLLDIGGNTGRWATRCVQYNQEVEVTIVDLPQQLEMMRKQTADVPGAERIHGHGANLLDRTVPFPSGFDTVWMSQFLDCFSEEEATSILSRVAQSASKDSKVYIMETLWDRQPYETGSYCLTQISLYFTAMANGNSKMFHSDDLIRCIKNAGLEVEEIHDNIGLGHSILQCRLK(SEQ ID NO.42) >c_ASMT2: MKPYNKEESKRIQIRRMFNNIAPYYDRICHILSFNIDRLWRKRLVELVAERKPTNIVDIATGTGDVAISMAKKIPTSQIVGIDLSEEMLKVAQQKIEHFSIKPNIRLLCEDAENLSLSSNTFDVVTISFGIRNFENIVKGLSECHRILKEGGSLFIMEFSTPKHKVFNVLYRIYSKHILPLIGQVISNDDKAYKYLPESIVEFNREHEFTELLLNNGFDSCISISLSNGIAHIYIGVKSKM(SEQ ID NO.43) >c_ASMT3: MRNKHLLNSILQNTSCPKGFWGRMILWGMNRFHASLARRGMKQVNWQPQWTVLDIGCGGGANLNRLLKQCPQGKVYGIDLSEESVVFASKYNVKELNKRCFIQQGSVCSLPYKDGTFDAVTAFETVYFWSPIEIALAEVVRVLRKGGCFLVGLEASSPELGKMWTERIKGMVVYTAGDLKDLLIEAGFSTIQVVHNKEEMYIIARK(SEQ ID NO.44) >c_ASMT4: MNYPQEKIKPYNADEKKSVQVEKMFDNIAPAYDQLNHALSWNIDKSWRRKAINWLKPFHPQHIMDVATGTGDFAIQACQTLHPQELIGTDISEGMMNVGREKVKAAGLDSRISFAKEDCTALTFPDKRFDAITVAFGVRNFEDLDKGLREMHRVLKDNGKLVILELSEPDWFPMKQLYAVYSKIVIPTLGKLLSKDRSAYTYLPQSIKAFPQGEIMTDIIRKAGFNQVSFKRLTLGICTLYLATK(SEQ ID NO.45) >c_ASMT5: MKPYNKEESKRIQIRRMFNNIAPYYDRICHILSFNIDRLWRKRLVELVAERKPTNIVDIATGTGDVAISMAKKIPTSQIVGIDLSEEMLKVAQQKIEHFSIKPNIRLLCEDAENLSLSSNTFDVVTISFGIRNFENIVKGLSECHRILKEGGSLFIMEFSTPKHKVFNVLYRIYSKHILPLIGQVISNDDKAYKYLPESIVEFNREHEFTELLLNNGFDSCISISLSNGIAHIYIGVKSKM(SEQ ID NO.46) >c_ASMT6: MNPVVSYIKQTLQGYYPDTELVPMAKLLLTQVFGMSVVELYAGKDTTFSVNEQKRLDDILVRLQKLEPIQYIIGTEEFYGLTFEVNKHVLIPRPETGELVDWIIREHKYGRVKILDIGTGSGCIAVSLTKNLEEAEVVSWDVSEKALQVAERNCRRNGVRVTLEQRDVLLASPAGEQFDVIVSNPPYITEKEKANMSANVLEWEPELALFVPDDSPLLFYRKIAELGRDMLVSGGRLYFEINQEYGRETVDMLAGLGYKNIELRKDLFQNDRMIKAEK(SEQ ID NO.47) >c_ASMT7: MPQCFTIMNNDNNHNTIQEFDVNLICDFFLNTNRQGPGSPEATLKALSFINNLTVQSLIADLGCGTGGQTMVLAQHTPGSITGLDFFPEFIECFNRNAEKLNLQDRVKGVVGSMDALSFEKESLDLIWSEGAIYNIGFERGLNEWRNYLKPGGYLAVSESVWFTDHRPAEIHDFWMNAYTEIDTIPNKVVQIQKAGYIPVATFILPENCWTEHYYVPQAKAKEMFIKKYAGNKTAEKLMASICHEAELYSKYKEYYGYAFFIAKKINPFQG(SEQ ID NO.48) >c_ASMT8: MIEEKNYYGNLCTEMYEILHAEAPQDELNFYLSYAEKGKKILEPLCGSGRFLVPFVERGLDISGIDLSNEMLQKLKQKLPEAKVVQADIIKYSPREKFDYIFISSGSVSLFTDTDLCKQILCRIKEWLSPMGRFVFAVDTIANRCTDDNDYAIAVSVKTKENFELVLKSKNHYDEQSQTQLSPGIYEMYSDTKLIQSEFMDFQTHLYKYGEMEEYLKEVGFTQVKTYSSFDKEIAINDRCEMFLFECSL(SEQ ID NO.49) >c_ASMT9: MINKQLITRRFSRAVESYNREAVVQKQIAYRMSDMLNHYLPRPCGRILEIGSGTGFLTRRLMETLHPEKLVLNDICQEMSTCFTDLLGSGRAIFLAGDAESLPFPKGQDLIVSCSALQWFVSPELFFERCNTLLKQKGYFAFTTFGRDNLKEVASVTGSGLHYRSLEELEEALRIHYEIVKAHEERICLTFGTPLEVLYHLKHTGVAAVRQQAWTKRNLQDFCDKYARLFSDGRSVTLTYHPIYIIAKKRQ(SEQ ID NO.50) >c_ASMT10: MQKRHQDRQCYFNELANTSRSFYIDYVKQFISLSSSTHILEIGCGEGGNLLPFAELDCKVTGIDRAASRIHQAQSFFAASGYKGEFRTMDFFNFSSVSRYQLILIHDVIEHISNKEEFFRCLSPLLAKGGIIFWGFPSWQMPFGGHQQICHNRFVSSLPFIHLCPGIFYRFLLRCFHETPSCIEELSDIKRCRMTIDTFEQLAEKYGYEIIDRQLWFINPHYQQKFHLRPRKLYPVLAQLKHIRNYFSTSCFYITRHRDLHP(SEQ ID NO.51) >c_ASMT11: MKTNLLSPDKDPMGAAIADYFNHRKADKLRVFSSQFEEDEIPMNQLFRPYDEMPELEQIALQIAEGNILDVGAGSGCHALALQEMGKEVCAIDISPLSVEVMEKRGIKNVRQINLFDTHFLETFDTIIMLMNGSGIIGKLENMAAFFQKMKQLLRPGGCILMDSSDLRYLFEDEDGSFLIDLAGDYYGEIDFRMQYKNIKGDSFDWLYVDFQTLSLYAAGNGFEAELIKEGEHYDYLTRLRWKG(SEQ ID NO.52) >c_ASMT12: MAYKKVYLKPGKEESLKRFHPWVFSGAIQRIEGEPEEGEIVDVYTSKKEFIACGHFQIGSIAVRVLSFKEGEIDHEFWKHKLEVAYDLRRSLGLAGNPANNTYRLVHGEGDNLPGLIIDVYDHTAVMQAHSAGMHVYRMEIADALSEVMGDVVRNIYYKSETTLPFKADLGPENGFIKGGSPENIAMEYGLKFHVDWLKGQKTGFFVDQRENRHLLERYAKGRNVLNMFCYTGGFSFYAMRGGANLVHSVDSSAKAIDLTNMNVELNFPGDPRHKAYAEDAFKYLDRMGDQYDLIILDPPAFAKHKDALRNALRGYTKLNAKAFEKIKPGGILFTFSCSQVVDKMNFRNAVFTAAAQSGRSVRILHQLTQPGDHPVNIYHPEGEYLKGLVLYVE(SEQ ID NO.53) >c_ASMT13: MKQRHKFESIVAETLLIPLYYRAKESRRKNPILNDKVAEGLVDSLEYDYSRFDGAKLSEVGCVVRGWFFDRAVRRFIGKHADAVVVNVGCGLDTRFQRIGDGKAVFYDMDLPEVIALRRELIPEQPGNPYIAASLLDAGWLDDLRRRHPDAHFIFVVEGVLMYFYEKQVKTFLHHVAERFGGGELWFDVCGTMMSRHGVKPDSLREHEAQIRSGLSDGRLVEQWEPRLRLLEQANYMKFFRPRWGFFFGQILGRMTRLCYKFSSMLGYEIVSNSRDTAAILHER(SEQ IDNO.54) >c_ASMT14: MKYLEVTFTTHPCNETVNDVVSALAGEIGFESFVECEAGVQAYIQQTLFDEEALKEMIASFPLPDTTVTYTIKEAEDKNWNEEWEKNFFQPIVIGDRCCIHSTFHKDTPKTEYEILINPQMAFGTGHHETTSSIISELLEADLKGKSVLDMGCGTSILAILASMRGANPVTAIDIDDWCVNNSKDNIALNHIHNITVELGDANLLKGRKPFDVIIANINRNILLADLPHYAACMHPGSEIFMSGFYMQDIPFIREKAESLGMEFVHHREKNNWAAVKFIMK(SEQ ID NO.55) >c_ASMT15: MNVKILVCCHKLDIWASDSPYVPIHVGKELHPELDLKIQEDNAGDSISNKNDSYCELTGMYWAWKNLKNVDVIGLCHYRRYFDFHKQCKWPYPITSFTTSQFKLLDFSIPQSVVDRILKGGIVVPRSVIYPIPLFYDYASCHIGSDMRIIQQIIEEKGDAKFINAFEDVMFRNNKLIHYNMFIMKWDDFNNYCNWLFPILKEAEKRIDISHYNVIQKRIWGYMAERLMNVWLYAEKKNLLFYPVIWINNNSSNMETRIKYLCRTLRYNLINKLATLQLTFKTK(SEQ ID NO.56) >c_ASMT16: MLYYCTLFDSNYISKGIALYLSLERHTDDFLLYVMAMDRKCEKMLKNMDFAHMHVECIEDIETGSLLEAKQNRSRAEYCWTCGSVVTEHFFRTRMLPEITYLDSDLMFFCSPKCVDNELKKANASVGLTPHFINNTIFGVYCVQYVYFKRDEDGEKCLNWWKNECLKWCY SKLEDGKYGDQKYLDYMKNMFSNVYAIENRGVGIANWNIEQYDYHFNEGFLIHQGRQWPIVFFHYNGVNVKVENGKLIFVYSKYLTKVVVETFIKQYAELLTFVYINYLGIKIDGFEMYPQNKVKVILNGLWLYVRGNKIAMRLEQKFMEHKYIQRVSPYSERKNR (SEQ ID NO.57) c_ASMT17: MKCLHIITPVKDSIDSTLETVKAIMESDILVPFTYTVYNDFSTEENTHRLEEAARQWNFRLVNLSDLTDHPSPNYLLVLQTAQKEAIEADAGLLIVESDVIVKKNTLQSLYNGALQQKQCG IAAAVTTTDEKGIINYPYLHAKGHENQVYPEKKHCSFCCSLLTPRLLKAFDFQTLDPSKNWYDVTISHESLKRGFQNYLFTTLPVWHRPHSSRPWKQLKYKNPLKYYWLKYTKGLDKI (SEQ ID NO.58) The nucleic acid sequences of the above 28 DDC enzymes are shown in SEQ ID NO.59~86, the nucleic acid sequences of the above 13 AANAT enzymes are shown in SEQ ID NO.87~99, and the nucleic acid sequences of the above 17 ASMT enzymes are shown in SEQ ID NO.100~116.

[0036] These candidate genes were synthesized and inserted into the pET28a vector, and then transformed into... E. coli BL21 (DE3) was cloned and expressed. Successfully transformed cells were selected. E. coli BL21 (DE3) clones were cultured overnight, followed by induction of target protein expression with 0.25 mMIPTG. The clones were then transferred to LB medium containing 0.5 mg / mL of the metabolic substrate (5-HTRP, 5-HT, or NAS). After culturing for 12-16 hours, the supernatant of the bacterial culture was collected and used for LC-MS detection of the concentrations of metabolic substrates and products, thereby testing the activity of metabolic enzymes. E. coli BL21 (DE3) clone. Results showed that clones expressing the DDC2 protein possessed DDC activity (…). Figure 1 Clones expressing the AANAT6 protein possess AANAT activity. Figure 2 Clones expressing the ASMT1 protein possess ASMT activity. Figure 3 These results indicate that the DDC2 protein (SEQ ID NO.2) and AANAT6 protein (SEQ ID NO.34) found in Bifidobacterium adolescentis are metabolically active DDC enzymes and AANAT enzymes, and the ASMT1 protein (SEQ ID NO.42) found in Bacteroides dysplasiae is a metabolically active ASMT enzyme.

[0037] Compared with reported human gut bacteria Ruminococcus gnavus and Clostridium sporogenes Sequence alignment of the DDC enzymes from the source revealed that the DDC enzyme reported in this invention has extremely low (< 2%) sequence similarity. Figure 4 The other two intestinal bacterial-derived AANAT and ASMT enzymes reported in this invention have not been previously reported.

[0038] 2. Activity assays of DDC, AANAT, and ASMT enzymes derived from intestinal bacteria.

[0039] Expressing DDC2 protein E. coli Cloning and expression of AANAT6 protein E. coli Cloning and expression of ASMT1 protein E. coli Clones were cultured overnight, followed by induction of target protein expression with 0.25 mM IPTG, and then purified. The purified DDC, AANAT, and ASMT proteins were co-incubated with different concentrations of metabolic substrates (5HTRP, 5-HT, or NAS), and the concentrations of substrates and products were subsequently detected by LC-MS. The results showed that the DDC enzyme Km from Bifidobacterium was 938.5 uM, and the AANAT enzyme Km was 683.3 uM. (Previously reported (PMID: 25263219)) Ruminococcus gnavus and Clost ridium sporogenes Compared to DDC enzymes from other sources, the DDC enzyme reported in this invention has a higher substrate affinity (Km = 2.8 ± 0.0 mM). Figure 5 ).

[0040] Using the identified DDC and AANAT sequences as templates, genomes of other intestinal species were compared. Species containing homologous sequences (sequence similarity ≥60%, coverage ≥80%) were found to originate from the genus *Bifidobacterium*. Single-cell cultures of these *Bifidobacterium* species were incubated with corresponding metabolic substrates. LC-MS analysis revealed that these *Bifidobacterium* species all exhibited corresponding DDC or AANAT enzyme activities. Figure 6 ).

[0041] 3. Gut bacteria or engineered bacteria that express melatonin synthase can increase melatonin levels in the host.

[0042] The identified enzyme genes were synthesized and inserted into probiotics using CRISPR-Cas9 technology. E. coli Nissle In the 1917 genome, a heterologous expression AANAT enzyme was constructed. E. coli -AANAT) and ASMT enzyme ( E. coli -ASMT) E. coli Nissle The 1917 engineered strain was used in in vivo experiments on mice.

[0043] Experiments were conducted using 8-week-old C57BL / 6J germ-free mice, with Bifidobacterium adolescentis (Ba) alone or together with Bacteroides davidiana (Bd) as... Germ-free mice were inoculated via gavage at a dose of CFU / 0.2 mL. Feces and serum were collected one week after colonization for metabolic analysis. It was found that inoculation with *Bifidobacterium adolescentis* alone significantly increased NAS levels in feces and serum. Compared with *Bifidobacterium adolescentis* colonization alone, co-colonization with *Bacteroides davidiana* reduced NAS levels in feces and serum and increased melatonin levels. This confirms that the combination of *Bifidobacterium adolescentis* and *Bacteroides davidiana* can utilize 5-HT as a substrate to metabolize and produce melatonin, thereby increasing melatonin levels in the host. Figure 7 (ab).

[0044] Engineered E. coli expressing melatonin synthase was administered via gavage to 8-week-old C57BL / 6J standard SPF mice. E. coli -AANAT (expresses AANAT enzyme) E. coli -ASMT (expressing the ASMT enzyme) was used to sacrifice mice one week later, and fecal, cecal contents, and serum samples were collected for metabolic analysis. The results showed that it significantly increased melatonin levels in the intestines and serum. Figure 8 (j).

[0045] By administering melatonin synthase-expressing intestinal bacteria *Bacteroides davidiana* to 8-week-old C57BL / 6J standard SPF mice via gavage... B.doreiWT was found to significantly increase melatonin levels in the gut and serum. Figure 8 (kq); When administered orally, intestinal bacteria lacking melatonin synthase, Bacteroides davidiana ( B.dorei When KO), its effect on increasing melatonin levels disappears.

[0046] 4. Gut bacteria or engineered bacteria that express melatonin synthase can improve related diseases by increasing melatonin levels in the host.

[0047] A mouse model of inflammatory bowel disease was established by administering 2.5% DSS in the drinking water to 8-week-old C57BL / 6J mice for one week. Simultaneously, the mice were administered DSS via gavage. CFU / 0.2 mL inoculum E. coli -AANAT bacteria, E. coli -ASMT bacteria, control group administered the same amount by gavage E. coli One week later, the mice were sacrificed, and fecal, cecal contents, and serum samples were collected for metabolic analysis. The results showed that, compared to the control group, gavage administration... E. coli -AANAT bacteria can promote the conversion of 5-HT to NAS, significantly increasing the level of NAS metabolism in the host gut, via gavage. E. coli -ASMT bacteria can further promote the conversion of NAS to melatonin, significantly increase melatonin levels in the host gut, alleviate DSS-induced intestinal inflammatory response, improve weight loss and colonic shortening in mice, and reduce colonic tissue damage. Figure 9 ).

[0048] A mouse model of inflammatory bowel disease was established by administering 2.5% DSS-containing drinking water to 8-week-old C57BL / 6J mice for one week. Simultaneously, the mice were administered 2×10⁻⁶ DSS via gavage. 8 Mice were administered CFU / 0.2 ml of *Bacteroides davidiana*, while the control group received an equal volume of PBS via gavage. One week later, mice were sacrificed, and fecal, cecal, and serum samples were collected for metabolic analysis. Results showed that, compared to the control group, gavage administration of *Bacteroides davidiana* significantly increased melatonin levels in the host gut, alleviated DSS-induced intestinal inflammation, improved weight loss and colonic shortening, and reduced colonic tissue damage in mice. When *Bacteroides davidiana*, an intestinal bacterium lacking melatonin synthase, was administered via gavage... B.dorei When KO occurs, its improvement effect disappears. Figure 10 ).

[0049] 5. Intestinal bacteria or engineered bacteria that express melatonin synthase can be used for the biosynthesis of melatonin.

[0050] In in vitro testing, Bifidobacterium adolescentis ATCC 15703 alone or Bacteroides multifiliis ( Bacteroides doreiDA75 (accession number: CGMCC 46612) was inoculated together with a medium containing 1 mM 5-HT. After 48 hours of culture, the supernatant was collected for LC-MS analysis to determine the concentrations of metabolic substrates and products. The results showed that *Bifidobacterium adolescentis* cultured alone could metabolize 5-HT to produce NAS, but could not produce melatonin. However, when co-cultured with *Bacteroides davidiana*, they could work together to convert 5-HT into melatonin. Figure 11 ).

[0051] Figure 12 This refers to the metabolic pathway from tryptophan to melatonin. Figure 13 This invention describes a functional enzyme derived from gut bacteria that was discovered in the melatonin metabolic synthesis pathway.

[0052] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A melatonin synthase, wherein the melatonin synthase is selected from one or more of DDC enzyme, AANAT enzyme, and ASMT enzyme, wherein, The DDC enzyme comprises the amino acid sequence shown in SEQ ID NO.2 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.2; the AANAT enzyme comprises the amino acid sequence shown in SEQ ID NO.34 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.34; and the ASMT enzyme comprises the amino acid sequence shown in SEQ ID NO.42 or its active fragment, or an analogue with more than 80% similarity to the amino acid sequence shown in SEQ ID NO.

42.

2. A nucleic acid molecule encoding the melatonin synthase of claim 1, wherein, The sequence of the nucleic acid molecule encoding the DDC enzyme is shown in SEQ ID NO. 60; Preferably, the sequence of the nucleic acid molecule encoding the AANAT enzyme is shown in SEQ ID NO.92; Preferably, the sequence of the nucleic acid molecule encoding the ASMT enzyme is shown in SEQ ID NO.

100.

3. A pharmaceutical preparation comprising the melatonin synthase of claim 1.

4. A bacterial agent comprising a first bacterium expressing the AANAT enzyme of claim 1, a second bacterium expressing the ASMT enzyme of claim 1, and a third bacterium expressing the DDC enzyme of claim 1.

5. The microbial agent according to claim 4, wherein the microbial agent comprises intestinal bacteria or engineered bacteria; Preferably, the first and / or third bacteria are Bifidobacteria; Preferably, the second bacterium is Bacteroides multifiliis; Preferably, the Bifidobacterium is selected from Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium animalis.

6. The microbial agent according to claim 5, wherein, The aforementioned Bacteroides multifiliis is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46612.

7. A strain of Bacteroides multifiliis, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46612.

8. A method for synthesizing melatonin or melatonin synthesis-related metabolites in vitro, the method comprising: Add the melatonin synthase of claim 1, the pharmaceutical preparation of claim 3, the bacterial agent of any one of claims 4-6, or the Bacteroides strain of claim 7 to a system containing a melatonin synthesis substrate; The melatonin synthesis substrates include one or more of 5-HTRP, 5-HT, and NAS; The melatonin synthesis-related metabolites include 5-HT and / or NAS.

9. The use of the melatonin synthase of claim 1, the nucleic acid molecule of claim 2, the pharmaceutical preparation of claim 3, the bacterial agent of any one of claims 4-6, or the Bacteroides dysporidis strain of claim 7 in any one of the following: (1) Prepare products that promote melatonin synthesis; (2) To prepare drugs for the prevention or treatment of diseases related to melatonin secretion or function; (3) Prepare foods or health products that help improve sleep; Preferably, the health product includes probiotic supplements, and more preferably, the probiotic supplement includes probiotic sticks and probiotic gummies.

10. The application according to claim 9, wherein, The diseases related to melatonin secretion or function include sleep disorders, nervous system disorders, mental health disorders, ophthalmological disorders, or inflammatory bowel disease; preferably, the sleep disorders include insomnia or delayed sleep phase syndrome. Preferably, the neurological diseases include Alzheimer's disease or Parkinson's disease; Preferably, the mental health-related illnesses include depression; Preferably, the ophthalmological diseases include age-related macular degeneration and glaucoma; Preferably, the drug can also be used to relieve intestinal inflammatory reactions.

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