Engineered bacteria and methods for synthesizing perillaldehyde

Abstract

This invention provides an engineered microorganism and a method for synthesizing perillaldehyde. The engineered microorganism comprises a gene for perillaldehyde synthase and a gene for Bayer-Villiger monooxygenase. The perillaldehyde synthase comprises: (a1) a protein having any sequence in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and possessing perillaldehyde synthase activity. The Bayer-Villiger monooxygenase comprises: (b1) a protein having any sequence in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and possessing Bayer-Villiger monooxygenase activity. This invention solves the problem of inefficient biosynthesis of perillaldehyde in existing technologies and is applicable to the field of synthetic biology.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and more specifically, to an engineered bacterium and a method for synthesizing perillaldehyde. Background Technology

[0002] Ambergris, a precious fragrance, originates from the grayish-white soft stones found in the stomachs of sperm whales. Due to its scarcity and difficulty in obtaining, research has largely focused on developing synthetic methods to replace natural resources. Ambroxol is a high-strength, long-lasting, and highly stable ambergris-based ingredient, widely used in the production of high-end perfumes and fragrances. In my country, ambroxol has been approved as a food flavoring (S0280) for addition to various foods. Sclareol glycol (molecular formula C...) 16 H 30 O2 is a key precursor to the precious fragrance ambroxol. The synthesis of ambroxol from perillyl glycol requires only one chemical step. However, both the traditional production of perillyl glycol and its existing biosynthetic routes face significant bottlenecks, severely restricting its large-scale production and application. Currently, perillyl glycol is mainly produced from perillyl alcohol via oxidation with potassium permanganate to obtain perillyl lactone, followed by further reduction. However, this process has many problems, such as significant environmental pollution, and its yield and product separation efficiency cannot yet meet industrial needs. Compared with traditional chemical synthesis processes, biotechnology has advantages such as mild reaction conditions, good stereoselectivity, and less environmental pollution. Therefore, the use of biotechnology to prepare natural fragrances has become one of the most popular research hotspots in the fragrance industry.

[0003] Methods for producing perillaldehyde by combining biotransformation and chemical catalysis have been reported: one method utilizes fungi. Cryptococcus albidus ATCC 20918 describes the fermentation of perillyl alcohol as a raw material to produce perillyl lactone (US5212078A, 1993), followed by chemical conversion to synthesize perillyl diol, which is then chemically converted to ambroxol; another method involves using fungi... Hyphozyma roseoniger ATCC 20624 describes the production of perillyl alcohol (US4798799A, 1989) from perillyl alcohol, followed by the chemical production of ambroxol from perillyl alcohol. Rosehip yeast ( Hyphozyma roseonigra ATCC 20624 is currently the only known microorganism capable of synthesizing perillyl alcohol using perillyl alcohol as a single carbon source. However, perillyl alcohol is cytotoxic to *Rhodotorula rubiginii*, making it difficult to achieve an economically viable substrate concentration, and its metabolic modification is also challenging, hindering the construction of highly efficient biocatalytic systems. In addition, Li Shengying et al. from Shandong University synthesized perillylone via a chemical method using *Rhodotorula rubiginii* (ATCC 20624). Hyphozyma roseonigraBaeyer-Villiger oxidase from ATCC 20624 converts perillone to perillyl glycol acetate, which is then used to synthesize perillyl glycol via a chemical method (CN114921428A, 2022). M. Schalke et al. of Vermenescher GmbH observed the formation of perillyl alcohol oxide during co-expression of alcohol dehydrogenase and enaldehyde cleavage peptides in Escherichia coli; this perillyl alcohol oxide may be spontaneously synthesized from perillone (CN 114630905 A).

[0004] However, there are currently no methods for synthesizing perillaldehyde in easily modified model strains using biotransformation, especially no reports of directly synthesizing perillaldehyde using a simple and inexpensive carbon source (glucose). Summary of the Invention

[0005] The main objective of this invention is to provide an engineered microorganism and a method for synthesizing perillaldehyde, so as to solve the problem of the difficulty in efficiently biosynthesizing perillaldehyde in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, an engineered bacterium is provided, comprising a gene for perillaldehyde synthase and a gene for Bayer-Villiger monooxygenase, wherein the perillaldehyde synthase comprises: (a1) a protein having any sequence in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity; wherein the Bayer-Villiger monooxygenase comprises: (b1) a protein having any sequence in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0007] Furthermore, the aforementioned engineered bacteria include yeast; the aforementioned yeast includes Saccharomyces cerevisiae or Yersinia lipolytica.

[0008] Furthermore, the aforementioned engineered bacteria also include those derived from rockrose. Cistus creticus The gene for lysine pyrophosphate diol synthase.

[0009] Further, the above-mentioned lysine pyrophosphate diol ester synthase includes: (c1) a protein having the sequence shown in SEQ ID NO: 51, or; (c2) a protein having more than 70% homology with the amino acid sequence defined in (c1) and having lysine pyrophosphate diol ester synthase activity.

[0010] Furthermore, the engineered bacteria also include genes for an enzyme used to synthesize geraniol pyrophosphate.

[0011] Furthermore, the above-mentioned synthetic gerany-gerany-pyrophosphate includes the above-mentioned engineered bacteria synthesizing gerany-gerany-pyrophosphate using glucose and / or pyruvate as precursors.

[0012] To achieve the above objectives, according to a second aspect of the present invention, a method for preparing an engineered bacterium is provided, the method comprising: knocking in a gene into a starting engineered bacterium to obtain the engineered bacterium; the knocked-in gene includes a gene for perillaldehyde synthase and a gene for Bayer-Villiger monooxygenase, wherein the perillaldehyde synthase comprises: (a1) a protein having any sequence in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity; wherein the Bayer-Villiger monooxygenase comprises: (b1) a protein having any sequence in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0013] Furthermore, the knock-in genes also include those derived from rockrose. Cistus creticus Genes for lysine pyrophosphate synthase and / or genes for enzymes used to synthesize geraniol pyrophosphate.

[0014] To achieve the above objective, according to a third aspect of the present invention, a method for synthesizing perillaldehyde is provided, the method comprising: using the above-mentioned engineered bacteria, or engineered bacteria prepared by the above method, to ferment and synthesize the above-mentioned perillaldehyde in a fermentation medium.

[0015] Furthermore, the above fermentation includes shake flask fermentation or fermenter fermentation.

[0016] To achieve the above objective, according to a fourth aspect of the present invention, a method for catalyzing the production of perillaldehyde from 8-hydroxy-hemisin-13-en-15-aldehyde is provided, the method comprising using perillaldehyde synthase to catalyze the production of perillaldehyde from 8-hydroxy-hemisin-13-en-15-aldehyde to obtain perillaldehyde; wherein the perillaldehyde synthase comprises: (a1) a protein having any of the sequences in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity.

[0017] Furthermore, the above method includes: using yeast cells capable of expressing the above perillaldehyde synthase to catalyze the above 8-hydroxy-hemiflora-13-en-15-aldehyde to obtain the above perillaldehyde.

[0018] To achieve the above objective, according to a fifth aspect of the present invention, a method for catalyzing the production of perillaldehyde from perillaldehyde diacetate is provided, the method comprising catalyzing the perillaldehyde using a Bayer-Villiger monooxygenase to obtain the perillaldehyde diacetate; wherein the Bayer-Villiger monooxygenase comprises: (b1) a protein having any sequence in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0019] Furthermore, the above method includes: using yeast cells capable of expressing the above-mentioned Bayer-Villiger monooxygenase to catalyze the above-mentioned perillaldehyde to obtain the above-mentioned perillaldehyde diol acetate.

[0020] Furthermore, the perillaldehyde in the above method is the perillaldehyde obtained by the above-described method of catalyzing the production of perillaldehyde from 8-hydroxy-heliotropin-13-ene-15-aldehyde.

[0021] By applying the technical solution of this invention, an engineered bacterium containing the genes for perillaldehyde synthase and Bayer-Villiger monooxygenase was obtained. This engineered bacterium can be used to achieve efficient and environmentally friendly synthesis of perillaldehyde diol, thereby meeting the growing market demand. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 A de novo synthetic route for perillaldehyde according to an embodiment of the present invention is shown.

[0024] Figure 2 The following are gas phase diagrams of fermentation broths with different exogenous additions of (E)-lysanthin-13-ene-8,15-diol to yeast according to Example 2 of the present invention.

[0025] Figure 3 The integration of *Yersinia lipophila* according to Example 3 of the present invention is shown. SCOT Gas phase diagram of fermentation broth.

[0026] Figure 4 The integration of *Yersinia lipophila* according to Example 4 of the present invention is shown. BVMO Gas phase diagram of fermentation broth.

[0027] Figure 5A The integration of *Yersinia lipophila* according to Example 4 of the present invention is shown. BVMO NMR chromatogram of perillaldehyde in fermentation broth.

[0028] Figure 5B The integration of *Yersinia lipophila* according to Example 4 of the present invention is shown. BVMO NMR chromatogram of perillaldehyde in fermentation broth. Detailed Implementation

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0030] As mentioned in the background section, there is an urgent need in the field to find a market-competitive synthetic route for perillaldehyde that meets the requirements of green chemistry, utilizing synthetic biology and biocatalysis techniques, in order to meet the ever-growing demand for perillaldehyde. Therefore, in this application, the inventors have attempted to develop an engineered bacterium capable of producing perillaldehyde, and based on this, have proposed a series of protection schemes for this application.

[0031] In a first typical embodiment of this application, an engineered bacterium is provided, comprising a gene for perillaldehyde synthase (SCOS) and a gene for Bayer-Villiger monooxygenase (BVMO). The perillaldehyde synthase (SCOS) comprises: (a1) a protein having any sequence in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity. The Bayer-Villiger monooxygenase (BVMO) comprises: (b1) a protein having any sequence in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0032] In this application, the inventors discovered that (E)-lysanthin-13-en-8,15-diol can synthesize 8-hydroxy-hemisanthin-13-en-15-aldehyde under the action of yeast endogenous alcohol dehydrogenase ADH. The aforementioned SCOS and BVMO can be used as precursors to synthesize perillaldehyde. The engineered bacteria contain genes capable of expressing SCOS and BVMO, thus enabling the expression of SCOS and BVMO, thereby achieving the synthesis of perillaldehyde using (E)-lysanthin-13-en-8,15-diol as a precursor.

[0033] Engineered strains integrate the above-mentioned SCOT and BVMOThis gene effectively solves the problems of environmental pollution, low yield, and insufficient product separation efficiency in the traditional chemical synthesis of perillaldehyde. Specifically, SCOS catalyzes the conversion of 8-hydroxy-hemisperid-13-en-15-aldehyde to perillone, while BVMO further converts perillone to perillaldehyde acetate. Perillaldehyde acetate is unstable in yeast and is hydrolyzed to perillaldehyde by intracellular esterases. This process is achieved in engineered bacteria, including but not limited to Saccharomyces cerevisiae or Yersinia lipolytica, which not only avoids the toxic organic reagents used in chemical synthesis and reduces environmental pollution during production, but also improves the purity and separation efficiency of the product. Although SCOS and BVMO are known enzymes in the prior art, many enzymes with similar functions exist in actual experiments, but not all enzymes with similar catalytic activity can achieve the specific catalytic reaction described in this application.

[0034] In exploring the inventive features of this invention, it should be noted that although SCOS and BVMO have been extensively studied and reported in existing literature and patents, and numerous enzymes with similar catalytic activities exist, achieving efficient and selective catalytic reactions in specific biosynthetic pathways, especially for the synthesis of sagebearing diol, is far from easy. This specific catalytic conversion process requires not only enzyme activity but also the enzyme's ability to precisely identify and convert specific substrates, thereby avoiding side reactions and ensuring the efficient formation of the target product, sagebearing diol.

[0035] The specific SCOS and BVMO enzymes screened and expressed in this invention (such as SEQ ID NOs: 1-25 and SEQ ID NOs: 26-50) have been thoroughly validated and shown to exhibit excellent conversion efficiency in *Saccharomyces cerevisiae* and *Yarrowia lipolytica* chassis cells for exogenously added (E)-lysantango-13-en-8,15-diol. Furthermore, the de novo synthesis of perillaldehyde can be achieved with the addition of enzymes related to the precursor material. In contrast, enzymes with similar activities reported in the prior art (including but not limited to those shown in SEQ ID NO: 52 and SEQ ID NO: 53; only these two enzymes are listed here for comparison due to space limitations) have failed to catalyze the actual reaction of (E)-lysantango-13-en-8,15-diol or perillaldehyde. This demonstrates that for specific substrates and catalytic reactions, not all enzymes with similar activities, or classified as belonging to the same category or having the same name, can complete the catalytic reaction. This fact further supports the inventiveness of the invention, as it demonstrates that the specific amino acid sequence of an enzyme is crucial for the construction of efficient biosynthetic pathways.

[0036] In summary, this invention demonstrates a high degree of innovation in enzyme selection, successfully solving the bottleneck problem in the biosynthesis of perillaldehyde in the prior art, providing strong technical support for subsequent microbial fermentation production, and possessing significant industrial application prospects and environmentally friendly advantages.

[0037] SEQ ID NO: 1 (MtSCOS):

[0038] MSDELARKRFTEFKERTDTIADAELDDYWASLPPATIDGMLGEWKGGEFVTGHRMNGLLDKARWFGKTFTSRTEVQPLVCLDADGNKFSNVELGKGEASLWLEEFRGEVTATMVYDGQPTHDHFKRIDDTAVLGIMNGKGVLDNGRYFYFYLERV.

[0039] SEQ ID NO: 2 (SpSCOS):

[0040] MSREYFDQLMCQDATTEGELLTLFDMLEPVACEDILSRWRGGGFNTGHWLLPALVKLRWFGKWFVSPSDAKPMVCWDDTGALSSSQAMNGEASLAMMAFRGKISATVVYDGVPMFGHLRRVDDETLLGAVSGKAIGSAELLPDGKHQFFFLDRISAWPAPLT.

[0041] SEQ ID NO: 3 (RhTSCOS):

[0042] MDIDQARSTFHELRRRDTGVSPDELDAVWAALDTVRAEDILGSWKGDDFATGHRLHDKLVASRWHGKTFHSLEDAKPLICRDANGNLYSDVEGGNGEASLWNVEFRDEVTATMVYDGAPIFDHFKEVDDSTLMGIMNGKSALVLDGGRHYYFLLERD.

[0043] SEQ ID NO: 4 (RoSCOS):

[0044] MVRKLSDLPVRDETRGTTMDIDQARSTFHALRQHDTGVSPDELDVVWAALDTVRAEDILGNWKGDDFATGHRLHDKLVASRWHGKTFVSLEDAKPLICRDADGNLYSDVEGGNGEASLWNIEFRGEVTATMVYDGAPIFDHFKKVDDSTLMGIMNGKSALVLDGGRHYYFLLERD。

[0045] SEQ ID NO:5(XcSCOS):

[0046] MNTKQIFDELRSREKIEDESELLNFFDSLPEVRVGEILSKWKGGDFNTGHWGHESLIEMNWFGKWFKSKFDAIPLVCFNNDGKLFSNHIMKGEASLWEVEFRGKSSATMIYDGIPIFDHFRKVDDNTLLGVMNGKPIEGFPDIVFNGKYYYFYLERLADFPVEFTEEK。

[0047] SEQ ID NO:6(RhCSCOS):

[0048] MSDTAAPERLRELLAMESGVETTELDQLWSRLDTVRAEDILGSYKGSAFATGHGLCRALPASNWYGKDFRSITDAKPLICRAEDGTLFSNVELGQGEASLWNIEFRGEVTATMVYDGKAVFDHFKRIDSKSLMGIMNGKPELVLSRGEFFYFALELC。

[0049] SEQ ID NO:7(RhMSCOS):

[0050] MDPLALDEVWAALPTVHIDELEGRWRGSGFETGHRTGRLLARSGWYGKNFGSRFDVQPMLCRGPDGELYSDVAAGKGEASLWMVEFRGECTATMVYDGMAVFDHFKRVDDRTLMGIMNGKDVLDGGEHFYFALVKDETG。

[0051] SEQ ID NO:8(SiSCOS):

[0052] MSQEQDQIDVAPRPPGARLVRPTVVERWRALREASGPVDIGALDALWADLAVVEASSLLGSWRGFALPTGHPLERVLARSRWHGKRFVALDDAKPLICRGPGGGLYSDTAAGKGEASLWNVEFRGEVTATMVYDGMAVFDHFKRVDENTLMGVMNGKPHLVLAEGRHFYFGLEREHVGPGSADFTHGREPGRDRG。

[0053] SEQ ID NO:9(ApSCOS):

[0054] MDAATARTRIAEIRAAGGKTTIDELDDLWAALPAVTPDDILGSWRGSEFVSGHRFEGYLPKIRWHGKRFTSRTDVAPLVCRDDNGNLFDDTERAKGGASLWTVEFRGEPTATMVYDGQPILDHFKRIDDHSLLGVMNGKGVLDDGRHYYFVLELSRPETANPSHPEALGRSRTEILDRPRTEALARPRTEILDRPRTEALGRSHSETE。

[0055] SEQ ID NO:10(StSCOS):

[0056] MDILEARARFQDLREKDGPVDPSELDEIWAALATVRPEEILGEWKGDEFRTGHPLNGALAKAGWYGKTFVAVHDAKPLICRNAAGELYSDRELGKGEASLWTVEFRGEPTATMVYDGQPVFDHFKQVDDTTLIGIMNVKGTPAEGPFFYFFLERVPEVPHGGPHGGPHGGPHGGPRAGEGS。

[0057] SEQ ID NO:11(TcSCOS):

[0058] MTAERTVRGLIDEPQHSEPAELAGLFAQLEPVTIDFMLGDWHGGELPSGHPMDGALGKARWYGKSFRSANDVQPLVCLDDAGEKYSNVALGKGEASLRLIDFGGTVTASMVYDGQPVIDHFAKVDDDTVMGVMTGKKLAAPYFYFYLERDAASGTSGGCLADR。

[0059] SEQ ID NO:12(GoSCOS):

[0060] MIADTPCGRIGYGENSAPSPGAPNRREAFVTASSQAPIENDVDARREAAQARFAELRTARTPVADADLDAVWADLDTIRPADMLGRWHGGEFVTGHAMNGLLTKIGWYGKNFISTSEVQPLVCRNDAGELYSNTEVGKGEASLWAVEFRGEVTASMVYDGQPVIDHFKRVDDTTVMGIMNGSGGLIGGRHFYFYLERDS。

[0061] SEQ ID NO:13(AaSCOS):

[0062] MDVHEARSLFRELTERDGRVDPGELDAIWAVLPTVRPEEMLGAWKGGEFDTGHPLNGMLEKAGWYGKTFHSPHDAKPLMCRNETGELYSNTELGQGEASLWTVEFRGESTATMVYDGRPVFDHFKWVDEDTLLGIMNGKGVPAEGPFYYFYLERAPESGAASGAVPGAVPGAGEGA。

[0063] SEQ ID NO:14(SinSCOS):

[0064] MFSRAFSKTAFSIPRVTSRRFITTFADLAKSRKATTQQCWEAFDALEPVKVDELVGYRWKGYEIYTDHPWCGLLDANNWFGKEYFSSEHGNPLLTYATDEKSGNDVFPAEPLKYFALTAEGTNILGDLRGQIESPEGVGCRLRTILFRGVNTASMFYDQLPINDTFKKVDDNTVFGVMDCKMVPDPPYFFVLEKYLKI。

[0065] SEQ ID NO:15(WlSCOS):

[0066] MNNDKAVATFFELKESQAISEPADLDELWAALEPIGRDFMLGAWSGGELPSGHAMDGQLKQVNWHGKTFDAYHDVKPLVCRDDEGNLYSNKELGKGEASLWAVEFRGETTAAMVYDGQPVIDHFKKVDDTHVMGIMNGKTSLVRDKHFYFYLERE。

[0067] SEQ ID NO:16(MxSCOS):

[0068] MPGKTVPGAIKLASNETVFGPLPSVRAAIEQATDVVNRYPDTGCTELKSALARHLNTVGDTGGWASEHVAVGCGSVSLCQQLIQITASVGDEVLFGWRSFELYPIQVRVAGATAVQVPLADHTFDLHAMLAAVTDRTRLVFVCNPNNPTSTVVDPDALVRFVEAMPSHVLIAIDEAYVEYVRDNMLPDSLGLVRSHPNVVVLRTFSKAYGLAGLRIGYAVGHPDVIAALDKVYVPFTVTNVSQAAAMASLEAADELLARTDAVVAERAGSVPGCAMRGLRCRRRKPTSSGYRWGAHIRLRRTGRQRAHRGPPVRPRRGAGHDRCTGRKRRAAAIRGQLDSRRTPMNDALARKKFTEFKERSDRISDTALDEYWASLTPATIADMIGEWQGGEFDTGHRMNGRLEKARWFGKTFNSATDVQPLVCLDADGNKFSNVEMGKGEASLWMEEFRGEITATMVYDGQPVHDHFKKIDDHAVMGIMNGKGVLDNGRYYYFYLERV。

[0069] SEQ ID NO:17(MaSCOS):

[0070] MPNQLVWQYGWLSKPTDSGVLVNIETARSRFAELRGQSDVDTAELDEIWSALDTVRAEDVVGHWKGDEFHTGHKMNGQLQAARWYGKFFDSLNDVEPIVCYDDEGQLFSNHQLSRGGASLWDIEFRGETTATMVYDGQPIFDHFKRVDGNTLMGIMNGKRQRTADKLFYFLLERDQ。

[0071] SEQ ID NO:18(CbSCOS):

[0072] MATLRSYLHTTVPAPLDTDGVAVVDLRSYLAKIDREYATQRYAETDAPLLYIRIQIGKATCSALIDCGASRNFISQAFLARAGFGPRVRRKTQPTQVTLADDHTQKSIDRCVDGVPVYFAVLACEPVSFDVLDTKFDMIlgMSWLHSADHPVNHFDRTVHIRDRNGVLVPCTVATSHTSIACHVVSVARIRDAIARNDVEEMSLARKQQHTYLMEDEADSTRWKSAKKKR VKKKKKKKKKKKKKKQKQKQKQKKKKKKKKKKKKKKKKKKKKKTKKTKKKKKRRRRRIPHCRCLRMEGAIERMNEDEARARFHDRLRKEKDGVAPRELDRIWASLPPLDAGDIRGLWKGAAFQTGHALCQKLTVHWYGKLFESSTDVKPLMCYNEQGELYHYTEQSMGDASLWNVQFRGEVTAMVYDGQPIFDHFKKVDDSLMGIMNGKVNFVFDKGEHFYFILERS.

[0073] SEQ ID NO: 19 (AmSCOS):

[0074] MNPRDRFAEIRADGGKATPDELDQLWAALPAVRPEDILGSWQGSEFVSGHPFEGQLATRWHGKTFTSLTDVTPIVCLDDEGNRYANQEWSRGGASLWTVEFRGEPTATMIYDRRPILDHHFKRIDENTLLGVMNGKGVLSDGRHYYFILTKEAEARESEAKEAETKEAA。

[0075] SEQ ID NO: 20 (NoSCOS):

[0076] MEALREGPTAYDDLERLWADATPVRAEDLGTRGSDFPAAGHPVSGMLEGARWYGKRFESLTEAHPLICRDDAGELVSDTGLGKGIASLWDIAFRGEVTASMVYDGQPIIDHFKALDERTLIGVMNGKGVLAKGRHYFFVLERDD.

[0077] SEQ ID NO: 21 (RrSCOS):

[0078] MTRTALAAVVREYGADPQRCRRGSTHLRPSGVTMTVADELAALRRRTDTISPRELDDLWARLEPARIDDLVGYRWRGFSFDTGHRTHTLLGKARWYGKQFAAAHDVQPLMCRDASGELYSDVETGRGEASLWEVRFRGEVTATMVYDGMPVFDHFKKADDDTLVGVMNGKGGLVFDAGEHYWFGLERDIAL。

[0079] SEQ ID NO:22(MySCOS):

[0080] MGEWQGGEFDTGHRANGFMQKLNWFGKTFHSATDAKPLVCLDAEGNKFSNTEAMNGEASLWMEEFRGELVASMVYDGRPVHDHFKAVDDNAVVGIMNGKGALDTRSGTPRHLYFYLERN。

[0081] SEQ ID NO:23(RtSCOS):

[0082] MAVPEEFAELVRSGHRLDSAELDALWARLDVAETGLFTGLWRGGAFDTGHRAAAMLGKSGWYGKRFDALMDVKPLICRDEDGALYSDTTAGRGEASLWMVEFRGEVTATMVYDGMAVFDHFKKVDEDTVMGIMNGKGFALDGGKPFYFWLARD。

[0083] SEQ ID NO:24(FrSCOS):

[0084] MARARWAELRAAGGPLEPAELDDLWARLAPVAAADILGEWRGFAFPTGHRLEQVLARSHWYGKRFTALDDVQPLLCRGADGELFSDVKSGRGEASLWNVEFRGEITATMVYDGMAVLDHFKQVDDRTLMGIMNGKPAVVLDGGKHFYFGLERA。

[0085] SEQ ID NO:25(AxSCOS):

[0086] MSALERWRELRAATGPIDPDLLDALWADLEVVDAAAILGSWRGFAFPTGHPIEKALGASRWHGKRFDALDDAKPLICRAEDGTLHSDTTTGGGEASLWNVEFRGEVTATMVYDGRPVLDHFKRVDENTLMGVMNGKPRLVLADGRHFYFGLERE。

[0087] SEQ ID NO:26(FpBVMO1):

[0088] MSGNKLPIHSERHMRIVCIGFGASGLCFAYKLQRSFSNFSLTVFEKNPEVSGTWYENRYPGVACDVQAHNYTWSFAPKHDWSRVYAPAQEIYSYFNDFMATHGLEKYCKTSHKVVGATWDDQKGIWKLQVEDLAHQRIEYHECDILINASGILNNWRWPAIPGLEKYKGTLVHSANWDESLDLTGKHVGLIGNGSSGIQILPAIQPIVDQLTTFIREPTWISPPFGTEQRIYTEEEKERFKNDPEVLTLLRKANETNNNSMLGIYFPDHELQKKTRADFDRQMREKLKDVPWLQERLIPTWGVGCRRLTPGINYLETLSKPNVKVVYGEIESITEKGCRCDDGIEYPVDVLICATGFDTTFKPRFPLYGLEGKNLQDEWALEPRSYLGLAAPYMPNYFQFLGPHCPIGSGPVLIAIEAQADYMLSFCDRWQTENIHSMSPKNEAVTDFLEHCQEFMQRTVWIEECRSWYKSGSAAGRVSALWPGSTLHYLEALKEPRGDDWEIKYSGNRFSWLGNGFSQTELDDEADLGYYIRSHDDSPYASRRKRREAITHTGFNARALQANGDVNAGKDLLTTVEEYVRDREAGQPHL。

[0089] SEQ ID NO:27(FpBVMO2):

[0090] MSDVYLPEFNADKQPTVLDKVVGLPDAPIHAERHIKVICIGAGASGLLFAYKLQRSFYNFDLTIYEKNEDIAGTWYENRYPGCACDIPAHTYVWSFEPKTDWSSVYAGSSEIYQYFDNFSRKYDLRKYCKMQHQVIGATWDDDRGVYIVKVKDLASGKETIDYCNILVNAGGILNAWRYPAIPGLNKYKGTLLHTAAWDNSVDLSGKVVGLIGNGSSGIQVLPTIQPHVDKVITFIREPTYVSPVQGLSQHVYTADEKKQFTEQEGTLTKYRKQIERDTNKLFNLFIKDTPSQKENRAYITGLMKEKIANPYLEERLIPEWSLGCRRLTPGIGYLESLSKPNVEVVYGEITEITEKGCICDNGQEYPVDVLICATGFDTSFRPRFPVIGPNKKNLQDVWGKQPHSYLGLAAAEFPNYLVFLGPNCPIGNGPVLIAIEAQADWMMSVIDRYQTTNIHSFSPKQEAVDDFIEYKDKFMQKTIWSEPCRSWYKGAEIDAPITALWPGSTLHYLEALNQVRWEDFNVRFNGNRFAWLGNGYSQTELDPLADTAYYIREKDDSPPASTRAKREALTRTGEVGSTAPRRVGAPQQQSHL。

[0091] SEQ ID NO:28(VlBVMO):

[0092] MADLLLPETMHAGERDASRRLDGPIHSEKHIRVICVGAGASGLLVAYKMQKHFQNFSLAVYEKNEAVSGTWWENKYPGCACDVPSHNYTWSFEPKLDWSAVYPPAKEIFAYFEGFARKYALYQYVKLQHQVVESSWNHQDGGYDVKIKDLATGAIVEDHCDVLINAGGILNNWKWPAIPGLDKYKGVLLHTANWDESVSLEGKHVGLIGNGSSGIQVLPAIRDQCKKVTTFIREPTWVSPVQGLEQHRFSEEEKREFVENPAALLEYRKNIESGLNGQFGIFLKNNRINRDTHTYMTQQMKEKLGDAYLEEKLIPDWSVGCRRLTPGVDYLESLTKPNVSVVYGEIKSLGERGPIGEDGREHPVDVLICATGFDTSFRPRFPIIAPSGENLQDKWSGTPESYLGIGVAGFPNYFNMLGPNCPIGNGPVLSAIEAQADWMLRVIDRLQSTNAVQIAPKEQAVRDFVEYKEWFMTKTVWSDPCRSWYKARADGPVVALWPGSTLHYIEALKEVRFDDLDIVHAGNRFAWLGNGYSQTELDNSADWAFYIRNRDDDAPLTTGGRRKVLSKSGTVKSRDIVVFSGKRDEAEAQKETARFSFKHTMADSDDHNDERDPPNDGYRGIRSCSTHVEALAAMVEDLDCRKTGENAELKSEVALLLKDFYDKIDKYAKPPTAHRDIVYVRSIKKALEAAIQYREAGEAFSHRLPSSAQMLALRYTHQSGTLEEDVCAVLVDYQVKYGAPVPFLGFGYTAKPVHELLLATPVQPTHVIDEHEHLSERHATPFEHMKDQVERCEKQQSEQNDVASVQSEAPCPTISVEEDPSLNFRGPDAEFDQMDFNAWNLKHTMKLRQDLQGLREMTAEMTTAQRAEIDALKMDREENAYLLSILLRQMQECRRELRALKVQLAVPTPPHSQHSKKRSVDATVLEDETRKKARTEEYDAQ。

[0093] SEQ ID NO:29(AcBVMO):

[0094] MRVIVIGAGASGIYMAYKLKYNFTDFVLDIYEKNADIGGTWFENRYPGCACDVPAHNYTYTFDPKCDWSSTYSSSREIFQYFSDFVDKHELRQYIASHHEVTETRWDENASEWVVQVYNREIDSTFERRCDFLINACGVLNSWRWPTIPGIQSFKGPLLHTAAWDDNVDLTGKRVGLIGNGSSGIQILPQIQKVAKHVTTFVRKPTWVIPTFGSEMREHTDEERQQFRENPEEYLKMRKATEKVLTGVYPLFINDWPIQTQTAEYMKASMKEKINNEELAEQLIPDFPVGCRRLTPGVNYLESLTLPNVTTLYGEITEITPTSCITSTGTETELDILICATGFDTTFRPRFPLIGRSGHNLQDEWAHEPRSYLGVAASGFPNYFMFLGPNSPVGSGPSIISIEVQGSYIAEFLNRWQKEDIKAFDPKREAVDDFITQKDLFMEQTVWNSNCKTWYKSPVTGKVTALWPGSVLHYIETMAKPRYDDYDVTYASKNRFAYLGNGFSQMEHRPDADTSYYIRARDDGASVADSWGH。

[0095] SEQ ID NO:30(FpBVMO3):

[0096] MDTQNLPEYMKRPALQPRRLRIVGIGAGASGLLLAYKIQRNFINVDLTIFEKNEGVGGTWWENNYPGCACDNFSHTYTYSFEPKTDYEGTYSTSQEIQGYFESFCHKYNLDRYIQLSHAVTRCEWDEVGATWTVTTNDLRSGRSKKTVCDILVNATGVLNSWKWPDVPGLDKFQGQLVHSAHWDPQVDLDQKTVGLVGNGSSGIQILPAILPQVNRVVHVIRSPAWVVTPFGNSMPRKFTEEEKNEFADHPETHLLLRKRIEATNNSFFQIFLKNTPECRAAKQKFTEQMREQLGSFDLANKLIPKWPLGCRRLTPGIGYLQALQDPKTELIYDALAEVTEAGLRTTSGREIPVDIIICATGFDTSFKPKYPVVGPKGQDLRDVWSDQPKGYLGLAVPEFPNYFTFLGPNCPIGNGPVLCAVEAQGDYICKFIYRMQTEDIRTVTPKMAAADEFMKFKDTFFQTTVWSEDCQSWYKLRHNNKISAVWTGSTIHYLRAVEHPRYEDWDYTYLYDNRWSFLGNGLAPDDVDPKADLAFYVRQFDDAPIIGSKKIYEGPWVANRGESAIGLKDTEYKEPEEVLPELPPAGPRL。

[0097] SEQ ID NO:31(FpBVMO4):

[0098] MADTILPDLRSSGTQPGGSKPIKSQRHLRVICVGAGASGLLFAYKLQRSFDDFSLVLYEKNGELGGTWFENRYPGCACDIPAHTYTWSFEPKHDWSAVYAGSREIYQYFNDFSDKYKLRRYVRVKHQVTGARWDEKEGTWDVTVQDLLSGKTFPDRCDIFINAGGILNAWQWPRIPGLQSFKGPLLHSANWDDKTVLEGKTVGLIGNGSSGIQILPAIRPIAKKVTTFIREPTWVSPVQGLEGRTYSREERESFVRDKGKLLEYRKGIESALNSQFAIFLQNTQVQRDTRQHMQKQMSEKLHDPVLEDKLIPKWSVGCRRLTPGIGYLESLKADNVEVIYGEILKITPAGCVCDDGREHSVDILICATGFDTSFKSRFPVLGTKGKNLQDEWAEEPASYLGIAAPGFPNYFMFLGPNCPIGNGPVLTAIEAQADYILKLINRWQTENIHSMSPKLEAVKDFIAYKDSFMSKTVWHEECRSWYKNNSASGKIAALWPGSTLHYLEAVSDVRGDDWEIEYKGNRFAFLGNGFSQTEVDDTADFAYYIRDHDDGPFLSFKKNLQALNKSGTMAGQAKLNIMVKRESNL。

[0099] SEQ ID NO:32(FpBVMO5):

[0100] MSPIATADLEAPPTNGHVPTDSETRCRRAPIHANRHMRVVCIGAGASGIYMAYKLNHSFTDFSLDVYEKNPDISGTWYENRYPGCACDVPAHNYTYSFEPKSDWSANYATSKEIFKYFADFVDKYDLRKYITCNHEVVGAHWDRNNHEWVVRVRRPSQNVFEQRCDFLINAAGILNHWRWPAIPGLHSFKGDLVHSAAWDEKLDVTGKHVGLIGNGSSGIQILPKMQEQARHVTTFIREPTWVSPVRGMEYHEYSDEEKEMFRLKPEVWLEMRKRAEKAMALVFPLMLKGSASQEAAAAYMKSQMVQKINNDELAQKLIPDFAVGCRRLTPGINYLENLTAKNVTTLYGEIRAITPTGCVTEDGKEHELDVLICATGFDTTFKPRFPLIGRTDKDLAKEWEIEPRSYLGMAAAGYPNYFMYLGPNCPIGNGPIIFSIEIQGEYIAHFMNRWQKEDILTFDPKPEAVDDFIHQKDLFMQETVWNSSCLSWYKNPHTGKITALWPGSTLHYMETLANPRYDDYNVEYRGRRFAYLGMGFSQAELDPNVDVTYYIRNQDDDAPLCRPLFSTRNAKDITPRMTTLAEAGL。

[0101] SEQ ID NO:33(SsBVMO):

[0102] MASNVTPVGNSPQARDAARRLEAPIHSERHIRVICVGAGASGLLFAYKMRKHFTNFSLTIYEKNPAVAGTWFENRYPGCACDVPSHNYTWSFEPKLDWPAVYPPAKDIFDYFEGFATKYDLRQYVKLQHQVIGAYWDAQKGGYDVKVKDNANGVVVSDHCDILVNASGILNNWRWPAIPGLDKYKGTLLHTANWDPNTVLDGKHVGLIGNGSSGIQVLPAIREKCSKVTTFIREPTWVSPVQGLEQHIYSPEERAEFANKPGALLKYRKEIETGLNGQYGIFLKKSKINEDTRAYMIAQMKEKLNNDALAEKLIPDWSVGCRRLTPGVNYLESLTKPNVEVVYGEISAVTEKGCLCDNGQEYPVDVLICATGFDTSFRPRFPVVTPTGDNLQDTWAVDPASYFGVAAAGVPNYLVFLGPNCPIGNGPVLSAIEAQADWMCQLIDRYQTTNMATFAPSAQSVRDFVEFKEYYMRRTVWADPCRSWYKQRPDGPITALWPGSTLHYIEAVKELRFDDFDITYAGNRFAWLGNGYSQTELDATADWAYYIREEDDGAPLSTAGKRKLLTKSGTIQSRNAVSWSSGGGEGQSDRPRELRL。

[0103] SEQ ID NO:34(FpBVMO6):

[0104] MSSTSVSTGLWAKYPAINQVKRTLKVVCIGAGASGLLVAYKLQKHFDNLDLTIFEKNPDISGTWFENRYPGCACDVPAHCYTFSFEPKPDWNANYASSWEIHRYFTDFATKYRLGRFIKLNHKVVGAIWSVDRAEWDVCVEDLTTRKNFTVSCHCLVNAGGILNVWKYPPIPGLLDKFQGTVVHSAAFPEDLDVNGKVVGLIGNGSSGIQILPAIAPQVAQLKTFIREATWVSPPAGEEFRTYSDEDKARFASDPDLHLQVRKHAEEVMNSQFSLFHRSSEAQNQIRAYMVSEMKRKINNAKLEKVLIPEWSVGCRRLTPGPNYLESLSRDNVEVAFGEITHVSEKGPVLDDGSEHPVEVLICATGFDTTFKPRFPLVGDSGKELASEWKDEPEGYLGIAVPGYQNYFMLLGPNCPAGNGPLLIAVEAQVDYLVKMLSRFQKENWSSFEVKVEPTKDFNEWKNEYMKLTIWTEECRSWYKSGSTSGNVVALWPGSTLHYLEAVKDVRWEDWDIKHQPGHNRWEFLGNGHSTAEARTRDLSYYIRQYDDEPVDPCLKATSQTEEAASAKSSGPVMFERPLETKL。

[0105] SEQ ID NO:35(FpBVMO7):

[0106] MALTVEQIPMAKKVESQQPDVAVKVRFPKDDLVHPKNSAISELKSESPITASGSSALQPPSSIEDILEQPLGTCDHVRVIIIGAGASGLNMIRSMRKHLEDFELTVYEKNPEVGGTWYENRYPGCKCDIPSHNYQFSWRPNPTWSAFFAGAPEIQKYLKTLTEEEQLQTSIKLGYQVVRADWNEGDGLWNVKVKNLATGEIIDDHCNFLLNASGILNNWKWPDIKGLHSYQGQLIHSANWPREYDFTGKTVAVIGNGSSGVQIVPAIQPEVGKLIHFIRSPTWIAPSHLERMSLSPKGSVLTEIELQGEAFSQQQIDQFTKDPAYYHEFVKAVEAYTNAKFKGTVNHLPEATMAKELMTAHMIERLGDDRRLIDFLIPDFPVGCRRITPGVGYLESLRKPNVQVVTDSIAEITPEGLLTSTGEEFKVDVIVCATGFDVSFCPRFPVIGRDGKNLQDLWKEQLPAAYMSCLIPEMPNYFVFLGPNAPIGHGSVLTITEHVAKFMIKIIRKCQTQGIKAISPKKDAVEEFLEHVQKFMPRTAWSGNCRSWFKNGRATGPVTALHPGSRIHWFHMLEDFRGEDFDYVRRTRNRYAYLGNGFSIREMEGGDQTWYLDHPDVLF。

[0107] SEQ ID NO:36(EnBVMO):

[0108] MTVHYVHEGPEPQESRYSIPQHTTWMDPNNRRLRVITIGAGFSGILMAYQIQKQCANIEHVVYEKNHDIGGTWLTNRYPNAGCDVPSHAYTYRFALYPDWPRYFSYASDIWEYLDKVCAAFKLRQYMQFRTEVIKACWNEEEGQWKVRLRRQRPGQEPEEFDDHCHILLNACGVLSNPKWPDTPGLHDRFKGRVIHTAAWPDDYGEVQWNSDRVAVIGSGASSIQAVAGIQPHVGHLDIFVRTGVWFGVLAGNTGAPTKIYSEAERAQFRSNPSALVEHTKSIEAEVNGMWGAFYRDSMAQKGASAFFRQRMASIIKDDRLAKGFTPTFGFGCRRITPGDPYMHAIQQANVDVHFTAVASCTEDGIVGADGIERLVDTIVCASGFDNTYRPQFPIIGRRGVDLRDKWKTNPEAYLGLAVPDMPNYITFIGPSWPIQNGSVMAPLHSVSEYAIQFLKKMQNENIRAWAPRQQITDRFNEHVQEWVKHTVWSDQCRSWYKNNETGRVNAIWPGSSLHYQAVIERPRYEDFEISYADANPWAHLGMGWTMLDRAGGKQADVSPHLCLENIDPVWFKSIGGDVDILRKQLEKGHTLPNNASHAEA。

[0109] SEQ ID NO:37(CiBVMO1):

[0110] MGSIDRYKFAQEPIHQPQRKRLICIGAGLAGIGAAYQYQQQLENVDFVIYEKNHDVGGTWLENRYPGCACDIPAHGYTYSWEGNPNWSRFYVSAEEIFDYFKGCAVKWNCMQYIKLSHRVIEARWSEPNNQWFLKIENSADGTIFEDQCDVLLSATGILNKWKWPDIPGLSSFKGKLLHSARWDQEYNFEGKTVAVVGAGSSAIQIVPSLQPVVKKLVNFIRSPTWITPEFSQSLAKDGRDTRFTPEEIERFNKDKSHFLQYRKLVQNTGSSSFSLYYKGSDLQEQSMTKFANLMRERLDYNEELCEKLIPKFAVGCRRFTPGNGYLESLIKPNVQVVLTGIEEITTNGIRAADRTEHAVDAIVCATGFDCSHRPAFPVVGRNGLDLAEYWKEQPRHYMSVAAPGFPNYFILGGPNSPIANGSLISGLETEISYAFACLRKMQTENIASMDVKEEAVDDFLEHRDSLMDAMVWSGGCRSWYKNGTVNGPVIGPWIGSTWHFNEALQTPRFEDYNMVYSARNRFAYLGNGRTIGELTGADMTAYMREPGA。

[0111] SEQ ID NO:38(PfBVMO1):

[0112] MAPSAITEPQVVINGEGKPLDNTVPATNGAVKEVKTDESVSSAIQYHYPPTIPEGTEYSVLKQYHSKPTKLRVACIGAGASGLCLAYKMERMMVPDSWELTLFDKNPQFGGTWYENTYPGVACDIPSPLYTFTWDPKPDWSHYFAYGDEIRRYFEDFAERHGSKQYMKLNTKVVEAQWDEEKGIWQLTLEDQVNKGTWKDWCHCLVNGTGILNNWKWPDIDGVHDFAGPKMHSANWDHSVDFQGKTVGVIGTGSTSVQIVPALQKEVQHLKVFMRSSTWISPPFGGGVLEEDLRKNDNGAAAQPGKRQYTFTDADKKKFSEDHEYYLTFRKRIEAEINSLFGMYQQGSEMSETFRKAITEEMHRRIGPGNEKLKEFIIPTWAPGCRRISPGDGYLEALVQPNVQPIYGGIQRVVPEGIVTEDGELHKVDILVCATGFNVAFRPAFKLINAAGNTLHEDWGDSVNLYMGVSTPRFPNYYTIVGPGATWSSGTLLPSIETTVEYSIKMMKKIQHENIRSLDVKQDALDDIYKHFDKFHETTVFQEQCRSWFKDGKIKNRIYLWPGCTIHFLKTIKEPRLEDYNIRYRYGNRFAYLGNGEVKANVTKDVKGLSTYIRDSDHEWSVE。

[0113] SEQ ID NO:39(PfBVMO2):

[0114] MFTIKDSPVENHRKLKIRVIGAGYSGIYLGIRIPQRLRNVDLRIYEKEDQIGGTWWVNKYPGCACDVPSHSYQYSFEPKPDWSSMYAPQHEICAYLQGVAEKFGVTRFVKQQHEVTACTWNATTKQWILDVKDVQSKTTFRDEADVVISARGFFTTPSWPDISGLRTFEGQIMHSASWDTSYDFGDKNIGVIGNGSSAIQIVPELEKIGGTKLSCFVRSKTWITNPFGDSAMLELGLDPTRPKFSDEQRNAFLNDPEKLLAFRKTLERHGNTVHEVAHRDSDLQKMAVTMSTAAMRERLSSKPEIADFLIPSFGVGCRRATPGPGYLEALGRPNVDFITDPIKEVNSKGILLKTGRQIDLDCIVCATGFNTSGVPQFQVHGSNGTTLAQRFSPNSEAYLSLAVDGFPNLFFMLGPNAGVGSGALTIIIESAGDYIIKCIRKLQKEDYSTMNVKKERVSDWVEHCQAYFKKTVYTDQCKSWYKGSGGDGSHIIGLWPGSTLHALEALRSPRWEDFEWESLRNSGNKMRWLGNGYSITHTKSSSDDGQYGGDPAWYIDPMFQDKPLPGRPEDDMRYKMRPFSH。

[0115] SEQ ID NO:40(AuBVMO):

[0116] MGSITPTSCQHARAGYRIRETPMGTRRPMKVIFMGMGAAGINFAHSVATQTHNVSLTIYEKSSDLGGTWLENRYPGCACDIPSVCYQFSWQRKPDWSKYYAGSREIFGYFKDVAVSNGLERFAKFNHQIVGAEWLDSISKWRITVVRDQDPTTAFDDYADFFLNGGGHLNTWKWPEIRGLDTFNGPRLHSANWDDAVELDGKRVLVIGAGSSAVQIVPNILPSVDHLHIVARSPTWITAGFAPKYAGAGGANFEYSETTKQQFRDDPELYLRYCKAIESELNVRFRLVVNDSPEALQARKFSEAQMKEKLAKKSDLIDKLMPKTFGIGCRRPTPGNGFLEALTEDKTTVWTEDIKAITQTGFVSADGQHHEVEIIVCATGFDTTFRPRFPLIANGRNVQDEPADAPAYLGLNLPEIPNYFMFSAPYGPLGHGSALPMIEAFTKYILHIISKAQVEDIRRIQVSRQAAEEFTRHADLYMKRTAWSGPCSSWFKAGQVGRKPKLWPGSRIHYLTVLQAPRYEDYEISYLTSNRFNYLGDGFEVREYDGRDLTWYYGLVDGQDVQPAEVPSPMY。

[0117] SEQ ID NO:41(TaBVMO1):

[0118] MGSLDHQMPPTQDDFASYEGYSIPDTPMGTARYLKIICIGAGASGLNLAFQIRKNMRNVDFVIFEKNEDVGGTWYENRYPGCACDIPAHNYQFSWAANPNWSEFYAKSSEILEYLRATSYKYDLRRYIRFQNRILDCTWDEESGMWNLHIQDETSGQVITESCHFLFNGGGYLNHWKWPSIAGLDTFGGDLVHTANWKEGIDLRGKRVGVIGNGSSGMQVLPAIYPDVESLVSFSRSPTWVTPTFASEFAGPNGANFGYSEEQKRQFSEDPEKFLEYRKALEKTMTGNFKLNLKDSEMQKETQQFLKDYMKGVLREDRLIKPLIPEFGVGCRRPTPGLGYLETLTKPNVRLVTDAISEIVPQGIKLETGEIIELDVIVCATGFDYSWIPRFPVTGRNNVTLSSLWKDRPTAYFGLAVAELPNYFVFLGPSSALSHGSAIPTIEAVTKYMLRIVWKAQTETYKAVVPSTEAVNDFILHNDTFHQRTIWGTKCRSWLKGGKEDGKVLTHPGSRVQNIHCLLNPRFEDWTWTPLAKNRFAFMGNGMTVLEEDGRDVTWYINDSNTGYEGIFY。

[0119] SEQ ID NO:42(TaBVMO2):

[0120] MTILDEQFYSELARVESSVPSNRLRWYLSAIACLAGLNYSEEIPPLYQVLLKSYIHKDRQFEETRIIREGLTKALGVIGAAKTGVALRALAQATPDILRDSKCYRENENPDIASRRGKDLVKSIYGKDPDTDPNPTRIAAADYDYIVLGSGYSGLMMAIIVKEKFREANLEFQVYEKNHDMGGTWLVNRYPGCQCDIPAHNYQYSFEPNPYWKNYYATSEQIHQYMKDTAKKYDCEEYFAYNHRVVKARWEENDKKWHLTVEASGSTFVDVCDIFINAGGVLDNWKWPDIEGIGSFKGKLMHSANWDQEYDFKGKKAGVIGIGSSGIQILPQVAKVADHTTLFARSETWITPDPGVSQPGVGDPEVDEAYNYTSKELNRFIEDPEYLLAHRKSLQNARIQGFKQFFLGSREAEESFLHFKKTMEERLGFSKKGRTIAEQLIPKFPVGCRRLTPGQGFLESLLQDNVILEWKNLDRIIDSGIITKDGRHIPCDVICCATGFDTSFKPVFPIIGRNGVNLATKWENEAPEAYFGITVSGFPNYFSFIGPNSPISNGSLVQAIQMTGIYIAKCISKLQTQSIRSMDISLDAQQEYNEHSQRFLEKTVWSAPCSSWYKQGMANGRITAIYGGSSYHFIQALKEPRWEDYSFEYVGSNRFTYLGNGMTKREVFGKAIGDTQTLDFESYWQLFNLPPILE。

[0121] SEQ ID NO:43(CiBVMO2):

[0122]

[0123] SEQ ID NO:44(CiBVMO3):

[0124] MVSELGYKVEPQWHSKPNYIRVICVGAGAAGLLVAYKMKKDFKNYEFICYEKLVLTFRCACDVPAHAYTYSFEPNPEWSSFYAYAPEIKQYFEKFADKYDLHPSIKLNSRVQSATWVEDKGIYEVEVDAGGRKINDWCHVIINGTGFLNDWKWPKIEGLHDFEGKLLHSANWDTSVDYTDKTVAVIGTGSSAIQIVPQVQKKAKHLITFMRSVTWISPPVGAQVLEEDKSHSSDSSQTQAPQAQYWYTEEDKKKFREDPEALLEYRKKLESSVNNLFDMFIAGSETSKWAEKLMREEMHRRIGPGHEELKERLIPKWAPGCRRITPGDGYLEALVKDNVTPIHNEIVKVVPEGLIDDAGRLHKVDILVCATGFNLAFAPPFKVLGVNGVSMADEFNPEPHVYLALTVPKFPNYFVVNGVRGNWASGTSLPSHEVQVEYILQCMKRMQEENIRALEVKMEPVKQLYEHIDEWHKGSVWNTECKSWYKNNIIGGKLWIWGGSALHYMKTIKFVRWEHYDFRYNNKNMWSFLGNGRVEAEVMKDTSRLAPYMRNEDSHYVERETWLQICFDRSLASKDLYMMKAARYHGQRDLRVEEVDVPQIGDGQVLVQVEWCGICGSDLHEYLVGPAVIPRKEAPHPLTGAVLPVTMGHEFCGRVSKVGPNSKLQIGQAVMVDPRVFCSSCHSCNIGDTNICNSWGFLGLQSNDGGGYSEYVAVKEDMCYVLPASVSLSEAALIEPLTVARHATKKSGFDDYRDKTVLVLGGGPVGLALIFVLKASGVKKLIVSEPTAKRQEQAAKFVDVVLNPKEVNVPEKCRELTDGRGVDIVFDCAGIMPGLKDGMDALRRGGTYVNVAGWERECIVPMGHFMLKEIIFRASMSYTEEDFKQTVDEFVAGKFKGFEKLVTARIALDDVVAKGFEELVNHKDDHVKILVTPKRELLAH。

[0125] SEQ ID NO:45(SmBVMO1):

[0126] MPPSTENGVEEVDHQAATEPWIYVPKPVKHVRVICIGAGCSGIYMSYAIPRKLKNVSLKVYEKNADLGGTWLENRYPGCACDVPSHGYSWSFALNPEWSRFYSQAEEIWKYQKSVAKRFDCEKYMTFNTKINEAVWDDKRGVWTLYGINEVTYDRVKDECEILISGMGALNTWRWPNIEGLDLFEGKLMHTALWDTKYSLEGKRVIVIGTGASSVQTVPTIQPIVSELILFQRHAGWIPPQADATGMAQGSNLEYTEEQKKYWRENPDKHKEFYENFNEGYETFFKTFMMNSPENVNWQRDVTNYYQTIIKDPSIRERLLPSYEIGCRRITPHTKYLQALQQKNVKLVTAGIKEITETGVRTVDGKLYEADVLVTATGFDTSFIPRVKIVGKNDISIQEAWMNVAEGYLGTAVCGFPNYYTILGPNTPIGQASLLPYMESQGDYIIQMISKMQDDAIKSFDPKKQVQKEFNEWTQYFLLRTVWQGGCASWYKTDTGANTAIWPGSSGHFRNFIVKPRFEDYEIVYDHKNRFEHLLGCGLTR。

[0127] SEQ ID NO:46(FmBVMO1):

[0128] MVSELGYKVEPQWHSKPNFIRVICVGAGAAGLLVAYKMKKNFKNYELVCYEKNSGIGGTWFENRYPGCACDVPAHAYTYSFEPNPEWSTFYAYAPEIKQYFEKFADKYDLHPNIKLNSKVLSATWVEDKGIYEVEVDVEGRKINDWCHVIINGTGFLNDWKWPKIEGLHDFKGKLLHSANWDSSVDYTDKTVAVIGTGSSAIQIVPQVQKKAKHLTTFMRSVTWISPPVGGQVLEEDKSHSSDSKQAQAPQAQYWYTEEDKKKFREDPQALLEYRRKLESSVNNLFDMFIAGSETSQWAEKLMREEMHRRIGPGHEELKEKLIPKWAPGCRRITPGDGYLEALVKDNVTPVHNEIVKVVPEGLIDDAGRLHKVDILVCATGFNLAFAPPFKVLGVNGVSMADEFNPEPQVYLALTVPKFPNYFVVNGVRGNWAAGTALPSHEVQVEYILQCMKRMQEENIRALEVKMEPVKQLYEHIDEWHKGSVWNSDCKR。

[0129] SEQ ID NO:47(SmBVMO2):

[0130] MAATEAKMLERGNLVASEAESLIERLSRQSISDVGSSKWTRQHQDLERLNIQAHYNVLNQNEEFITEALITFDKIPTLIHELLVINLWKTKVLPLIANSLPKEHAVKLYFVMYHEAILVNLLEIVMYSKSSCLAAGDTILDVIDYCAKKFALLSTWTDSSDPMEGKTSTQDMLQVSEEEHHVAIARDLDYAIATSTMSIFRYLTDYITDLSLTAMTRILNTNDMICSCVSVMERAPWLRKTQQHGMQRFEDGKWKNVTVEDVSVLGKVEAQMWIALYNMLLEPECRKKYEYTTSRQSKILRLRDFITESLVDQLPVLVGLQRALEELSIMQPPDDPSVHRRGAMIEQVSELYDEIIKGVDWKKTAHAYKKHAMQETDASRMETAKSLASMYDLSELDALLDDPKCAKCGNPAEKRCSRCRHEWYCSRACQVKSWAGHKAICDILYKEVLHANEDGDEGEGQQQQLVSWSGINVAVQLQKIGFTNFTLYEKNPHLGGTWFENRYPGCACDVPSHLYNFSYERNPDWSSTYSSWDEILQYTIKVATKYNVIPFIKVNHKVIGATWNESTGTYRVEIDNNGTIVTDTCNALILGTGALNSPRIPDVPGADTFPVTLHTGRWDPNVKLEGKRVGVIGNGASGIQLIPQIVPKVGNLDFFMRTPPWILDYAISGKYTDEEKRKFREEPGVLDKYQEGLWNDLMKAWPLFLTGSRMQNRIRQRSLAKLEAEVKDPVLRKKLTPNFAVGCRRLTMSATFLSAVQQPNVSVITDTIERVTPEGIVLKTGELRKLDVLIYATGFVTDFKPRFPIIGRGGKSIAEEFADDAEGYLGVVTKTFPNMFFMMGVNAPWSFGSAPPAIEAVTMYVVQTIQFMMKNRVKSVEVKESVQRKWNDDALSFFPTTVWSLACGNWYKSKNGRFTAIWPGTWEEFIKAMEAPNINDFNIERWPQPASARL。

[0131] SEQ ID NO:48(TsBVMO):

[0132] MAAESLSKGVQNDPCIIENRAVDDARPIKVRVMGAGISGIITCIRLMQRITNLDLSVWEKNDDIGGTWYENRYPGCACDIPSHTYQATFEPNLEWSHFYATSKEIHQYWKKVAQKYGAMKHIHVNHKVLAAHWNDKAAKWDLKVQVADGSVQEESCDVFISCAGSLNNWKWPAIPGLHDFEGKLLHTAAWDESYDYKNKRIAVIGNGSSGIQVVPAMLPDVTHIDHYARGRTWLSPTFARHKLDEIGGKNLDNIAFSPETIAGFKANRAAYHKFRKDIEHDLQNGFWVTIKDTPEQLAGSEFFKENMKRRLTKKPELLDQIVPDFPPGCRRLTPGPGYLEALTDEKVEVIKTEISSVDATGIRTADGVHREVDVIVCATGFDTSYLPRFPMTGRNGLSLAEKWKEIPETYISLATNEFPNYFICLGPNAALGHGSLTLLIEKEIDYITQCVAKIQRDNIRSMAPRKEAVERFTKHCEQHFSKTVFSTKCRSWYKGGKEDGRVIAVWPGSSLHALKTFSNPRWEDFEYEYINDNPNGWIGDGWTTAEKLRDFNVDYLDDNQIDFPTPFEVEIEIAVEAAIKRNEEEVQRGKEVGTEAAEFKEEDGQAMKNSVVGDGSKNSSAVHIETVATEVPV。

[0133] SEQ ID NO:49(FmBVMO2):

[0134] MAIEAATNGVHDTSKNGVVNGTKSTYQYSSHYMWKPRPIRIIVVGCGVSGIAAVKIFKERFPGKPVELVIYEKNAGVAGTWFENRYPGCSCDVPSHAYSFSWEGNPYWSRAYVDWKELLEYFTGRAKAYGVYDFVHLEHRVSKAVWNDSTGQWEVEIDNLKTGEKIVDKGEVLVNATGFLNNWKWPDIPGLQSFKGVLAHSAHYDTSLKLDDRVVGVIGSGSSAIQIVPQAQRGQSPTWITPELVAELAPQGRETVFTKEQQEAWASDPDGFLRHRKRAESTMNHFFDIQVKNSSLQKQAAEETRRQMIDQLSKKKELIPKLVPEFALGCRRITPGHGYLEALCSDNVTVQTEEIREVVPDGLILDDGTHIKLDVLICATGFNTSFCPPFTLVGEGGQVINEIWKDEPRSYLGFGAAGFPNYFITSGPNSPTANGALIPCFEFCLRFAYNAAEKLMTENIKSITPKQEAVDDFQEHKDSVMKDLVWTSSCRSWYKNGTIDGTVWGPWPGSAVHFLELITTPRWEDFNFKYRSSNRFEFLGRGRTARELEGGDLAWYLDQPGASDAARKEIVHVNDPIMASDPEESIGADGLYAVYDSDNHRVEDIAGIGAAVVRSLAARGADVCFNYTSESSITKSETLVAEIQAEYGTRAVCVRADLGQPDAGDKIVEAARRSLTPPEGGRFQIDIVVNNAGVCPADLLPDITTKSFYDSYAINVLGPILVMQAVLPYLPHDRSGRVINISSISATIGTLGHTVYGGTKAALDAMTRTWSRELAERATVNALNVGPVLTDMQPIPYVAEKVALMNTMTPLGPIRDTDTDEMKAIAKSFNGRVAYDYEIGDAVALHCMPEARWTTGMIIGVNGGFDFIR。

[0135] SEQ ID NO:50(GlBVMO):

[0136] MACAFRRLSHCGRVYARPIQPFRRGEYWKQWAHRFLSVSSTHPQQQPLDTGRSSDTSNPSLKRQLSSGPRNAVDSSLSSPTRTVYKIKEQPLGGNHKKLRVIIIGGGVSGLNMLIQLKKHISGVTPIIYEKNAETGGTWFENRYPGCASDDPSHSYQFSHTPNPKCNSLFSPAAEIQKYLIGVCEKHNLRDEIKCSHSVVRAEWEEESGMWVVDVKDEDKNRTFKDRCHFLMDASGIFNHYKWPDIKGISIFEGDLIHTAHWPADFKYESKAFAVIGNGASGVQIIPELQPGVGKLVHIIRGNSWIAPPSAAVEMCSALTEKGTKKYNNLQINKFSEDPTAYREFMKNIEAFSNQRFRMILSGSPDAEIAQERAESYMRSCLKSNPSLASSLIPSFPIGCRRITPGKGYLKSLLASNVTVISEPVQVSSRGIKLKGGGIINLDAIICATGFDCSFVPRCQLIREHGNLQDIWKTRTPEAYMSCMIEGVPNYFNFLGPNGPLAHGAIPLVAEQLAKYIIRHLQKFQLEHIHSAWPLPAAIDDYSEHIKGFMPRTSWSKPCGSWYKGATKDGPVFALHPGSQSHFFHMLEKPRWEDFEWRTKAGGGGNRFGYLGNGFSIREEEGEDTTWYLNDPHGS。

[0137] SEQ ID NO:51(CcLPPS):

[0138] MAFTFTSAHLFLPVTENHSVHVNYSIPPGNWRLWSTAKGGSNKLDIRRLRCSARRTPEPLAQGSNGGRDGVEAIQRLQTIADDKIDGGANELGIVVWDLIRDGVDAVKSMFDSMGDGDISISAYDTAWVALVKDVNGSGGPQFPSSLQWIVDNQLPDGSWGDSEVFSAYDRLLKTLACVVALKSWNIRPDKCQKGLKFFRDNISKLEKENVEASAQMLSGFEVVFLSLIEVARRLDIQIPLHSPVFEDLIARRNLKFAKIPLDLMHNVPTSLLNSLEGMTGVELDWEKLLKLQSQDGSFITSPSSTAFALMQTNDTKCLGYLKFVVQKFNGGAPGQYPVEIFERIWVVDRLQRLGISRYFQLEIKECCLDYAFKHWTQYGSSWARNTPVYDLDDTCMAFRILRLHGYDVSAEAFRHFEKNGVFFCFGWETTQSVTVNFNLYRATQVAFPGENILKEAKQFSFNFLMKKQAAREFQDKWVILKDFPGELKYALEFPWYASLPRVETRFYVEQYGGDNDVWIGKTLYRMPYINNNVYLELAKLDFNNCQALHRKEWETMQKWFMESKLDEFGVSSKTLLESYFLAAASIFEPERSTERLAWAKTAFLMETIGSYFDDEMNSKDLRKAFVQEFKNIYERRMEAKGTKWNLIIILLTTLNHLTEVCGRDINSYLCHSWEKWMMMWEPEGDRYKGAAELLSNSINLSSGRLFSNDTLSHPNYEKLVTLSNKLCHQLGNSRRGNHNEDSDIKDTKIEIAMQELVQLVHQNSSDDISMDLKQTFFAVVRSFYYAAHCDRGTINSHIVKVLFESVV。

[0139] SEQ ID NO:52(SCH94-3944):

[0140] MNLNEARTAFARLRAAENGLSPAELDEVWAALETVAAEEILGEWKGDDFATGHRLHEKLSASRWYGKTFNSVEDAKPLICRDEDGNLYSDVKSGNGEASLWNIEFRGEVTATMVYDGAPIFDHFKKVDDSTLMGIMNGKSALVLDGGQHYYFLLERA。

[0141] SEQ ID NO:53(HrBVMO):

[0142] MTTPTMTMPSDLGAATNDTQCRNPKIDDDFVRAALEEASAPALRLALLQVTGDQQLENMQVHKTPIRGGVLNDYTLSDTDAKLVKEKAYKYLAHLDEVQEVPKPVSKKRAFQLMDLYSDAPMYTTPNEPSFDYEEGYEELAFEDYPRDVNWTGAQPSPTELSKWKVMIVGAGISGIAAAIPLKRLGIPFEIVERQSGIGGTWLLNTYPDCRVDTLVYLFQYKFEKKYKWKDFFSSREDLQQYIEYVATKWGVKDNITYDREVVAATWDEKTKLWSMTLKHKNGNEEVKTCNAIISAAGLFSTPNLPDIAGIHDFKGPLFHTAQWDHSKDYHGKNVALIGTGSTGTQLTPMVAEGARHLSVYQRTPNWIASYEGYRAKVTDHMHWLCDAMPYYWNWYCYSAWFRSLQLANTQYHDPEWRAQGGLINKRNDFLRTSLTKFINEKFADRPDLIAKVTPKQAPMVRRLVVDNGFYDALKRDNVSLITDSIERITEKGIMTKDGNEIEYDMIVLGAGFKTSQYLWPVNYTGTDGMTLAKAWAKDGARSYLGMTMPNYPNLFTLYGPNHQPRGGSLYSYGEMWARYAVASIVGMIERGASSMEIKKDVFDKYQAALDKGNKRIIWESEGAAYYVNEHGRQAVNMPWTTAEYHPMIAKVNFDDYNLTYDDKKQNGVHKANGINGHQTNGHSHSHTHGHTNGHANGHKESWLSNH。

[0143] In a preferred embodiment, the engineered bacteria include yeast; preferably, the yeast includes Saccharomyces cerevisiae or Yersinia lipolytica.

[0144] Preferably, the brewing yeast used in this application is brewing yeast CEN.PK2-1C (Taisto Biotechnology Co., Ltd., TS989763), and Yersinia lipolytica is Yl590. The accession number of Yersinia lipolytica Yl590 is CGMCC NO.30855, the accession date is June 4, 2024, and the accession address is No. 3, Datun Road, Chaoyang District, Beijing. It has been disclosed in patent publication number CN118879517B. Those skilled in the art, under the guidance of the prior art (including but not limited to CN119859588A), can also flexibly select other strains in the prior art to achieve the biosynthesis of perillaldehyde. This biosynthesis is based on (E)-lysopran-13-en-8,15-diol, which includes, but is not limited to, exogenously added (E)-lysopran-13-en-8,15-diol, or (E)-lysopran-13-en-8,15-diol prepared by the strain itself through biosynthesis.

[0145] The *Yersinia lipolytica* Yl590 can spontaneously generate geranyl-geranyl pyrophosphate from a basic carbon source. Therefore, after introducing the aforementioned pyrophosphate lysenoside diol ester synthase into *Yersinia lipolytica* Yl590, geranyl-geranyl pyrophosphate can further synthesize (E)-lysenoside-13-en-8,15-diol. (E)-lysenoside-13-en-8,15-diol, under the action of the endogenous alcohol dehydrogenase ADH, generates 8-hydroxy-hemisperidin-13-en-15-aldehyde. Furthermore, by continuing to introduce the aforementioned perillaldehyde synthase and Bayer-Villiger monooxygenase into *Yersinia lipolytica* Yl590, the de novo synthesis of perillaldehyde diol can be achieved. However, it should be noted that the specific chassis strain used in this application is not necessarily the only choice among the aforementioned engineered bacteria. Those skilled in the art can flexibly select strains from the prior art as chassis cells and obtain engineered bacteria capable of producing perillaldehyde by introducing genes encoding the corresponding enzymes into the chassis cells.

[0146] In a preferred embodiment, the engineered bacteria also include those derived from *Rosa rockosa*. Cistus creticus The gene for lysine pyrophosphate diol ester synthase (LPPS); preferably, the lysine pyrophosphate diol ester synthase comprises: (c1) a protein having the sequence shown in SEQ ID NO: 51, or; (c2) a protein having more than 70% homology with the amino acid sequence defined in (c1) and having lysine pyrophosphate diol ester synthase activity.

[0147] The engineered bacteria described above also include genes capable of expressing LPPS. LPPS in the strain is used to synthesize (E)-lysantango-13-en-8,15-diol from geraniol-geraniol pyrophosphate as a precursor. It should be noted that the LPPS mentioned above includes enzymes known in the art capable of catalyzing the production of (E)-lysantango-13-en-8,15-diol from geraniol-geraniol pyrophosphate, and is not limited to the protein shown in SEQ ID NO: 51.

[0148] In a preferred embodiment, the engineered bacteria further includes a gene for an enzyme used to synthesize geraniol geraniol pyrophosphate (GGPP); preferably, the synthesis of geraniol geraniol pyrophosphate includes the engineered bacteria synthesizing geraniol geraniol pyrophosphate using glucose and / or pyruvate as precursors (also known as substrates).

[0149] When the engineered bacteria further incorporate genes for an enzyme used to synthesize geraniol-geraniol pyrophosphate, this feature serves to provide the upstream key substance required for the perillaldehyde biosynthesis pathway—geraniol-geraniol pyrophosphate. Geraniol-geraniol pyrophosphate is a direct precursor for the synthesis of (E)-lysanthan-13-en-8,15-diol. By expressing specific enzyme genes, the engineered bacteria can efficiently synthesize geraniol-geraniol pyrophosphate using inexpensive and readily available glucose or pyruvate as substrates, thereby promoting the biosynthesis of perillaldehyde. This design not only increases the self-sufficiency of the biosynthetic pathway but also avoids the need to add expensive precursor compounds, reducing production costs and improving the economic efficiency of the biosynthetic process.

[0150] In a preferred embodiment, the gene for the enzyme used to synthesize geraniol pyrophosphate includes any one or more of the following: BTS1 , ERG20 , IDI , MVD1 , ERG8 , ERG12 , HMG1 , ERG13 , ERG10 , PDH .

[0151] Optionally, the above ERG10 It can also be replaced with MvaE The above HMG1 It can also be replaced with MvaE The above ERG13 It can also be replaced with MvaS The above BTS1 It can also be replaced with CrtE .

[0152] In the prior art, the routes and related enzymes for the biosynthesis of gerany-gerany pyrophosphate are well understood, as disclosed in CN119859588A. Those skilled in the art can flexibly select the desired enzymes or combinations of enzymes based on the prior art, using inexpensive compounds such as glucose and pyruvate, or products that engineered bacteria can express themselves, as precursors to achieve the biosynthesis of gerany-gerany pyrophosphate, and even de novo synthesis (synthesis starting from a carbon source). This application provides an optional de novo route for the synthesis of gerany-gerany diol, as follows: Figure 1 As shown. Some of these enzymes are endogenous proteins from the chassis cells, eliminating the need for additional introduction of exogenous genes into the engineered bacteria.

[0153] In the aforementioned engineered bacteria, the applicant has established a biosynthetic route for perillaldehyde, enabling the biosynthesis of perillaldehyde in a single strain with a clear reaction route. This allows for the biosynthesis of perillaldehyde using common chassis cells (including but not limited to yeast) found in existing technologies, particularly for de novo synthesis.

[0154] In a second typical embodiment of this application, a method for preparing engineered bacteria is provided. The method includes: knocking in a gene into a starting engineered bacterium to obtain the engineered bacterium; the knocked-in gene includes a gene for perillaldehyde synthase and a gene for Bayer-Villiger monooxygenase, wherein the perillaldehyde synthase (SCOS) comprises: (a1) a protein having any sequence in SEQ IDNOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity; wherein the Bayer-Villiger monooxygenase (BVMO) comprises: (b1) a protein having any sequence in SEQ IDNOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0155] In a preferred embodiment, the knock-in gene further includes: a gene derived from rockrose. Cistus creticus Genes for lysine pyrophosphate diol ester synthase (LPPS) and / or genes for enzymes used to synthesize geraniol geraniol pyrophosphate.

[0156] In a preferred embodiment, the above-mentioned lysine pyrophosphate diol ester synthase comprises: (c1) a protein having the sequence shown in SEQ ID NO: 51, or (c2) a protein having more than 70% homology with the amino acid sequence defined in (c1) and having lysine pyrophosphate diol ester synthase activity.

[0157] In a preferred embodiment, the gene for the enzyme used to synthesize geraniol pyrophosphate includes any one or more of the following: BTS1 , ERG20 , IDI , MVD1 , ERG8 , ERG12 , HMG1 , ERG13 , ERG10 , PDH Optionally, the above ERG10 It can also be replaced with MvaE The above HMG1 It can also be replaced with MvaE The above ERG13 It can also be replaced with MvaS The above BTS1 It can also be replaced with CrtE The enzymes used to synthesize geraniol pyrophosphate can be found in the above description of the prior art.

[0158] Using the above preparation method, an engineered strain for the biosynthesis of perillaldehyde can be prepared. By further knocking in genes known in the prior art for the synthesis precursor, an engineered strain for the de novo synthesis of perillaldehyde can be obtained.

[0159] The engineered bacteria mentioned above include, but are not limited to, yeast; preferably, the yeast includes, but is not limited to, Saccharomyces cerevisiae or Yersinia lipolytica.

[0160] In the above preparation method, for the knock-in of genes in engineered bacteria, those skilled in the art can flexibly choose known gene editing or recombination methods from the prior art, all of which can achieve the corresponding effects. The aforementioned "knock-in" includes inserting a foreign gene into the genome of the chassis cell, and also includes having the foreign gene exist in the chassis cell in the form of a free plasmid.

[0161] In a third typical embodiment of this application, a method for synthesizing perillaldehyde is provided, the method comprising: using the engineered bacteria described above, or engineered bacteria prepared by the above method, to ferment and synthesize perillaldehyde in a fermentation culture medium.

[0162] This application provides a method for the biosynthesis of perillaldehyde. The core of this method lies in utilizing specific perillaldehyde synthase and Bayer-Villiger monooxygenase to catalyze the synthesis of perillaldehyde in engineered bacteria (including but not limited to engineered Saccharomyces cerevisiae or Yersinia lipolytica). This is achieved by overexpressing the perillaldehyde synthase gene. SCOT and Bayer-Villiger monooxygenase gene BVMO These engineered strains were able to efficiently synthesize sagebearing diol with the exogenous addition of (E)-lysantango-13-en-8,15-diol. This process not only avoids the environmental pollution caused by traditional chemical synthesis, but also improves the synthesis efficiency and product purity.

[0163] Furthermore, by expressing lysparganide pyrophosphate synthase in engineered bacteria, (E)-lysparganide-13-en-8,15-diol can be synthesized using geraniol-geraniol pyrophosphate as a substrate without the exogenous addition of (E)-lysparganide-13-en-8,15-diol. Furthermore, the above method can be further improved by introducing an enzyme for synthesizing geraniol-geraniol pyrophosphate, enabling the direct synthesis of geraniol from simple carbon sources such as glucose and pyruvate. This greatly simplifies the fermentation process and reduces production costs. Therefore, this invention provides a simple and environmentally friendly biosynthetic route for geraniol, laying the foundation for the microbial fermentation production of geraniol and showing promising industrial application prospects.

[0164] In a preferred embodiment, the fermentation includes shake flask fermentation or fermenter fermentation.

[0165] In a preferred embodiment, the shake-flask fermentation includes: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium for shake-flask fermentation for expansion culture to obtain the above-mentioned perillaldehyde diol.

[0166] In a preferred embodiment, the fermentation in the fermenter includes: inoculating the genetically engineered bacteria cultured overnight into the fermenter for scale-up culture to obtain the above-mentioned perillaldehyde diol.

[0167] The fermentation process described above includes, but is not limited to, shake flask fermentation or fermenter fermentation. Those skilled in the art can flexibly choose commonly used fermentation methods and culture media in the prior art to achieve the fermentation of perillaldehyde. Choosing between shake flask fermentation and fermenter fermentation allows for flexible adjustment of the fermentation method according to different production scales. Shake flask fermentation is suitable for small-scale synthesis in the laboratory, facilitating optimization of conditions and preliminary screening of highly efficient expression strains; while fermenter fermentation is more suitable for large-scale industrial production, enabling automated process control, increasing yield, and reducing production costs. This preferred fermentation method allows engineered bacteria to efficiently express the target gene under suitable conditions, thereby promoting the biosynthesis of perillaldehyde. This not only simplifies the production process but also improves synthesis efficiency, providing strong support for the industrial production of perillaldehyde.

[0168] Optionally, the fermentation method includes: inoculating recombinant yeast (engineered bacteria) into YPD liquid medium, culturing overnight at 25-30℃ and 180-220 rpm to obtain seed liquid, and inoculating the above seed liquid into fresh fermentation medium at an inoculation rate of 10%-20%, culturing at 25-30℃ and 180-220 rpm for 96-236 h to produce perillyl glycol.

[0169] Optionally, the YPD medium consists of 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. The fermentation medium also includes 2.5-5 g / L (NH4)2SO4, 15-20 g / L KH2PO4, and 0.5-2 g / L MgSO4. 7H2O, 30 g / L glucose, 10 g / L yeast powder, 10 mL / L trace element stock solution, 12 mL / L vitamin stock solution, 1.2 mL / L biotin, and add precursor (E)-lysanthan-13-ene-8,15-diol as needed, pH 3-6.

[0170] The processing method for perillaldehyde diol samples was as follows: 5 mL of fermentation broth was taken, an equal volume of ethyl acetate was added, and the perillaldehyde diol was extracted by shaking for 5 min. The mixture was then centrifuged at 12000 rpm for 2 min, and the upper organic phase was collected for gas chromatography analysis.

[0171] In a fourth typical embodiment of this application, a method for catalyzing the production of perillaldehyde from 8-hydroxy-hemisin-13-en-15-aldehyde is provided. The method includes using perillaldehyde synthase to catalyze the production of perillaldehyde from 8-hydroxy-hemisin-13-en-15-aldehyde to obtain perillaldehyde. The perillaldehyde synthase includes: (a1) a protein having any of the sequences in SEQ ID NOs: 1-25, or; (a2) a protein having more than 70% homology with the amino acid sequence defined in (a1) and having perillaldehyde synthase activity.

[0172] In a preferred embodiment, the method includes: using yeast cells capable of expressing the above-mentioned perillaldehyde synthase to catalyze the above-mentioned 8-hydroxy-heliotropin-13-en-15-aldehyde to obtain the above-mentioned perillaldehyde.

[0173] In a preferred embodiment, the yeast includes, but is not limited to, Saccharomyces cerevisiae or Yersinia lipolytica.

[0174] In a fifth typical embodiment of this application, a method for catalyzing the formation of perillaldehyde from perillaldehyde diacetate is provided. The method includes using a Bayer-Villiger monooxygenase to catalyze the perillaldehyde to obtain the perillaldehyde diacetate. The Bayer-Villiger monooxygenase includes: (b1) a protein having any of the sequences in SEQ ID NOs: 26-50, or; (b2) a protein having more than 70% homology with the amino acid sequence defined in (b1) and having Bayer-Villiger monooxygenase activity.

[0175] In a preferred embodiment, the method includes: using yeast cells capable of expressing the above-mentioned Bayer-Villiger monooxygenase to catalyze the above-mentioned perillaldehyde to obtain the above-mentioned perillaldehyde diol acetate.

[0176] In a preferred embodiment, the yeast includes, but is not limited to, Saccharomyces cerevisiae or Yersinia lipolytica.

[0177] In a preferred embodiment, the perillone in the above method is the perillone obtained by the above-described method of catalyzing the production of perillone from 8-hydroxy-heliotropin-13-ene-15-aldehyde.

[0178] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0179] Example 1: Modification of strain Yl590

[0180] The wild-type Yl590 strain was modified, and the gene was obtained by PCR using the Yersinia lipolyticis genome as a template and URA-Up-F / R and URA-Down-F / R primers. URA3 The upstream and downstream homologous arm fragments were obtained, and the two fragments were fused by PCR to obtain the URA3-Up+Down fragment. The fusion fragment was transformed into strain Yl590 using the lithium acetate (LiAC) method. The revived bacterial culture was plated on 5-FOA plates and incubated at 30°C for 2 days. After that, single colonies were picked and spotted onto SD-URA and YPD solid plates for preliminary screening. The obtained mutant strains were then verified by colony PCR, and the mutant strains were successfully obtained. URA3 Defective strain Yl590ΔURA3. URA3 The primers used for gene knockout are shown in Table 1.

[0181] Table 1

[0182]

[0183] Example 2: Synthesis of 8-hydroxy-hemerocallis-13-en-15-aldehyde using (E)-lysanthin-13-en-8,15-diol as a precursor

[0184] Yersinia lipolytica Yl590, Yersinia lipolytica PO1f (Taisto Biotechnology, TS153400), Saccharomyces cerevisiae CEN.PK2-1C (Taisto Biotechnology, TS989763), Saccharomyces cerevisiae BY4742 (Miaoling Biotechnology, T0081), and Saccharomyces cerevisiae W303a (Miaoling Biotechnology, T0126) were inoculated into YPD medium and cultured overnight to obtain seed culture. The seed culture was then inoculated at a 10% inoculation rate into 50 mL of fermentation medium containing 1 g / L (E)-lysanthan-13-ene-8,15-diol and fermented at 30℃ and 220 rpm for 144 h.

[0185] An equal volume of ethyl acetate was added to the fermentation broth for complete extraction. The upper organic phase was collected by centrifugation and analyzed. The formation of two new substances was observed in both cases (experimental results are shown in the figure). Figure 2 As shown in the figure, this is because yeast has a widespread endogenous presence of alcohol dehydrogenase (ADH). The 8-hydroxy-hemisin-13-en-15-aldehyde synthesized by alcohol dehydrogenase (ADH) from (E)-lysenoside-13-en-8,15-diol is unstable. After dehydration and rearrangement, it synthesizes two isomers of (+)-8,13-epoxy-hemisin-15-aldehyde, which is consistent with the report in the prior art CN114630905A.

[0186] Example 3: Synthesis of perillone using (E)-lysanthan-13-ene-8,15-diol as a precursor

[0187] In Example 2, Yersinia lipolytica Yl590 and Saccharomyces cerevisiae CEN.PK2-1C were catalyzed by the addition of exogenous (E)-lysandon-13-en-8,15-diol to 8-hydroxy-hemisandro-13-en-15-aldehyde under the action of endogenous alcohol dehydrogenase (ADH) in yeast. In Example 3, 8-hydroxy-hemisandro-13-en-15-aldehyde was further used to synthesize perillaldehyde.

[0188] Homologous recombination technology was used to introduce 25 different strains of yeast from different sources into Yersinia lipolyticis Yl590ΔURA3. SCOT The genes and SCH94-3944 (CN 114630905 A, SEQ ID NO: 52) were used to construct strains SGY01-SGY26;

[0189] Twenty-five different sources were introduced into Saccharomyces cerevisiae CEN.PK2-1C. SCOT Using the gene and SCH94-3944 (CN114630905 A, SEQ ID NO: 52), the SGS01-SGS26 strains were constructed. The construction information of the recombinant strains is shown in Table 2.

[0190] Each of the following strains was inoculated into YPD medium and cultured overnight to obtain seed culture: Yersinia lipolytica Yl590, Saccharomyces cerevisiae CEN.PK2-1C, recombinant Yersinia lipolytica SGY01-SGY26, and recombinant Saccharomyces cerevisiae strains SGS01-SGS26. The seed culture was then inoculated at a 10% inoculation rate into 50 mL of fermentation medium containing 1 g / L (E)-lysanthan-13-ene-8,15-diol and fermented at 30°C and 220 rpm for 144 h.

[0191] An equal volume of ethyl acetate was added to the fermentation broth for complete extraction. The supernatant organic phase was collected by centrifugation and analyzed by gas chromatography, yielding results consistent with existing technology CN114630905A. When perilla ketone synthase (…) was introduced into the yeast… SCOT Following this, in addition to the formation of two isomers of (+)-8,13-epoxy-hemisin-15-aldehyde, the formation of perillaldehyde oxide was also observed. The experimental results are as follows: Figure 3 As shown. This is because perillone is unstable and will be converted into perillyl oxide under mild conditions (Barrero et al., Tetrahedron 49, (45) 1993, 10405-10412; Hua et al., Tetrahedron 67 (6) 2011, 1142-1144) (CN 114630905 A). Among them, SGY01-SGY26 and SGS01-SGS26 synthesized different amounts of perillyl oxide. The amount of perillyl oxide synthesized by recombinant Yersinia lipolytica was better than that of recombinant Saccharomyces cerevisiae, and 25 different sources SCOT The catalytic effects of the gene were superior to those of SCH94-3944 in CN114630905A. No perillaldehyde oxide was detected in the control yeast Yl590 and Saccharomyces cerevisiae CEN.PK2-1C. The experimental results are shown in Table 2.

[0192] Table 2 Recombinant Yeast SCOT Genetic information and fermentation results

[0193]

[0194] Note: In Table 2, - represents no synthesized product; + represents a synthesis amount of 10-20 mg / L of perillyl oxide (excluding the endpoint value of 20 mg / L); ++ represents a synthesis amount of 20-100 mg / L of perillyl oxide (excluding the endpoint value of 100 mg / L); +++ represents a synthesis amount of 100-300 mg / L of perillyl oxide (excluding the endpoint value of 300 mg / L); ++++ represents a synthesis amount of 300-500 mg / L of perillyl oxide (excluding the endpoint value of 500 mg / L); +++++ represents a synthesis amount of 500 mg / L or more of perillyl oxide.

[0195] Example 4: Synthesis of sagediol from (E)-lysanthan-13-en-8,15-diol as a precursor

[0196] In Example 3, perillone was synthesized by converting (E)-lysantho-13-ene-8,15-diol using SGY05 and SGS05. Perillone is unstable and is converted into perillyl alcohol oxide. In this Example 3, perillone was further used to synthesize perillyl diol acetate.

[0197] Homologous recombination technology was used to introduce 25 different strains of yeast from different sources into Yersinia lipolytica SGY05. BVMO Genes and HrBVMO(CN114921428A, SEQ ID NO: 53), the SGY27-SGY52 strain was constructed;

[0198] Twenty-five different sources were introduced into Saccharomyces cerevisiae SGS05. BVMO Genes and HrBVMO (CN114921428A, SEQ ID NO: 53) The SGS27-SGS52 strain was constructed, and the construction information of the recombinant strain is shown in Table 3.

[0199] Each of the following strains was inoculated into YPD medium and cultured overnight to obtain seed culture: Yersinia lipolytica SGY05, Saccharomyces cerevisiae SGS05, recombinant Yersinia lipolytica SGY27-SGY52, and recombinant Saccharomyces cerevisiae SGS27-SGS52. The seed culture was then inoculated at a 10% inoculation rate into 50 mL of fermentation medium containing 1 g / L (E)-lysanthan-13-ene-8,15-diol and fermented at 30°C and 220 rpm for 144 h.

[0200] An equal volume of ethyl acetate was added to the fermentation broth for complete extraction. The supernatant organic phase was collected by centrifugation and analyzed. BVMO Subsequently, a new peak of perillaldehyde glycol acetate (CN114921428A, 2022) was detected, along with perillaldehyde glycol. The test results are as follows. Figure 4 As shown in the figure. Perillaldehyde was extracted from the fermentation broth and identified by NMR. The results confirmed the successful synthesis of perillaldehyde, as shown in the figure. Figure 5A and Figure 5B As shown. Both SGY27-SGY52 and SGS27-SGS52 synthesized different amounts of perillaldehyde acetate and perillaldehyde diol, and from 25 different sources... BVMO The catalytic effects of the genes are superior to those of existing technologies. HrBVMO (CN114921428A) The recombinant *Yersinia lipolytica* synthesized more perillaldehyde than the recombinant *Saccharomyces cerevisiae*. Acetate is unstable in yeast cells and is hydrolyzed into alcohol by intracellular esterases. Perillaldehyde was not detected in *Yersinia lipolytica* SGY05 and *Saccharomyces cerevisiae* SGS05. The experimental results are shown in Tables 3 and 4.

[0201] Table 3 Recombinant Lipolytic Yeast BVMO Genetic information and fermentation results

[0202]

[0203] Note: In Table 3, - represents no synthesized product; + represents a synthesis amount of 50-100 mg / L of perillyl glycol acetate (excluding the endpoint value of 100 mg / L); ++ represents a synthesis amount of 100-300 mg / L of perillyl glycol acetate (excluding the endpoint value of 300 mg / L); +++ represents a synthesis amount of 300 mg / L or more of perillyl glycol acetate. The representative amount of styracidium is 5-100 mg / L (excluding the endpoint value of 100 mg / L). The representative amount of styracidium is 100-200 mg / L (excluding the endpoint value of 200 mg / L). The amount of styracidium synthesized is above 200 mg / L.

[0204] Table 4 Recombinant Saccharomyces cerevisiae BVMO Genetic information and fermentation results

[0205]

[0206] Note: In Table 4, - represents no synthesized product; + represents a synthesis amount of 50-100 mg / L of perillyl glycol acetate (excluding the endpoint value of 100 mg / L); ++ represents a synthesis amount of 100-300 mg / L of perillyl glycol acetate (excluding the endpoint value of 300 mg / L); +++ represents a synthesis amount of 300 mg / L or more of perillyl glycol acetate. The representative amount of styracidium is 5-100 mg / L (excluding the endpoint value of 100 mg / L). The representative amount of styracidium is 100-200 mg / L (excluding the endpoint value of 200 mg / L). This indicates that the amount of styracidium synthesized is 200 mg / L or higher.

[0207] Example 5: Synthesis of (E)-Lysanthan-13-ene-8,15-diol by modification of strain Yl590

[0208] Homologous recombination technology was used to introduce [a novel gene] into Yersinia lipolyticis Yl590ΔURA3. Cistus creticus Source CCLPPS Genes were used to construct strain SG01, which successfully synthesized 2.55 mg / L (E)-lysantango-13-en-8,15-diol de novo. (E)-lysantango-13-en-8,15-diol was not detected in the control strain Yl590. This indicates... CCLPPSThe introduction of the gene can effectively synthesize the precursor lysine pyrophosphate (GGPP) produced in vivo into lysine pyrophosphate diol ester (8OH-CPP), which can spontaneously convert into (E)-lysine-13-en-8,15-diol and the byproduct sagerol (CN119859588B).

[0209] The fermentation method for the above-mentioned recombinant Yersinia lipolytica strain and the detection method for (E)-lysanthan-13-ene-8,15-diol are as follows:

[0210] Yersinia lipolyticis Yl590ΔURA3 and recombinant Yersinia lipolyticis strain SG01 were inoculated into YPD medium and cultured overnight to obtain seed culture. The seed culture was then inoculated into 50 mL of fermentation medium at a 10% inoculation rate. Fermentation was carried out at 30℃ and 220 rpm for 144 h. An equal volume of ethyl acetate was added to the fermentation broth for complete extraction, and the upper organic phase was collected by centrifugation for gas chromatography analysis.

[0211] Example 6: Synthesis of Perilla frutescens ketone by modifying strain Yl590

[0212] Introduced into strain Yl590ΔURA3 Cistus creticus Source CCLPPS The gene (encoding the enzyme shown in SEQ ID NO: 51) was used to successfully construct strain SG01. Strain SG01 successfully synthesized 2.55 mg / L (E)-lysantango-13-en-8,15-diol de novo. (E)-lysantango-13-en-8,15-diol was not detected in the control strain Yl590, indicating that... CCLPPS The introduction of the gene can effectively synthesize the precursor lysine pyrophosphate (GGPP) produced in vivo into lysine pyrophosphate diol ester (8OH-CPP), which can spontaneously convert into (E)-lysine-13-en-8,15-diol and the byproduct styrosine.

[0213] Twenty-five different sources were introduced into strain SG01. SCOT Genes were successfully used to construct the SG02-SG26 strain. The construction information of the recombinant strain is shown in Table 5.

[0214] Each of the recombinant *Yarrowia lipolytica* strains SG01-SG26 was inoculated into YPD medium and cultured overnight to obtain seed culture. The seed culture was then inoculated at a 10% inoculation rate into 50 mL of fermentation medium and fermented at 30°C and 220 rpm for 144 h. An equal volume of ethyl acetate was added to the fermentation broth for complete extraction, and the supernatant organic phase was collected by centrifugation for gas chromatography analysis. The recombinant strains synthesized different amounts of perillaldehyde oxide. No perillaldehyde oxide was detected in SG01, while trace amounts were detected in SG11, SG14, and SG25. The results are shown in Table 5.

[0215] Table 5 Introduction of Recombinant Yeast SG01 SCOT Genetic information and fermentation results

[0216]

[0217] Note: In Table 5, + indicates that the amount of perillaldehyde oxide synthesized is 0.1-0.5 mg / L (excluding the endpoint value of 0.5 mg / L); ++ indicates that the amount of perillaldehyde oxide synthesized is 0.5-1 mg / L (excluding the endpoint value of 1 mg / L); +++ indicates that the amount of perillaldehyde oxide synthesized is 1 mg / L or more.

[0218] Example 7: Synthesis of Perillaldehyde by Modifying Strawberry Strain Yl590

[0219] Introduced into strain Yl590ΔURA3 Cistus creticus Source CCLPPS The gene (encoding the enzyme shown in SEQ ID NO: 51) was used to successfully construct strain SG01, which successfully synthesized 2.55 mg / L (E)-lysanthan-13-ene-8,15-diol de novo.

[0220] Introduced in SG01 Xenorhabdus cabanillasii Source XcSCOS The gene (encoding the enzyme shown in SEQ ID NO: 5) was used to successfully construct strain SG06;

[0221] Introduced in SG01 Rhodococcus sp. CUA-806 source RhCSCOS The gene (encoding the enzyme shown in SEQ ID NO: 6) was used to successfully construct strain SG07;

[0222] Introduced in SG01 Sungouiella intermedia Source SinSCOS The gene (encoding the enzyme shown in SEQ ID NO: 14) was successfully used to construct SG15;

[0223] Introduced in SG01 Mycobacterium xenopis Source of 4042 MxSCOS The gene (encoding the enzyme shown in SEQ ID NO: 16) was used to successfully construct strain SG17.

[0224] Introduced in SG01 Nocardioides sp. Source WE KNOW The gene (encoding the enzyme shown in SEQ ID NO: 20) was used to successfully construct strain SG21.

[0225] To further synthesize perillaldehyde, styrax diol was introduced into strain SG06. Fonsecaea pedrosoi Source FpBVMO1 Gene (encoding the enzyme shown in SEQ ID NO: 26) Aspergillus campestris Source AcBVMO Gene (encoding the enzyme shown in SEQ ID NO: 29) and Sporothrix schenckii Source SsBVMO The gene (encoding the enzyme shown in SEQ ID NO: 33) led to the successful construction of strains SG27, SG28, and SG29.

[0226] Introduced into SG07 strain respectively Fonsecaea pedrosoi Source FpBVMO1 Gene, Aspergillus plain Source AcBVMO Genes and Sporothrix schenckii Source SsBVMO The genes were used to successfully construct the corresponding SG30, SG31 and SG32 strains;

[0227] Introduced into SG15 strain Fonsecaea pedrosoi Source FpBVMO1 Gene, Aspergillus plain Source AcBVMO Genes and Sporothrix schenckii Source SsBVMO The genes were used to successfully construct SG33, SG34 and SG35 strains;

[0228] Introduced into SG17 strain Fonsecaea pedrosoi Source FpBVMO1 Gene, Aspergillus plain Source AcBVMO Genes and Sporothrix schenckii Source SsBVMO The genes were used to successfully construct SG36, SG37, and SG38 strains.

[0229] Introduced into SG21 strain Fonsecaea pedrosoi Source FpBVMO1 Gene, Aspergillus plain Source AcBVMOGenes and Sporothrix schenckii Source SsBVMO The genes were used to successfully construct SG39, SG40, and SG41 strains.

[0230] exist FpBVMO1 Gene or AcBVMO Gene or SsBVMO Under the influence of genes, *Yarrowia lipolytica* can convert perillone into perillyl acetate and perillyl glycol. Strains SG27, SG28, SG29, SG30, SG31, SG32, SG33, SG34, SG35, SG36, SG37, SG38, SG39, SG40, and SG41 all synthesized varying amounts of perillyl glycol, while control strains SG06, SG07, SG15, SG17, and SG21 did not synthesize perillyl glycol. The experimental results are shown in Table 6.

[0231] Table 6. Genotypes and fermentation results of recombinant yeast synthesizing perillaldehyde.

[0232]

[0233] Note: In Table 6, + indicates that the amount of styracidium synthesized is 0.1-0.15 mg / L (excluding the endpoint value of 0.15 mg / L); ++ indicates that the amount of styracidium synthesized is 0.15-0.3 mg / L (excluding the endpoint value of 0.3 mg / L); +++ indicates that the amount of styracidium synthesized is 0.3 mg / L or more.

[0234] The fermentation method for the above-mentioned recombinant Yersinia lipolytica strain and the detection methods for perillaldehyde and perillyldiol are as follows:

[0235] Recombinant *Yarrowia lipolyticis* strains SG27, SG28, SG29, SG30, SG31, SG32, SG33, SG34, SG35, SG36, SG37, SG38, SG39, SG40, SG41, SG06, SG07, SG15, SG17, and SG21 were inoculated into YPD medium and cultured overnight to obtain seed culture. The seed culture was then inoculated into 50 mL of fermentation medium at a 10% inoculation rate. Fermentation was carried out at 30°C and 220 rpm for 144 h. An equal volume of ethyl acetate was added to the fermentation broth for complete extraction, and the supernatant organic phase was collected by centrifugation for gas chromatography analysis.

[0236] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present invention, by screening and expressing genes related to the biosynthesis pathway of perillaldehyde and integrating them into model strains *Saccharomyces cerevisiae* and *Yarrowia lipolytica*, achieves for the first time the biosynthesis of perillaldehyde using model strains, especially using a single strain, laying the foundation for subsequent rational modification to obtain high-yield perillaldehyde strains. Simultaneously, an engineered strain for de novo synthesis of perillaldehyde was constructed in *Yarrowia lipolytica*, eliminating the need for any precursors or organic reagents during fermentation, simplifying the fermentation process and reducing production costs. The engineered strain provided by the present invention has a simple fermentation process, good application prospects, and lays the foundation for the microbial fermentation production of perillaldehyde.

[0237] The engineered strains provided by this invention can synthesize perillaldehyde in a highly efficient and environmentally friendly manner. Compared with traditional chemical synthesis, this greatly reduces the impact on the environment, improves the separation efficiency and purity of the product, and directly utilizes simple and inexpensive carbon sources such as glucose through microbial fermentation, which greatly reduces production costs and simplifies the production process. This demonstrates the feasibility and superiority of microbial fermentation in the large-scale production of perillaldehyde.

[0238] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An engineered bacterium, characterized in that, The engineered bacteria include the genes for perilla ketone synthase and Bayer-Villiger monooxygenase. The perilla ketone synthase is a protein with any of the sequences shown in SEQ ID NOs: 1-25; The Bayer-Villiger monooxygenase is a protein with any of the sequences shown in SEQ ID NOs: 26-50; The engineered bacteria are yeast, specifically Saccharomyces cerevisiae or Yersinia lipolytica.

2. The engineered bacteria according to claim 1, characterized in that, The engineered bacteria also include those derived from rockrose ( Cistus creticus The gene for lysine pyrophosphate diol synthase.

3. The engineered bacterium of claim 2, wherein, The lysine pyrophosphate synthase is a protein with the sequence shown in SEQ ID NO:

51.

4. The engineered bacterium of any one of claims 1-3, wherein, The engineered bacteria also include genes for an enzyme used to synthesize geraniol pyrophosphate.

5. The engineered bacterium of claim 4, wherein, The synthetic gerany-gerany-pyrophosphate comprises the engineered bacteria synthesizing gerany-gerany-pyrophosphate using glucose and / or pyruvate as precursors.

6. A method for preparing engineered bacteria, characterized in that, The preparation method includes: knocking in a gene into *Saccharomyces cerevisiae* or *Yarrowia lipolytica* to obtain the engineered strain; The knock-in genes include the genes for perilla ketone synthase and Bayer-Villiger monooxygenase. The perilla ketone synthase is a protein with any of the sequences shown in SEQ ID NOs: 1-25; The Bayer-Villiger monooxygenase is a protein with any of the sequences shown in SEQ ID NOs: 26-50.

7. The preparation method according to claim 6, characterized in that, The knock-in gene also includes: Derived from rock rose ( Cistus creticus The gene for lysine pyrophosphate diol ester synthase, and / or Genes for the enzyme used to synthesize geraniol pyrophosphate.

8. A method for synthesizing perillaldehyde, characterized in that, The method comprises: using the engineered bacteria of any one of claims 1-5 or the engineered bacteria prepared by the method of any one of claims 6-7 to ferment and synthesize the sagediol in a fermentation medium.

9. The method according to claim 8, characterized in that, The fermentation includes shake flask fermentation or fermenter fermentation.

10. A method for catalytically generating perillaldehyde from 8-hydroxy-hemisens-13-en-15-aldehyde, characterized in that, The method includes using perillaldehyde synthase to catalyze the 8-hydroxy-heliotropin-13-en-15-aldehyde to obtain perillaldehyde; The perilla ketone synthase is a protein with any of the sequences shown in SEQ ID NOs: 1-25.

11. The method according to claim 10, characterized in that, The method includes: using yeast cells capable of expressing the perillaldehyde synthase to catalyze the 8-hydroxy-heliotropin-13-ene-15-aldehyde to obtain the perillaldehyde.

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