Recombinant yeast for producing bakuchiol, construction method and application thereof

Abstract

The application provides a recombinant yeast for producing bakuchiol, a construction method and application thereof. The recombinant yeast is Yarrowia lipolytica containing isopentenyl transferase, and a gene coding the isopentenyl transferase is derived from Psoralea corylifolia Linn. The recombinant yeast can be used to produce bakuchiol in a large yield through a biosynthetic method, and provides technical support for industrialization of the bakuchiol.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosynthesis, in particular to a recombinant yeast for producing bakuchiol, a construction method and application thereof. BACKGROUND

[0002] Bakuchiol is a kind of heteroterpene compound extracted from Psoralea corylifolia Linn. plant. It has attracted widespread attention due to its anti-wrinkle, antioxidant, anti-inflammatory and other physiological functions. In addition, bakuchiol not only has similar functions to retinol, but also has lower irritation and higher light stability than retinol, and is considered as the best substitute for retinol, which has attracted widespread attention in the field of cosmetics. The brands of cosmetics containing bakuchiol have also increased from 30 brands to about 150 brands in 2020. According to statistics, the global anti-aging market is expected to reach 191.7 billion US dollars in 2021, and the annual growth rate in the Chinese market is higher than that in the world, which further accelerates the demand for bakuchiol.

[0003] At present, the production methods of bakuchiol mainly include direct extraction from plants and chemical synthesis. Bakuchiol has a low content in natural plants, and obtaining bakuchiol from plants requires a large amount of Psoralea corylifolia Linn. plants. Excessive excavation will destroy the ecological environment and cause serious shortage of plant resources. Chemical synthesis of bakuchiol has the problems of complex synthesis route, low yield, large solvent consumption, and uneconomic and environmental protection.

[0004] With the development of synthetic biology and metabolic engineering, the fermentation of economic and renewable carbon sources (glucose, glycerol, sucrose) into high-value compounds by microorganisms has attracted more and more attention due to its low cost, high efficiency and environmental friendliness. However, the technology for biosynthesis of bakuchiol is not mature, therefore, how to find an efficient method for biosynthesis of bakuchiol is of great significance for the commercialization of bakuchiol. SUMMARY

[0005] The main purpose of the present application is to provide a recombinant yeast for producing bakuchiol, a construction method and application thereof, so as to solve the problem of low synthesis efficiency of bakuchiol in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present application, a recombinant yeast for producing bakuchiol is provided, and the recombinant yeast is Yarrowia lipolytica or Saccharomyces cerevisiae containing isopentenyltransferase; wherein the gene encoding isopentenyltransferase is derived from Psoralea corylifolia Linn.

[0007] Further, the amino acid sequence of the prenyltransferase tDN28194 is shown as SEQ ID NO: 1.

[0008] Further, the recombinant yeast further comprises a tyrosine ammonia-lyase, which is derived from Flavobacterium johnsoniae.

[0009] Further, the recombinant yeast further comprises any one or more of the following enzymes: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase or chorismate mutase.

[0010] Further, the recombinant yeast further comprises any one or more of the following enzymes: methylmalonyl-CoA synthase, methylmalonyl-CoA reductase, farnesyl pyrophosphate synthase, methylmalonyl-5-phosphate kinase, methylmalonyl-diphosphat decarboxylase, methylmalonyl kinase or isopentenyl pyrophosphate isomerase.

[0011] Further, the amino acid sequence of the tyrosine ammonia-lyase is shown as SEQ ID NO: 9.

[0012] Further, the amino acid sequence of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase is shown as SEQ ID NO: 10, and the amino acid sequence of the chorismate mutase is shown as SEQ ID NO: 11.

[0013] Further, the amino acid sequence of the methylmalonyl-CoA synthase is shown as SEQ ID NO: 2; the amino acid sequence of the methylmalonyl-CoA reductase is shown as SEQ ID NO: 3; the amino acid sequence of the farnesyl pyrophosphate synthase is shown as SEQ ID NO: 4; the amino acid sequence of the methylmalonyl-5-phosphate kinase is shown as SEQ ID NO: 5; the amino acid sequence of the methylmalonyl-diphosphat decarboxylase is shown as SEQ ID NO: 6; the amino acid sequence of the methylmalonyl kinase is shown as SEQ ID NO: 7; and the amino acid sequence of the isopentenyl pyrophosphate isomerase is shown as SEQ ID NO: 8.

[0014] To achieve the above object, according to a second aspect of the present application, there is provided a method for constructing the above-mentioned recombinant yeast, which comprises: introducing a gene encoding a prenyltransferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain a recombinant yeast; wherein the gene encoding the prenyltransferase is derived from Psoralea corylifolia Linn.

[0015] Further, the construction method comprises: introducing the genes encoding the prenyltransferase and the tyrosine ammonia-lyase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast.

[0016] Further, the construction method comprises: introducing the genes encoding the prenyltransferase and the tyrosine ammonia-lyase and the genes encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase or chorismate mutase.

[0017] Further, the construction method further comprises: introducing the genes encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast: methylmalonyl-CoA synthase, methylmalonyl-CoA reductase, farnesyl pyrophosphate synthase, methylmalonyl-5-phosphate kinase, methylmalonyl diphosphate decarboxylase, methylmalonyl kinase or isopentenyl pyrophosphate isomerase.

[0018] To achieve the above object, according to a third aspect of the present application, a preparation method of bakuchiol is provided, the preparation method comprising: performing fermentation culture by using the recombinant yeast for producing bakuchiol to obtain a fermentation liquid; performing centrifugation on the fermentation liquid to obtain a precipitated bacteria paste, and performing extraction on the precipitated bacteria paste, and the obtained solution is the bakuchiol.

[0019] Further, the fermentation culture comprises: placing the recombinant yeast in a YPD culture medium and a substrate to perform fermentation culture; wherein the substrate is selected from at least one of the following: p-coumaric acid, tyrosine or glucose.

[0020] By using the recombinant yeast of the present application, a large amount of bakuchiol can be prepared by a biosynthetic method, which is efficient and fast, and provides technical support for the industrialization of the subsequent bakuchiol. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application. In the drawings:

[0022] Figure 1 A schematic diagram of the related pathway for synthesizing bakuchiol is shown;

[0023] Figure 2 An HPLC detection graph of the recombinant Yarrowia lipolytica producing bakuchiol in Example 1 is shown;

[0024] Figure 3 An LC-MS detection graph of the recombinant Yarrowia lipolytica producing bakuchiol in Example 1 is shown;

[0025] wherein,Figure 1 The meaning of each abbreviation is as follows:

[0026] HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A, MvaE: mevalonate coenzyme A reductase, MvaS: mevalonate coenzyme A synthase, HMGR: HMG-CoA reductase, MVA: dimethylallyl phosphate, MVA-P: 5-mevalonate phosphate, ERG12: mevalonate kinase, ERG8: mevalonate-5-phosphate kinase, MVA-PP: 5-mevalonate pyrophosphate, MVD1: mevalonate diphosphate decarboxylase, IPP: isoprene pyrophosphate, IDI1: isopentenyl pyrophosphate isomerase, DMAPP: dimethylallyl pyrophosphate, ERG20*: farnesyl pyrophosphate synthase, GPP: geranyl pyrophosphate, E4P: erythrose-4-phosphate, PEP: phosphoenolpyruvate, ARO4: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, ARO1: multifunctional aromatic protein, ARO2: chorismate synthase, DHAP: 3-deoxy-D-arabino-heptulosonate-7-phosphate, CHA: chorismate, ARO7: chorismate mutase, ARO8: aromatic aminotransferase I, ARO9: aromatic aminotransferase II, PPA: prephenate, FjTAL: tyrosine ammonia lyase. DETAILED DESCRIPTION

[0027] It should be noted that the embodiments and features in the embodiments in the present application can be combined with each other without conflict. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] As mentioned in the background, the methods for preparing psoralen in the prior art are mostly chemical methods or natural extraction methods, and the yield of the prepared psoralen is small, and the operation is relatively complex. In order to obtain psoralen with high yield more efficiently, the present application provides a method for preparing psoralen by using recombinant yeast.

[0029] In a first typical embodiment of the present application, a recombinant yeast for producing bakuchiol is provided, which is Yarrowia lipolytica or Saccharomyces cerevisiae containing an isopentenyl transferase; wherein the gene encoding the isopentenyl transferase is derived from Psoralea corylifolia Linn. The above-mentioned genetically engineered bacteria have the beneficial effects of fast growth, good activity and less by-products. The use of the above-mentioned genetically engineered bacteria to synthesize bakuchiol helps to improve the production efficiency of bakuchiol. Yarrowia lipolytica is a single-cell eukaryote, and compared with Saccharomyces cerevisiae, it has a high acetyl-CoA supply capacity and can be applied to the preparation process of various acetyl-CoA derivatives and terpenoids. Since bakuchiol is mainly extracted from Psoralea corylifolia plant species, only the isopentenyl transferase derived from Psoralea corylifolia can catalyze the synthesis of bakuchiol from p-coumaric acid, and other sources of isopentenyl transferase do not have this activity. In order to further obtain bakuchiol with clear structure, in a preferred embodiment, the amino acid sequence of the isopentenyl transferase tDN28194 is shown in SEQ ID NO: 1.

[0030] SEQ ID NO: 1:

[0031] MEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR.

[0032] As Figure 1As shown, bakuchiol is obtained from the precursors p-coumaric acid and geranyl pyrophosphate catalyzed by the isopentenyl transferase tDN28194. Among them, geranyl pyrophosphate is synthesized from acetyl-CoA, an intermediate in the glycolysis pathway, under the continuous catalysis of the enzymes MvaE (mevalonate CoA-reductase), MvaS (mevalonate CoA-synthase), ERG12 (mevalonate kinase), ERG8 (mevalonate-5-phosphate kinase), MVD1 (mevalonate diphosphate decarboxylase), IDI1 (isopentenyl pyrophosphate isomerase) and ERG20* (farnesyl pyrophosphate synthase). The other precursor p-coumaric acid is synthesized from tyrosine under the catalysis of tyrosine ammonia-lyase FjTAL, and can also be synthesized from glucose under the catalysis of ARO4 (3-deoxy-D-arabinose-heptulosonate-7-phosphate synthase), ARO7 (chorismate mutase) and FjTAL.

[0033] Any enzyme involved in this synthesis pathway is suitable for the present application. In a preferred embodiment, the recombinant yeast further comprises a tyrosine ammonia-lyase TAL, which is derived from Flavobacterium johnsoniae. In a preferred embodiment, the recombinant yeast further comprises any one or more of the following enzymes: ARO4 (3-deoxy-D-arabinose-heptulosonate-7-phosphate synthase) or ARO7 (chorismate mutase). In a preferred embodiment, the recombinant yeast further comprises any one or more of the following enzymes: MvaS (mevalonate CoA-synthase), MvaE (mevalonate CoA-reductase), ERG20 (farnesyl pyrophosphate synthase), ERG8 (mevalonate-5-phosphate kinase), MVD1 (mevalonate diphosphate decarboxylase), ERG12 (mevalonate kinase) or IDI1 (isopentenyl pyrophosphate isomerase). Among them, MvaS / MvaE are derived from Enterococcus faecalis, ERG20, ERG8, MVD1, ERG12, IDI, ARO4 and ARO7 are derived from Yl590 (Yarrowia lipolytica), and TAL is derived from Flavobacterium johnsoniae.

[0034] In order to further efficiently obtain more bakuchiol, the specific sequences of the above enzymes are further limited, and in a preferred embodiment, the amino acid sequence of the tyrosine ammonia-lyase TAL is shown in SEQ ID NO: 9. In a preferred embodiment, the amino acid sequence of the 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase ARO4* is shown in SEQ ID NO: 10, and the amino acid sequence of the chorismate mutase ARO7* is shown in SEQ ID NO: 11. In a preferred embodiment, the amino acid sequence of the methylmalonyl-CoA synthase MvaS is shown in SEQ ID NO: 2; the amino acid sequence of the methylmalonyl-CoA reductase MvaE is shown in SEQ ID NO: 3; the amino acid sequence of the farnesyl pyrophosphate synthase ERG20* is shown in SEQ ID NO: 4; the amino acid sequence of the methylmalonyl phosphate kinase ERG8 is shown in SEQ ID NO: 5; the amino acid sequence of the methylmalonyl diphosphate decarboxylase MVD1 is shown in SEQ ID NO: 6; the amino acid sequence of the methylmalonyl kinase ERG12 is shown in SEQ ID NO: 7; and the amino acid sequence of the isopentenyl pyrophosphate isomerase IDI1 is shown in SEQ ID NO: 8.

[0035] wherein ERG20* is a mutant, and the mutation sites are F88W and N119W in the amino acid sequence of wild-type ERG20 (GenBank No. CAG79180.1); ARO4* is a mutant, and the mutation site is K221L in the amino acid sequence of wild-type ARO4 (GenBank No. CAG81841.1); and ARO7* is a mutant, and the mutation site is G139S in the amino acid sequence of wild-type ARO7 (GenBank No. CAG79658.1).

[0036] wherein the yeast into which the gene TAL encoding the tyrosine ammonia-lyase is introduced can synthesize bakuchiol using tyrosine as a substrate, i.e., can prepare bakuchiol without the need for p-coumaric acid. The yeast into which the gene TAL encoding the tyrosine ammonia-lyase, the gene ARO4* encoding the 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase, and the gene ARO7* encoding the chorismate mutase are introduced can synthesize bakuchiol using glucose as a substrate, i.e., can prepare bakuchiol without the need for p-coumaric acid and tyrosine.

[0037] SEQ ID NO: 2:

[0038] MTIGIDKISFFVPPYYIDMTALAEARNVDPGKFHIGIGQDQMAVNPISQDIVTFAANAAEAI LTKEDKEAIDMVIVGTESSIDESKAAAVVLHRLMGIQPFARSFEIKEACYGATAGLQLAKN HVALHPDKKVLVVAADIAKYGLNSGGEPTQGAGAVAMLVASEPRILALKEDNVMLTQDI YDFWRPTGHPYPMVDGPLSNETYIQSFAQVWDEHKKRTGLDFADYDALAFHIPYTKMGK KALLAKISDQTEAEQERILARYEESIIYSRRVGNLYTGSLYLGLISLLENATTLTAGNQIG LFSYGSGAVAEFFTGELVAGYQNHLQKETHLALLDNRTELSIAEYEAMFAETLDTDIDQ TLEDELKYSISAINNTVRSYRN.

[0039] SEQ ID NO: 3:

[0040] MKTVVIIDALRTPIGKYKGSLSQVSAVDLGTHVTTQLLKRHSTISEEIDQVIFGNVLQ AGNGQNPARQIAINSGLSHEIPAMTVNEVCGSGMKAVILAKQLIQLGEAEVLIAGGIEN MSQAPKLQRFNYETESYDAPFSSMMYDGLTDAFSGQAMGLTAENVAEKYHVTREEQDQ FS VHSQ LKAAQAQAEGIFADEIAPLEVSGTLVEKDEGIRPNSSVEKLGTLKTVFKEDG TVTAGNASTINDGASALIIASQEYAEAHGLPYLAIIRDSVEVGIDPAYMGISPIKAIQK LLARNQLTTEEIDLYEINEAFAATSIVVQRELALPEEKVNIYGGGISLGHAIGATGARLL TSLSYQLNQKEKKYGVASLCIGGGLGLAMLLERPQQKKNSRFYQMSPEERLASLLNEG QISADTKKEFENTALSSQIANHMIENQISETEVPMGVGLHLTVDETDYLVPMATEEPSV IAALSNGAKIAQGFKTVNQQRLMRGQIVFYDVADAESLIDELQVRETEIFQQAELSY PSIVKRGGGLRDLQYRAFDESFVSVDFLVDVKDAMGANIVNAMLEGVAELFREWFAE QKILFSILSNYATESVVTMKTAIPVSRLSKGSNGREIAEKIVLASRYASLDPYRAVTH NKGIMNGIEAVVLATGNDTRAVSASCHAFAVKEGRYQGLTSWTLDGEQLIGEISVPL ALATVGGATKVLPKSQAAADLLAVTDAKELSRVVAAVGLAQNLAALRALVSEGIQKGHM ALQARSLAMTVGATGKEVEAVAQQLKRQKTMNQDRALAILNDLRKQ.

[0041] SEQ ID NO: 4:

[0042] MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLSVVDTYQLLTGKKELDDEE YYRLALLGWLIELLQAFWLVSDDIMDESKTRRGQPCWYLKPKVGMIAIWDAFMLESGIYILLKKHFRQEK YYIDLVELFHDISFKTELGQLVDLLTAPEDEVDLNRFSLDKHSFIVRYKTAYYSFYLPVVLAMYVAGITN PKDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTDIQDNKCSWLVNKALQKATPEQRQILEDNYG VKDKSKELVIKKLYDDMKIEQDYLDYEEEVVGDIKKKIEQVDESRGFKKEVLNAFLAKIYKRQK.

[0043] SEQ ID NO: 5:

[0044] MTTYSAPGKALLCGGYLVIDPAYSAYVVGLSARIYATVSASEASTTSVHVVSPQFDKGEWTYNYTNGQLTAIGHNPFAHAAVNTVLHYVPPRNLHINISIKSDNAYHSQIDSTQRGQFAYHKKAIHEVPKTGLGSSAALTTVLVAALLKSYGIDPLHNTHLVHNLSQVAHCSAQKKIGSGFDVASAVCGSLVYRRFPAESVNMVIAAEGTSEYGALLRTTVNQKWKVTLEPSFLPPGISLLMGDVQGGSETPGMVAKVMAWRKAKPREAEMVWRDLNAANMLMVKLFNDLRKLSLTNNEAYEQLLAEAAPLNALKMIMLQNPLGELARCIITIRKHLKKMTRETGAAIEPDEQSALLNKCNTYSGVIGGVVPGAGGYDAISLLVISSTVNNVKRESQGVQWMELKEENEGLRLEKGFK.

[0045] SEQ ID NO: 6:

[0046] MIHQASTTAPVNIATLKYWGKRDPALNLPTNNSISVTLSQDDLRTLTTASCSPDFTQDELWLNGKQEDVSGKRLVACFRELRALRHKMEDSDSSLPKLADQKLKIVSENNFPTAAGLASSAAGFAALIRAVANLYELQETPEQLSIVARQGSGSACRSLYGGYVAWEMGTESDGSDSRAVQIATADHWPEMRAAILVVSADKKDTSSTTGMQVTVHTSPLFKERVTTVVPERFAQMKKSILDRDFPTFAELTMRDSNQFHATCLDSYPPIFYLNDVSRASIRVVEAINKAAGATIAAYTFDAGPNCVIYYEDKNEELVLGALKAILGRVEGWEKHQSVDAKKIDVDERWESELANGIQRVILTKVGGDPVKTAESLINEDGSLKNSK。

[0047] SEQ ID NO:7:

[0048] MDYIISAPGKVILFGEHAAVFGKPAIAAAIDLRTYLLVETTTSDTPTVTLEFPDIHLNFKVQVDKLASLTAQTKADHLNWSTPKTLDKHIFDSLSSLALLEEPGLTKVQQAAVVSFLYLYIHLCPPSVCEDSSNWVVRSTLPIGAGLGSSASICVCLAAGLLVLNGQLSIDQARDFKSLTEKQLSLVDDWSFVGEMCIHGNPSGIDNAVATQGGALLFQRPNNRVPLVDIPEMKLLLTNTKHPRSTADLVGGVGVLTKEFGSIMDPIMTSVGEISNQAMEIISRGKKMVDQSNLEIEQGILPQPTSEDACNVMEDGATLQKLRDIGSEMQHLVRINHGLLIAMGVSHPKLEIIRTASIVHNLGETKLTGAGGGGCAITLVTSKDKTATQLEENVIAFTEEMATHGFEVHETTIGARGVGMCIDHPSLKTVEAFKKVERADLKNIGPWTH。

[0049] SEQ ID NO:8:

[0050] MTTSYSDKIKSISVSSVAQQFPEVAPIADVSKASRPSTESSDSSAKLFDGHDEEQIKLMDEICVVLDWDDKPIGGASKKCCHLMDNINDGLVHRAFSVFMFNDRGELLLQQRAAEKITFANMWTNTCCSHPLAVPSEMGGLDLESRIQGAKNAAVRKLEHELGIDPKAVPADKFHFLTRIHYAAPSSGPWGEHEIDYILFVRGDPELKVVANEVRDTVWVSQQGLKDMMADPKLVFTPWFRLICEQALFPWWDQLDNLPAGDDEIRRWIK.

[0051] SEQ ID NO: 9:

[0052] MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPFVQKVKDHLINN.

[0053] SEQ ID NO: 10:

[0054] MSRSSSPNASSAEDVRILGYDPLLAPALLQTEVASTKNARETVSKGRKDSIDVITGKSDKLLCIVGPCSLHDPKAAMEYAQRLKELSDKLSGELVIVMRAYLEKPRTTVGWKGLINDPDMDESFNINKGLRLSRKVFCDLTDLGLPIASEMLDTISPQFLADLLSLGAIGARTTESQLHRELASGLSFPVGFKNGTDGTLGVAVDAVQAASHPHHFMGVTLQGVAAITTTKGNENCFIILRGGKKGTNYDAESVAECKKATESMLMVDCSHGNSNKDYRNQPKVSKAVAEQVAAGEKKIIGVMIESNIHEGNQKVPKEGPSALKYGVSITDACVSWETTVDMLTELANAVKERRNKN.

[0055] SEQ ID NO: 11:

[0056] MDFTKADTVLDLANIRDSLVRMEDTIVFNLIERAQFCRSEFVYKAGNSDIPGFKGSYLDWFLQESEKVHAKLRRYAAPDEQAFFPDDLPEAILPPIDYAPILAPYSKEVSVNDEIKKIYTDDIVPLVCAGTGDQPENYSSVMVCDIETLQALSRRIHFGKFVAESKFLSETERFTELIKNKDIAGIEAAITNSKVEETILARLGEKALAYGTDPTLRWSQRTQGKVDSEVVKRIYKEWVIPLTKKVEVDYLLRRLE.

[0057] In a second typical embodiment of the present application, a method for constructing the recombinant yeast is provided, and the method comprises the following steps: introducing a gene encoding isopentenyl transferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast; wherein the gene encoding isopentenyl transferase is derived from Psoralea corylifolia Linn.

[0058] URA3 encodes orotidine-5'-phosphate decarboxylase, which catalyzes a key reaction in the synthesis of RNA pyrimidine nucleotides in yeast. After knocking out URA3, the yeast cannot grow if no uridine or uracil is added in the culture medium, which can be used to screen the plasmid carrying URA3 gene, but has no effect on the fermentation production of bakuchiol by Yarrowia lipolytica. In a preferred embodiment, the Yarrowia lipolytica mentioned above is URA3-knocking-out Yarrowia lipolytica.

[0059] In the present application, the genes of relevant enzymes are introduced into the yeast by homologous recombination technology. In a preferred embodiment, the construction method comprises: introducing the genes encoding isopentenyl transferase and tyrosine ammonia lyase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain a recombinant yeast. In a preferred embodiment, the construction method comprises: introducing the genes encoding isopentenyl transferase and tyrosine ammonia lyase and the genes encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain a recombinant yeast: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase or chorismate mutase. In a preferred embodiment, the construction method further comprises: introducing the genes encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain a recombinant yeast: methylmalonyl-CoA synthase, methylmalonyl-CoA reductase, farnesyl pyrophosphate synthase, methylmalonyl-5-phosphate kinase, methylmalonyl diphosphate decarboxylase, methylmalonyl kinase or isopentenyl pyrophosphate isomerase.

[0060] Any gene capable of transcribing and translating the amino acids of the above-mentioned enzymes is suitable for use in the present application. In a preferred embodiment, the amino acid sequence of isopentenyl transferase tDN28194 is shown in SEQ ID NO: 1; the amino acid sequence of methylmalonyl-CoA synthase MvaS is shown in SEQ ID NO: 2; the amino acid sequence of methylmalonyl-CoA reductase MvaE is shown in SEQ ID NO: 3; the amino acid sequence of farnesyl pyrophosphate synthase ERG20* is shown in SEQ ID NO: 4; the amino acid sequence of methylmalonyl-5-phosphate kinase ERG8 is shown in SEQ ID NO: 5; the amino acid sequence of methylmalonyl diphosphate decarboxylase MVD1 is shown in SEQ ID NO: 6; the amino acid sequence of methylmalonyl kinase ERG12 is shown in SEQ ID NO: 7; the amino acid sequence of isopentenyl pyrophosphate isomerase IDI1 is shown in SEQ ID NO: 8; the amino acid sequence of tyrosine ammonia lyase TAL is shown in SEQ ID NO: 9; the amino acid sequence of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase ARO4* is shown in SEQ ID NO: 10; and the amino acid sequence of chorismate mutase ARO7* is shown in SEQ ID NO: 11.

[0061] In a third typical embodiment of the present application, a preparation method of bakuchiol is provided, which comprises: fermenting the recombinant yeast for producing bakuchiol to obtain a fermentation liquor; centrifuging the fermentation liquor to obtain a precipitated bacterial slurry, and extracting the bacterial slurry to obtain the bakuchiol.

[0062] By fermenting the recombinant yeast, a large amount of fermentation liquor can be obtained, and the bakuchiol in the bacterial slurry can be extracted to obtain more bakuchiol.

[0063] According to different types of enzymes introduced into the recombinant yeast, the recombinant yeast can synthesize bakuchiol with different substrates. In a preferred embodiment, the fermentation culture comprises: placing the recombinant yeast in a YPD culture medium and a substrate for fermentation culture; wherein the substrate is selected from at least one of: p-coumaric acid, tyrosine or glucose. The recombinant yeast into which the gene TAL encoding tyrosine ammonia-lyase is introduced can synthesize bakuchiol with tyrosine as the substrate. The recombinant yeast into which the gene TAL encoding tyrosine ammonia-lyase, the gene ARO4* encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and the gene ARO7* encoding chorismate mutase are introduced can synthesize bakuchiol with glucose as the substrate.

[0064] In order to synthesize bakuchiol more efficiently, in a preferred embodiment, the concentration of the bacterial solution of the recombinant yeast is OD600 of 0.1-0.3; the concentration of p-coumaric acid is 50-5000 mg / L; and the concentration of tyrosine is 50-5000 mg / L.

[0065] The present application will be further described in detail below in combination with specific embodiments, which should not be understood as limiting the scope of the present application.

[0066] The types and ingredients of the culture media used in the embodiments of the present application are as follows:

[0067] LB culture medium: 10 g / L of tryptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, and 20 g / L of agar powder.

[0068] YPD culture medium: 20 g / L of tryptone, 10 g / L of yeast powder, and 20 g / L of glucose.

[0069] SD-Ura culture medium (purchased from Solabio).

[0070] SD-His culture medium (purchased from Solabio).

[0071] The Yarrowia lipolytica used in the embodiments of the present application is Yarrowia lipolytica Y1590, the accession number of which is CGMCC NO. 30855, the preservation date of which is June 4, 2024, and the preservation address of which is No. 3 Datun Road, Chaoyang District, Beijing, which has been disclosed in the patent with the authorized publication number CN118879517B. The Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C (TS989763, Tesco Bio Limited).

[0072] Detection method:

[0073] 1. Extraction and concentration of bakuchiol:

[0074] Take 5 mL of fermentation broth, centrifuge at 1000 rpm for 2 min, discard the supernatant and collect the bacterial slurry. Add 500 μL of saturated sodium chloride and 0.1 g of quartz sand, mix well and then place in a multi-sample tissue grinder-96L (purchased from Shanghai Jingxin Industrial Development Co., Ltd.), set the parameters to 60 Hz and 120 S, and repeat the grinding and crushing of the bacterial cells twice. Then add 500 μL of ethyl acetate, shake for 15 minutes to extract bakuchiol, and transfer the ethyl acetate organic phase to a new centrifuge tube. After vacuum concentration, the product is dissolved in 100 μL of anhydrous ethanol for subsequent liquid chromatography and mass spectrometry detection.

[0075] 2. Liquid chromatography detection of bakuchiol:

[0076] The liquid chromatography test method is as follows: chromatographic column: Titank C18 3 μm 3×50 mm P / N: FMB-5559-YONU, column temperature: 40°C, flow rate: 1.0 mL / min, mobile phase A: H2O+0.1% H3PO4, mobile phase B: acetonitrile, detection time: 6.0 min, detection wavelength: 260 nm. The liquid chromatography gradient elution conditions are as follows: 0-4 min, 30%-90% B; 4-5 min, 90%-30% B; 5-6 min, 30% B.

[0077] 3. Mass spectrometry detection of bakuchiol: The mass spectrometry conditions are as follows: positive ion mode, electrospray ionization ion source (ESI), capillary voltage 3.0 KV, capillary temperature 500°C, cone hole voltage 30 V, and molecular weight scan range 0-500. The instrument used is Waters SQD-2.

[0078] Example 1

[0079] 1. Screening of isopentenyltransferase gene

[0080] The application screened 60 other sources of prenyltransferases, including prenyltransferases from Psoralea corylifolia Linn, Ulmus davidiana var japonica and Piper Longum, but only found that the prenyltransferase from Psoralea corylifolia Linn had the function of catalyzing the synthesis of bakuchiol.

[0081] The transcriptome data of Psoralea corylifolia Linn (Accession number: PRJNA938259), Ulmus davidiana var japonica (Accession number: PRJDB4728) and Piper Longum (Accession number: PRJNA580359) published by NCBI were downloaded and further assembled, predicted and annotated by Suzhou Jinyuzhi Biotechnology Co., Ltd. A prenyltransferase gene capable of catalyzing the synthesis of bakuchiol was screened and named as DN28194. The full-length of prenyltransferase DN28194 is 409 amino acids, and the full-length amino acid sequence is shown as SEQ ID NO: 12. Since the N-terminal signal peptide may affect the correct folding of the protein in yeast, leading to the decrease of the catalytic activity of the protein, the N-terminal signal peptide is truncated to obtain the amino acid sequence of prenyltransferase tDN28194, which is shown as SEQ ID NO: 1.

[0082] SEQ ID NO: 12:

[0083] MASMFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCED WARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAI LTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVY TINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKE HGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYM FMWKLFYIEYLMIPLIR.

[0084] 2. Construction of recombinant yeast

[0085] The gene URA3 in Yarrowia lipolytica Y1590 was knocked out by homologous recombination technology, and inoculated into YPD liquid medium for overnight culture. The next day, 1% inoculation amount was inoculated into 50 mL YPD liquid medium, and the yeast competent cells were prepared by using Zymogen FrozenEZ Yeast Transformation Kit II kit. The isopentenyl transferase encoding gene tDN28194 (nucleotide sequence as shown in SEQ ID NO: 13) was transformed into Yl-590ΔURA3 competent cells by homologous recombination technology, and plated and screened on SD-Ura3 medium, to obtain a recombinant Yarrowia lipolytica S1 producing bakuchiol.

[0086] SEQ ID NO: 13:

[0087]

[0088] Y. lipolytica Y1590AURA3 and recombinant Y. lipolytica S1 were inoculated into YPD medium overnight, Y1590 and S1 strains were inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid at initial cell concentration OD 600 = 0.2, 30°C, 220 rpm, and fermented for 72 h. 5 mL fermentation broth was centrifuged at 12000 rpm for 2 min, and the supernatant was discarded to collect the mycelium. After extraction and concentration, liquid chromatography and mass spectrometry were performed. The results of liquid chromatography are shown in Figure 2 , and the results of mass spectrometry are shown in Figure 3 .

[0089] After 72 h of fermentation, Y. lipolytica Y1590AURA3 did not detect the synthesis of bakuchiol, and recombinant Y. lipolytica S1 could synthesize 4.6 mg / L of bakuchiol.

[0090] Example 2 Construction of recombinant yeast for synthesis of bakuchiol with tyrosine as substrate

[0091] In order to be able to synthesize bakuchiol with tyrosine as substrate, the gene TAL (nucleotide sequence as shown in SEQ ID NO: 14) was integrated into the recombinant strain S1 to obtain the recombinant strain S2. Recombinant strains S1 and S2 were inoculated into YPD medium overnight, S1 and S2 were inoculated into 50 mL YPD medium containing 200 mg / L tyrosine at initial cell concentration OD 600 = 0.2, 30°C, 220 rpm, and fermented for 72 h.

[0092] SEQ ID NO: 14:

[0093]

[0094] The recombinant strain S2 was able to synthesize 3.1 mg / L of bakuchiol, while the recombinant strain S1 was unable to synthesize bakuchiol. It can be seen that the recombinant strain S2 was able to synthesize bakuchiol using only tyrosine in the presence of only tyrosine.

[0095] Example 3 Construction of a recombinant yeast for de novo synthesis of bakuchiol

[0096] In order to be able to synthesize bakuchiol de novo, the genes ARO4* (nucleotide sequence as shown in SEQ ID NO: 15) and ARO7* (nucleotide sequence as shown in SEQ ID NO: 16) were integrated into the recombinant strain S2 to obtain the recombinant strain S3. The recombinant strain S3 was inoculated into YPD medium and incubated overnight, and S3 was inoculated into 50 mL YPD medium at an initial bacterial liquid concentration OD 600 = 0.2, 30°C, 220 rpm, and fermented for 72 h.

[0097] SEQ ID NO: 15:

[0098]

[0099] SEQ ID NO: 16:

[0100] ATGGACTTCACTAAAGCCGACACCGTTCTGGATCTCGCCAACATCCGAGACTCGCTGGTCCGAATGGAGGACACTATTGTCTTCAATCTGATTGAGCGGGCTCAGTTCTGCCGTTCCGAGTTTGTGTACAAGGCCGGCAACTCGGACATTCCCGGCTTCAAGGGCTCTTACCTCGACTGGTTTCTGCAGGAGTCGGAAAAGGTGCACGCCAAACTGCGTCGGTACGCTGCCCCGGACGAGCAGGCCTTCTTCCCCGACGATCTACCCGAGGCCATTCTGCCCCCCATCGATTATGCGCCAATTCTGGCACCCTACAGCAAGGAGGTGAGCGTCAACGACGAGATTAAAAAGATTTACACCGACGACATTGTGCCCCTGGTGTGTGCTGGCACTGGAGATCAGCCCGAGAACTATTCGTCGGTCATGGTGTGCGACATCGAGACGCTGCAGGCGCTGTCGCGACGAATCCACTTTGGCAAGTTTGTGGCCGAGTCCAAGTTTCTGAGTGAAACCGAGCGATTCACCGAGCTCATCAAGAACAAGGACATTGCTGGTATTGAGGCGGCCATCACAAACTCCAAGGTGGAAGAGACGATTCTGGCCCGGCTGGGAGAAAAGGCACTGGCCTACGGCACAGACCCCACTCTCCGGTGGTCGCAGAGAACCCAGGGAAAGGTTGATTCCGAGGTTGTCAAGCGAATCTACAAGGAGTGGGTGATTCCACTCACCAAGAAGGTCGAGGTGGACTACCTGCTCCGGCGGTTGGAGTAG.

[0101] The recombinant strain S3 can synthesize 2.3 mg / L of psoralen, and it can be seen that the recombinant strain S3 can synthesize psoralen by using glucose only without adding tyrosine or p-coumaric acid.

[0102] Example 4 Construction of a recombinant yeast strain for high-yield production of psoralen using p-coumaric acid as a substrate

[0103] In order to further improve the production of bakuchiol, on the basis of the recombinant Yarrowia lipolytica S1 obtained in Example 1, recombinant strains S4-S8 capable of stably producing high yield of bakuchiol were constructed.

[0104] The gene MvaS (nucleotide sequence as shown in SEQ ID NO: 17) and the gene MvaE (nucleotide sequence as shown in SEQ ID NO: 18) were integrated into the recombinant strain S1 to obtain the recombinant strain S4;

[0105] The gene ERG20* (nucleotide sequence as shown in SEQ ID NO: 19) was integrated into the recombinant strain S4 to obtain the recombinant strain S5;

[0106] The gene ERG8 (nucleotide sequence as shown in SEQ ID NO: 20) and the gene MVD1 (nucleotide sequence as shown in SEQ ID NO: 21) were integrated into the recombinant strain S5 to obtain the recombinant strain S6;

[0107] The gene ERG12 (nucleotide sequence as shown in SEQ ID NO: 22) was integrated into the recombinant strain S6 to obtain the recombinant strain S7;

[0108] The gene IDI1 (nucleotide sequence as shown in SEQ ID NO: 23) was integrated into the recombinant strain S7 to obtain the recombinant strain S8;

[0109] SEQ ID NO: 17:

[0110]

[0111] SEQ ID NO: 18:

[0112]

[0113] SEQ ID NO: 19:

[0114]

[0115] SEQ ID NO:20:

[0116]

[0117] SEQ ID NO:21:

[0118]

[0119] SEQ ID NO:22:

[0120]

[0121] SEQ ID NO: 23:

[0122] ATGACGACGTCTTACAGCGACAAAATCAAGAGTATCAGCGTGAGCTCTGTGGCTCAGCAGTTTCCTGAGGTGGCGCCGATTGCGGACGTGTCCAAGGCTAGCCGGCCCAGCACGGAGTCGTCGGACTCGTCGGCCAAGCTATTTGATGGCCACGACGAGGAGCAGATCAAGCTGATGGACGAGATCTGTGTGGTGCTGGACTGGGACGACAAGCCGATTGGCGGCGCGTCCAAAAAGTGCTGTCATCTGATGGACAACATCAACGAGCGCGGTGAGCTGCTTCTGCAGCAGCGGGCGGCGGAAAAAATCACCTTTGCCAACATGTGGACCAACACGTGCTGCTCGCATCCTCTGGCGGTGCCCAGCGAGATGGGCGGGCTGGATCTGGAGTCCCGGATCCAGGGCGCCAAAAACGCCGCGGTCCGGAAGCTTGAGCACGAGCTGGGAATCGACCCCAAGGCCGTTCCGGCAGACAAGTTCCATTTCCTCACCCGGATCCACTACGCCGCGCCCTCCTCGGGCCCCTGGGGCGAGCACGAGATTGACTACATTCTGTTTGTCCGGGGCGACCCCGAGCTCAAGGTGGTGGCCAACGAGGTCCGCGATACCGTGTGGGTGTCGCAGCAGGGACTCAAGGACATGATGGCCGATCCCAAGCTGGTTTTCACCCCTTGGTTCCGGCTCATTTGTGAGCAGGCGCTGTTTCCCTGGTGGGACCAGTTGGACAATCTGCCCGCGGGCGATGACGAGATTCGGCGGTGGATCAAGTAG.

[0123] The recombinant strains S4-S8 were inoculated into YPD medium overnight, and S4-S8 were inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid at an initial cell concentration of OD600=0.2, 30°C, 220 rpm, and fermented for 72 h. 5 mL of the fermentation broth was centrifuged at 12000 rpm for 2 min, and the supernatant was discarded to collect the slurry. After extraction and concentration, liquid phase detection was performed, and the content of bakuchiol after 72 h of fermentation of the recombinant strains S4-S8 was shown in Table 1.

[0124] Table 1: Fermentation results of different yeast engineering strains constructed in Example 4

[0125]

[0126] Example 5: Effect of different concentrations of p-coumaric acid on the synthesis of bakuchiol

[0127] S8 was inoculated into 50 mL YPD medium containing p-coumaric acid at an initial cell concentration of OD600=0.2, 30°C, 220 rpm, and fermented for 72 h. 600 As shown in Table 2, S8 was inoculated into 50 mL YPD medium containing p-coumaric acid at different concentrations (as shown in Table 2), 30°C, 220 rpm, and fermented for 72 h. 5 mL of the fermentation broth was centrifuged at 12000 rpm for 2 min, and the supernatant was discarded to collect the slurry. After extraction and concentration, liquid phase detection was performed, and the content of bakuchiol after 72 h of fermentation under different conditions was shown in Table 2.

[0128] Table 2: Yield of bakuchiol synthesized by different concentrations of p-coumaric acid

[0129]

[0130] As shown in Table 2, with the increase of the concentration of p-coumaric acid, the content of bakuchiol also gradually increased, and the final content of bakuchiol reached 547.5 mg / L.

[0131] Example 6: Construction of recombinant yeast for high-yield production of bakuchiol with glucose as substrate

[0132] In order to improve the ability of the strain to synthesize bakuchiol from scratch, based on the recombinant Yarrowia lipolytica S3 obtained in Example 3, a recombinant strain S9 capable of stably producing high-yield bakuchiol was constructed.

[0133] The genes MvaS (nucleotide sequence as shown in SEQ ID NO: 17), MvaE (nucleotide sequence as shown in SEQ ID NO: 18), ERG20* (nucleotide sequence as shown in SEQ ID NO: 19), ERG8 (nucleotide sequence as shown in SEQ ID NO: 20), MVD1 (nucleotide sequence as shown in SEQ ID NO: 21), ERG12 (nucleotide sequence as shown in SEQ ID NO: 22) and IDI1 (nucleotide sequence as shown in SEQ ID NO: 23) were integrated into the recombinant strain S3 in sequence to obtain the recombinant strain S9;

[0134] The recombinant strain S9 was inoculated into YPD medium for overnight culture, and S9 was inoculated into 50 mL YPD medium at an initial bacterial liquid concentration OD600=0.2, 30°C, 220 rpm, and fermented for 72 h. The recombinant strain S9 could synthesize 89.3 mg / L of bakuchiol. It can be seen that the recombinant strain S9 can still synthesize more bakuchiol by using glucose only without adding tyrosine or p-coumaric acid.

[0135] Example 7 Construction of recombinant yeast for synthesizing bakuchiol by using different enzyme species by using Saccharomyces cerevisiae

[0136] The genes MvaS (nucleotide sequence as shown in SEQ ID NO: 17) and MvaE (nucleotide sequence as shown in SEQ ID NO: 18) were integrated into the wild-type Saccharomyces cerevisiae CEN.PK2-1C (TS989763) by using a homologous recombination system to construct the Erg021 strain. The gene tDN28194 (nucleotide sequence as shown in SEQ ID NO: 13) was introduced into the Erg021 strain to construct the recombinant strain Erg021-tDN28194. The gene BAK36(T1) (disclosed in the patent application file with publication number WO2023168043A1) was introduced into the Erg021 strain to construct the recombinant strain Erg021-BAK36(T1).

[0137] The recombinant strains Erg021-tDN28194 and Erg021-BAK36(T1) were inoculated into YPD liquid medium for overnight culture at the same time, and Erg021-tDN28194 and Erg021-BAK36(T1) were inoculated into 50 mL YPD medium at an initial bacterial liquid concentration OD600=0.2, 30°C, 220 rpm, and fermented for 72 h. 600=0.2 into 50 mL YPD medium containing 250 mg / L p-coumaric acid, 30 °C, 220 rpm, fermentation for 72 h. The recombinant strain S1 can synthesize 4.6 mg / L of psoralen, while Y1590ΔURA3-BAK36(T1) can only detect 0.05 mg / L of psoralen after grinding and concentrating 100 times.

[0138] Comparative Example 1

[0139] The gene BAK36(T1) (disclosed in the patent application document with publication number WO2023168043A1) was introduced into the Yarrowia lipolytica Y1590ΔURA3 strain constructed in the above Example 1 to construct the Y1590ΔURA3-BAK36(T1) strain. The recombinant strain Y1590ΔURA3-BAK36(T1) and the recombinant Yarrowia lipolytica S1 in Example 1 were inoculated into YPD liquid medium overnight, and Y1590ΔURA3-BAK36(T1) and S1 were inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid at an initial bacterial liquid concentration OD 600 =0.2 into 50 mL YPD medium containing 250 mg / L p-coumaric acid, 30 °C, 220 rpm, fermentation for 72 h. The recombinant strain S1 can synthesize 4.6 mg / L of psoralen, while Y1590ΔURA3-BAK36(T1) can only detect 0.05 mg / L of psoralen after grinding and concentrating 100 times.

[0140] Comparative Example 2

[0141] The gene tPcPT7 (disclosed in the patent application document with publication number CN118909814) was introduced into the Yarrowia lipolytica Y1590ΔURA3 strain constructed in the above Example 1 to construct the Y1590ΔURA3-tPcPT7 strain. The recombinant strain Y1590ΔURA3-tPcPT7 and the recombinant Yarrowia lipolytica S1 in Example 1 were inoculated into YPD liquid medium overnight, and Y1590ΔURA3-tPcPT7 and S1 were inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid at an initial bacterial liquid concentration OD 600 =0.2 into 50 mL YPD medium containing 250 mg / L p-coumaric acid, 30 °C, 220 rpm, fermentation for 72 h. The recombinant strain S1 can synthesize 4.6 mg / L of psoralen, while Y1590ΔURA3-tPcPT7 can only synthesize 0.15 mg / L of psoralen.

[0142] Comparative Example 3

[0143] Different strains and different substrates in Table 3 below were subjected to fermentation culture, and were inoculated into 50 mL YPD medium containing different concentrations and types of substrates at a concentration of OD600=0.2, 30°C, 220 rpm, and fermented for 72 h. 5 mL of fermentation liquid was centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the slurry was collected. After extraction and concentration, liquid phase detection was performed, and the content of bakuchiol after 72 h of fermentation of different strains was as shown in Table 3.

[0144] Table 3:

[0145]

[0146] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the recombinant Yarrowia lipolytica obtained by introducing the isopentenyltransferase encoding gene and other enzymes related to the synthesis of bakuchiol pathway in the present application can efficiently synthesize a large amount of bakuchiol, which lays an important foundation for the subsequent commercial production of bakuchiol by Yarrowia lipolytica, and has great application value.

[0147] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant yeast producing bakuchiol, characterized by, The recombinant yeast is Yarrowia lipolytica containing an isopentenyl transferase Yarrowia lipolytica ; wherein the gene encoding the prenyltransferase is derived from Psoralea corylifolia Psoralea corylifolia Linn ; The amino acid sequence of the isopentenyltransferase is shown as SEQ ID NO:

1. The Yarrowia lipolytica Yarrowia lipolytica selected from Yarrowia lipolytica Y1590.

2. The recombinant yeast of claim 1, wherein, The recombinant yeast further comprises a tyrosine ammonia-lyase derived from Flavobacterium johnsoniae Flavobacterium johnsoniae .

3. The recombinant yeast of claim 2, wherein, The amino acid sequence of the tyrosine ammonia-lyase is shown as SEQ ID NO:

9.

4. The recombinant yeast of claim 2, wherein, The recombinant yeast further comprises any one or more of the following enzymes: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase or chorismate mutase.

5. The recombinant yeast of claim 4, wherein, The amino acid sequence of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase is shown as SEQ ID NO: 10, and the amino acid sequence of the chorismate mutase is shown as SEQ ID NO:

11.

6. The recombinant yeast of claim 4, wherein, The recombinant yeast further comprises any one or more of the following enzymes: methylmalonyl-CoA synthase, methylmalonyl-CoA reductase, farnesyl pyrophosphate synthase, methylmalonyl-5-phosphate kinase, methylmalonyl diphosphate decarboxylase, methylmalonyl kinase or isopentenyl pyrophosphate isomerase.

7. The recombinant yeast of claim 6, wherein, The amino acid sequence of the methylmalonyl-CoA synthase is shown as SEQ ID NO: 2; The amino acid sequence of the methylmalonyl-CoA reductase is shown as SEQ ID NO: 3; The amino acid sequence of the farnesyl pyrophosphate synthase is shown as SEQ ID NO: 4; The amino acid sequence of the methylmalonyl-5-phosphate kinase is shown as SEQ ID NO: 5; The amino acid sequence of the methylmalonyl diphosphate decarboxylase is shown as SEQ ID NO: 6; The amino acid sequence of the methylmalonyl kinase is shown as SEQ ID NO: 7; The amino acid sequence of the isopentenyl pyrophosphate isomerase is shown as SEQ ID NO:

8.

8. A method of constructing a recombinant yeast of any one of claims 1 to 7, characterized in that, The construction method comprises: Introduction of a gene encoding a prenyltransferase into yarrowia lipolytica Yarrowia lipolytica The recombinant yeast is obtained. wherein the gene encoding the prenyltransferase is derived from Psoralea corylifolia Psoralea corylifolia Linn ; The Yarrowia lipolytica Yarrowia lipolytica selected from Yarrowia lipolytica Y1590.

9. The construction method of claim 8, wherein, The construction method comprises: introducing genes encoding the isopentenyltransferase and the tyrosine ammonia-lyase into the Yarrowia lipolytica to obtain the recombinant yeast.

10. The construction method of claim 8, wherein, The construction method comprises: introducing genes encoding the isopentenyltransferase and the tyrosine ammonia-lyase and genes encoding any one or more of the following enzymes into the Yarrowia lipolytica to obtain the recombinant yeast: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase or chorismate mutase.

11. The construction method of claim 10, wherein, The construction method further comprises: introducing genes encoding any one or more of the following enzymes into the Yarrowia lipolytica to obtain the recombinant yeast: methylmalonyl-CoA synthase, methylmalonyl-CoA reductase, farnesyl pyrophosphate synthase, methylmalonyl-5-phosphate kinase, methylmalonyl diphosphate decarboxylase, methylmalonyl kinase or isopentenyl pyrophosphate isomerase.

12. A method of preparing bakuchiol, characterized by, The preparation method comprises: fermenting the recombinant yeast for producing bakuchiol according to any one of claims 1-7 to obtain a fermentation broth; centrifuging the fermentation broth to obtain a precipitated bacterial slurry; and centrifuging the fermentation broth to obtain a precipitated bacterial slurry; and The precipitated bacterial slurry is extracted, and the obtained solution is the bakuchiol.

13. The method of claim 12, wherein, The fermentation culture comprises: The recombinant yeast is placed in a YPD culture medium and a substrate for the fermentation culture. The substrate is selected from at least one of p-coumaric acid, tyrosine or glucose.

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