Recombinant yeast for producing bakuchiol as well as construction method and application of recombinant yeast

Abstract

The invention provides recombinant yeast for producing bakuchiol as well as a construction method and application of the recombinant yeast. Wherein the recombinant yeast is Yarrowia lipolytica containing isopentenyl transferase, and the recombinant yeast is a Yarrowia lipolytica containing isopentenyl transferase. Wherein a gene for coding the isopentenyl transferase is derived from Psoralea coryllifelia Linn (Psoralea coryllifelia Linn), and the isopentenyl transferase is used for coding the isopentenyl transferase. By utilizing the recombinant yeast provided by the invention, the bakuchiol with high yield can be efficiently and quickly prepared through a biosynthesis method, and a technical support is provided for subsequent industrialization of the bakuchiol.

Description

Technical Field

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

[0002] Bakuchiol is a terpene compound extracted from the plant Psoralea corylifolia Linn. 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 photostability than retinol. It is regarded as the best substitute for retinol and has attracted widespread attention in the cosmetics field. The number of brands of cosmetics containing bakuchiol has also increased from 30 brands to about 150 brands in 2020. According to statistics, the global anti-aging market is expected to reach US$191.7 billion in 2021, and the average annual growth rate of the Chinese market is higher than that of the world, which has further accelerated the demand for bakuchiol.

[0003] At present, the production methods of bakuchiol are mainly direct extraction from plants or chemical synthesis. The content of bakuchiol in natural plants is low. Obtaining bakuchiol from plants requires the consumption of a large number of psoralea corylifolia plants. Excessive excavation will damage the ecological environment and cause a serious shortage of plant resources. The method of chemical synthesis of bakuchiol has problems such as complex synthesis route, low yield, large solvent consumption, and uneconomical and environmentally unfriendly.

[0004] With the development of synthetic biology and metabolic engineering, the use of microorganisms to ferment economical and renewable carbon sources (glucose, glycerol, sucrose) into high value-added compounds has attracted more and more attention due to its low cost, high efficiency and environmental friendliness. However, the technology for biosynthesis of bakuchiol is still immature. Therefore, how to find an efficient method for biosynthesis of bakuchiol is of great significance for the commercialization of bakuchiol. Summary of the invention

[0005] The main purpose of the present invention 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 object, according to the first aspect of the present invention, a recombinant yeast for producing bakuchiol is provided, wherein the recombinant yeast is Yarrowia lipolytica or Saccharomyces cerevisiae containing isopentenyl transferase; wherein the gene encoding the isopentenyl transferase is derived from Psoraleacorylifolia Linn.

[0007] Furthermore, the amino acid sequence of isopentenyl transferase tDN28194 is shown in SEQ ID NO:1.

[0008] Furthermore, the recombinant yeast also contains tyrosine ammonia lyase, and the tyrosine ammonia lyase is derived from Flavobacterium johnsoniae.

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

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

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

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

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

[0014] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a method for constructing the above-mentioned recombinant yeast is provided, the construction method comprising: 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.

[0015] Furthermore, the construction method includes: introducing genes encoding isopentenyl transferase and tyrosine ammonia lyase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast.

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

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

[0018] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a method for preparing bakuchiol is provided, the preparation method comprising: using the above-mentioned recombinant yeast for producing bakuchiol to carry out fermentation culture to obtain a fermentation broth; centrifuging the fermentation broth to obtain a precipitated bacterial sludge, extracting the precipitated bacterial sludge, and the obtained solution is bakuchiol.

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

[0020] By applying the technical solution of the present invention and utilizing the recombinant yeast of the present application, bakuchiol with a large yield can be prepared more efficiently and quickly through a biosynthetic method, thereby providing technical support for the subsequent industrialization of bakuchiol. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 A schematic diagram showing the relevant pathways for synthesizing bakuchiol in the present application is shown;

[0023] Figure 2 The HPLC detection chart of the production of bakuchiol by the recombinant Yarrowia lipolytica in Example 1 is shown;

[0024] Figure 3 The LC-MS detection diagram of the production of bakuchiol by the recombinant Yarrowia lipolytica in Example 1 is shown;

[0025] in, Figure 1 The meanings of the abbreviations are:

[0026] HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA, MvaE: mevalonate-CoA reductase, MvaS: mevalonate-CoA synthase, HMGR: HMG-CoA reductase, MVA: dihydroxymethylvaleric acid, MVA-P: 5-mevalonate phosphate, ERG12: mevalonate kinase, ERG8: mevalonate-5-phosphate kinase, MVA-PP: 5-mevalonate pyrophosphate, MVD1: mevalonate diphosphate decarboxylase, IPP: isopentenyl 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-arabinoheptulose-7-phosphate synthase, ARO1: pentafunctional aromatic protein, ARO2: chorismate synthase, DHAP: 3-deoxy-D-arabinoheptulose-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, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] As mentioned in the background technology, the methods for preparing bakuchiol in the prior art are mostly chemical methods or natural extraction methods, the yield of the prepared bakuchiol is low, and the operation is complicated. In order to obtain bakuchiol with a large yield more efficiently, the present application intends to provide a method for preparing bakuchiol using recombinant yeast.

[0029] In a first typical embodiment of the present application, a recombinant yeast for producing bakuchiol is provided, the recombinant yeast being Yarrowia lipolytica or Saccharomyces cerevisiae containing isopentenyl transferase; wherein the gene encoding the isopentenyl transferase is derived from Psoraleacorylifolia Linn. The above-mentioned genetically engineered bacteria have the beneficial effects of fast growth rate, good activity and few 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 unicellular eukaryotic organism, and compared with Saccharomyces cerevisiae, it has an efficient acetyl-CoA supply capacity and can be applied to the preparation process of various acetyl-CoA derivatives and terpenoid compounds. Since bakuchiol is mainly extracted from Psoralea corylifolia plant species, currently only the isopentenyl transferase from Psoralea corylifolia can catalyze the synthesis of bakuchiol from p-coumaric acid, and the isopentenyl transferases from other sources do not have this activity. In order to further obtain bakuchiol with a clear structure, in a preferred embodiment, the amino acid sequence of isopentenyl transferase tDN28194 is shown in SEQ ID NO:1.

[0030] SEQ ID NO: 1:

[0031] MEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAY FVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR.

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

[0033] Any enzyme that acts in the synthetic pathway is applicable to 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: 3-deoxy-D-arabinoheptulose-7-phosphate synthase ARO4 or chorismate mutase ARO7. In a preferred embodiment, the recombinant yeast further comprises any one or more of the following enzymes: mevalonate CoA synthase MvaS, mevalonate CoA reductase MvaE, farnesyl pyrophosphate synthase ERG20, mevalonate-5-phosphate kinase ERG8, mevalonate diphosphate decarboxylase MVD1, mevalonate kinase ERG12 or isopentenyl pyrophosphate isomerase IDI1. Among them, MvaS / MvaE are derived from Enterococcus faecalis, ERG20, ERG8, MVD1, ERG12, IDI, ARO4, and ARO7 are all derived from Yarrowia lipolytica Yl590, 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. In a preferred embodiment, the amino acid sequence of tyrosine ammonia lyase TAL is shown in SEQ ID NO: 9. In a preferred embodiment, the amino acid sequence of 3-deoxy-D-arabinoheptulose-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. In a preferred embodiment, the amino acid sequence of mevalonate CoA synthase MvaS is shown as SEQ ID NO:2; the amino acid sequence of mevalonate CoA reductase MvaE is shown as SEQ ID NO:3; the amino acid sequence of farnesyl pyrophosphate synthase ERG20* is shown as SEQ ID NO:4; the amino acid sequence of mevalonate-5-phosphate kinase ERG8 is shown as SEQ ID NO:5; the amino acid sequence of mevalonate diphosphate decarboxylase MVD1 is shown as SEQ ID NO:6; the amino acid sequence of mevalonate kinase ERG12 is shown as SEQ ID NO:7; and the amino acid sequence of isopentenyl pyrophosphate isomerase IDI1 is shown as SEQ ID NO:8.

[0035] Among them, ERG20* is a mutant, the mutation site is the 88th F mutation to W (F88W) and the 119th N mutation to W (N119W) in the amino acid sequence of wild ERG20 (the GenBank number of the protein is CAG79180.1), ARO4* is a mutant, the mutation site is the 221st K mutation to L (K221L) in the amino acid sequence of wild ARO4 (the GenBank number of the protein is CAG81841.1), and ARO7* is a mutant, the mutation site is the 139th G mutation to S (G139S) in the amino acid sequence of wild ARO7 (the GenBank number of the protein is CAG79658.1).

[0036] Among them, yeast introduced with the gene TAL encoding tyrosine ammonia lyase can synthesize bakuchiol using tyrosine as a substrate, that is, it can prepare bakuchiol without the need for p-coumaric acid. Yeast introduced with the gene TAL encoding tyrosine ammonia lyase, the gene ARO4* encoding 3-deoxy-D-arabinoheptulose-7-phosphate synthase, and the gene ARO7* encoding chorismate mutase can synthesize bakuchiol using glucose as a substrate, that is, it can prepare bakuchiol without the need for p-coumaric acid and tyrosine.

[0037] SEQ ID NO:2:

[0038] MTIGIDKISFFVPPYYIDMTALAEARNVDPGKFHIGIGQDQMAVNPISQDIVTFAANAAEAILTKEDKEAIDMVIVGTESSIDESKAAAVVLHRLMGIQPFARSFEIKEACYGATAGLQLAKNHVALHPDKKVLVVAADIAKYGLNSGGEPTQGAGAVAMLVASEPRILALKEDNVMLTQDIYDFWRPTGHPYPMVDGPLSNETYIQSFAQVWDEHKKRTGLDFADYDALAFHIPYTKMGKKALLAKISDQTEAEQERILARYEESIIYSRRVGNLYTGSLYLGLISLLENATTLTAGNQIGLFSYGSGAVAEFFTGELVAGYQNHLQKETHLALLDNRTELSIAEYEAMFAETLDTDIDQTLEDELKYSISAINNTVRSYRN。

[0039] SEQ ID NO:3:

[0040] MKTVVIIDALRTPIGKYKGSLSQVSAVDLGTHVTTQLLKRHSTISEEIDQVIFGNVLQAGNGQNPARQIAINSGLSHEIPAMTVNEVCGSGMKAVILAKQLIQLGEAEVLIAGGIENMSQAPKLQRFNYETESYDAPFSSMMYDGLTDAFSGQAMGLTAENVAEKYHVTREEQDQFSVHSQLKAAQAQAEGIFADEIAPLEVSGTLVEKDEGIRPNSSVEKLGTLKTVFKEDGTVTAGNASTINDGASALIIASQEYAEAHGLPYLAIIRDSVEVGIDPAYMGISPIKAIQKLLARNQLTTEEIDLYEINEAFAATSIVVQRELALPEEKVNIYGGGISLGHAIGATGARLLTSLSYQLNQKEKKYGVASLCIGGGLGLAMLLERPQQKKNSRFYQMSPEERLASLLNEGQISADTKKEFENTALSSQIANHMIENQISETEVPMGVGLHLTVDETDYLVPMATEEPSVIAALSNGAKIAQGFKTVNQQRLMRGQIVFYDVADAESLIDELQVRETEIFQQAELSYPSIVKRGGGLRDLQYRAFDESFVSVDFLVDVKDAMGANIVNAMLEGVAELFREWFAEQKILFSILSNYATESVVTMKTAIPVSRLSKGSNGREIAEKIVLASRYASLDPYRAVTHNKGIMNGIEAVVLATGNDTRAVSASCHAFAVKEGRYQGLTSWTLDGEQLIGEISVPLALATVGGATKVLPKSQAAADLLAVTDAKELSRVVAAVGLAQNLAALRALVSEGIQKGHMALQARSLAMTVGATGKEVEAVAQQLKRQKTMNQDRALAILNDLRKQ。

[0041] SEQ ID NO:4:

[0042] MSKAKFESVFPRISEELVQLLRDEGLPQDAVQWFSDSLQYNCVGGKLNRGLSVVDTYQLLTGKKELDDEEYYRLALLGWLIELLQAFWLVSDDIMDESKTRRGQPCWYLKPKVGMIAIWDAFMLESGIYILLKKHFRQEKYYIDLVELFHDISFKTELGQLVDLLTAPEDEVDLNRFSLDKHSFIVRYKTAYYSFYLPVVLAMYVAGITNPKDLQQAMDVLIPLGEYFQVQDDYLDNFGDPEFIGKIGTDIQDNKCSWLVNKALQKATPEQRQILEDNYGVKDKSKELVIKKLYDDMKIEQDYLDYEEEVVGDIKKKIEQVDESRGFKKEVLNAFLAKIYKRQK。

[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] MSRSSSPNASSAEDVRILGYDPLLAPALLQTEVASTKNARETVSKGRKDSIDVITGKSDKLLCIVGPCSLHDPKAAMEYAQRLKELSDKLSGELVIVMRAYLEKPRTTVGWKGLINDPDMDESFNINKGLRLSRKVFCDLTDLGLPIASEMLDTISPQFLADLLSLGAIGARTTESQLH RELASGLSFPVGFKNGTDGTLGVAVDAVQAASHPHHFMGVTLQGVAAITTTKGNENCFIILRGGKKGTNYDAESVAECKKATESMLMVDCSHGNNSNKDYRNQPKVSKAVAEQVAAGEKKIIGVMIESNIHEGNQKVPKEGPSALKYGVSITDACVSWETTVDMLTELANAVKERRNKN.

[0055] SEQ ID NO:11:

[0056] MDFTKADTVLDLANIRDSLVRMEDTIVFNLIERAQFCRSEFVYKAGNSDIPGFKGSYLDWFLQESEKVHAKLRRYAAPDEQAFFPDDLPEAILPPIDYAPILAPYSKEVSVNDEIKKIYTDDIVPLVC AGTGDQPENYSSVMVCDIETLQALSRRIHFGKFVAESKFLSETERFTELIKNKDIAGIEAAITNSKVEETILARLGEKALAYGTDPTLRWSQRTQGKVDSEVVKRIYKEWVIPLTKKVEVDYLLRRLE.

[0057] In a second typical embodiment of the present application, a method for constructing the above-mentioned recombinant yeast is provided, the construction method comprising: introducing a gene encoding isopentenyl transferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain 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 yeast RNA pyrimidine nucleotides. After URA3 is knocked out, if uridine or uracil is not added to the culture medium, the yeast cannot grow, and can be used to screen plasmids carrying the URA3 gene, but has no effect on the fermentation production of bakuchiol by Yarrowia lipolytica. In a preferred embodiment, the above-mentioned Yarrowia lipolytica is a Yarrowia lipolytica with the gene URA3 knocked out.

[0059] The present application utilizes homologous recombination technology to import the gene of relevant enzyme into yeast. In a preferred embodiment, the construction method includes: importing the gene encoding isopentenyl transferase and tyrosine ammonia lyase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast. In a preferred embodiment, the construction method includes: importing the gene encoding isopentenyl transferase and tyrosine ammonia lyase and the gene encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast: 3-deoxy-D-arabinoheptulose-7-phosphate synthase or chorismate mutase. In a preferred embodiment, the construction method also includes: importing the gene encoding any one or more of the following enzymes into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast: mevalonate coenzyme A synthase, mevalonate coenzyme A reductase, farnesyl pyrophosphate synthase, mevalonate-5-phosphate kinase, mevalonate diphosphate decarboxylase, mevalonate kinase or isopentenyl pyrophosphate isomerase.

[0060] Any gene that can be transcribed and translated to obtain the amino acids of the above enzymes is suitable for 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 mevalonate CoA synthase MvaS is shown in SEQ ID NO: 2; the amino acid sequence of mevalonate 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 mevalonate-5-phosphate kinase ERG8 is shown in SEQ ID NO: 5; the amino acid sequence of mevalonate diphosphate decarboxylase MVD1 is shown in SEQ ID NO: 6; the amino acid sequence of mevalonate 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. NO:9; the amino acid sequence of 3-deoxy-D-arabinoheptulose-7-phosphate synthase ARO4* is shown in SEQ ID NO:10; 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 method for preparing bakuchiol is provided, the preparation method comprising: using the above-mentioned recombinant yeast for producing bakuchiol to carry out fermentation culture to obtain a fermentation broth; centrifuging the fermentation broth to obtain a precipitated bacterial sludge, extracting the precipitated bacterial sludge, and the obtained solution is bakuchiol.

[0062] By conventionally fermenting and culturing the above-mentioned recombinant yeast, a large amount of fermentation liquid product can be obtained, and then the bakuchiol in the bacterial mud is extracted to obtain more bakuchiol in a simple manner.

[0063] Depending on the type of enzyme introduced into the recombinant yeast, the recombinant yeast can synthesize bakuchiol with different substrates. In a preferred embodiment, the fermentation culture includes: placing the recombinant yeast in a YPD medium and a substrate for fermentation culture; wherein the substrate is selected from at least one of the following: p-coumaric acid, tyrosine or glucose. Among them, the recombinant yeast introduced with the gene TAL encoding tyrosine ammonia lyase can synthesize bakuchiol with tyrosine as a substrate. The recombinant yeast introduced with the gene TAL encoding tyrosine ammonia lyase, the gene ARO4* of 3-deoxy-D-arabinoheptulose-7-phosphate synthase and the gene ARO7* of chorismate mutase can synthesize bakuchiol with glucose as a substrate.

[0064] In order to synthesize bakuchiol more efficiently, in a preferred embodiment, the bacterial solution concentration of the recombinant yeast OD600 is 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 is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0066] The types and components of the culture medium used in the examples of this application are as follows:

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

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

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

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

[0071] The Yarrowia lipolytica used in the examples of the present application is Yarrowia lipolytica Y1590, the deposit number of Yarrowia lipolytica Y1590 is CGMCC NO.30855, the deposit date is June 4, 2024, the deposit address is No. 3, Datun Road, Chaoyang District, Beijing, and has been disclosed in the patent with the authorization announcement number CN118879517B. The brewer's yeast is Saccharomyces cerevisiae CEN.PK2-1C (Testo Biotech, TS989763).

[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 sludge. Add 500 μL of saturated sodium chloride and 0.1 g of quartz sand, mix well and place in a multi-sample tissue grinder-96L (purchased from Shanghai Jingxin Industrial Development Co., Ltd.), set the parameters to 60 Hz, 120 S, and repeat the grinding and breaking of the bacteria twice. Then add 500 μL of ethyl acetate and shake for 15 minutes to extract bakuchiol. Transfer the ethyl acetate organic phase to a new centrifuge tube. After vacuum concentration, add 100 μL of anhydrous ethanol to dissolve the product for subsequent liquid phase detection and mass spectrometry detection.

[0075] 2. Liquid phase detection of bakuchiol:

[0076] The liquid phase 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: H 2 O+0.1% H 3 PO 4 , mobile phase B: acetonitrile, detection time: 6.0 min, detection wavelength: 260 nm. Liquid phase gradient elution conditions: 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 were positive ion mode, electrospray ionization (ESI), capillary voltage of 3.0 KV, capillary temperature of 500°C, cone voltage of 30 V, and molecular weight scanning range of 0 to 500. The instrument used was Waters SQD-2.

[0078] Example 1

[0079] 1. Screening for isopentenyl transferase genes

[0080] This application screened 60 isopentenyl transferases from other sources, including isopentenyl transferases from Psoralea corylifolia Linn, Ulmus davidiana var japonica and Piper Longum, but only the isopentenyl transferase from Psoralea corylifolia Linn was found to have 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 Jinweizhi Biotechnology Co., Ltd. A gene encoding an isopentenyl transferase that can catalyze the synthesis of bakuchiol was screened and named DN28194. The full-length isopentenyl transferase DN28194 is 409 amino acids, and the full-length amino acid sequence is shown in SEQ ID NO: 12. Since the presence of the signal peptide at its N-terminus may affect the correct folding of the protein in yeast, resulting in a decrease in the catalytic activity of the protein, the N-terminal signal peptide was truncated to obtain the amino acid sequence of isopentenyl transferase tDN28194, as shown in SEQ ID NO: 1.

[0082] SEQ ID NO:12:

[0083] MASMLFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITL CAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYLMIPLIR.

[0084] 2. Construction of recombinant yeast

[0085] The gene URA3 in Yarrowia lipolytica Yl590 was knocked out by homologous recombination technology, and inoculated into YPD liquid medium for overnight culture. The next day, it was inoculated into 50 mL YPD liquid medium at a 1% inoculation amount, and yeast competent cells were prepared using the Zymogen FrozenEZ Yeast Transformation Kit II kit. The isopentenyl transferase encoding gene tDN28194 (nucleotide sequence shown in SEQ ID NO: 13) was transformed into Yl-590ΔURA3 competent cells by homologous recombination technology, and plated on SD-Ura3 medium for screening to obtain recombinant Yarrowia lipolytica S1 producing bakuchiol.

[0086] SEQ ID NO:13:

[0087]

[0088] Yarrowia lipolytica Yl590ΔURA3 and recombinant Yarrowia lipolytica S1 were inoculated into YPD medium and cultured overnight. Yl590 and S1 strains were cultured at an initial bacterial concentration of OD 600 = 0.2 was inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid, and fermented at 30°C, 220 rpm, for 72 h. 5 mL of fermentation broth was taken, centrifuged at 12000 rpm for 2 min, and the supernatant was discarded to collect the bacterial sludge. After extraction and concentration, liquid chromatography and mass spectrometry were performed. The liquid chromatography test results are shown in Figure 2 The mass spectrometry results are shown in Figure 3 shown.

[0089] After 72 h of fermentation, no bakuchiol production was detected in Yarrowia lipolytica Yl590ΔURA3, while recombinant Yarrowia lipolytica S1 was able to synthesize 4.6 mg / L of bakuchiol.

[0090] Example 2 Construction of recombinant yeast for synthesizing bakuchiol using tyrosine as substrate

[0091] In order to synthesize bakuchiol using tyrosine as a substrate, the gene TAL (nucleotide sequence shown in SEQ ID NO: 14) was integrated into the recombinant strain S1 to obtain the recombinant strain S2. The recombinant strains S1 and S2 were inoculated into YPD medium and cultured overnight. S1 and S2 were cultured at an initial bacterial concentration of OD 600 =0.2 was inoculated into 50 mL YPD medium containing 200 mg / L tyrosine and fermented at 30°C, 220 rpm 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 when only tyrosine was added.

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

[0096] In order to synthesize bakuchiol from scratch, the genes ARO4* (nucleotide sequence shown in SEQ ID NO: 15) and ARO7* (nucleotide sequence 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 cultured overnight. 600 =0.2 was inoculated into 50 mL YPD medium and fermented at 30°C, 220 rpm for 72 h.

[0097] SEQ ID NO:15:

[0098]

[0099] SEQ ID NO:16:

[0100] .

[0101] The recombinant strain S3 was able to synthesize 2.3 mg / L of bakuchiol. It can be seen that the recombinant strain S3 was able to synthesize bakuchiol using only glucose without adding tyrosine or p-coumaric acid.

[0102] Example 4 Construction of recombinant yeast for high production of bakuchiol using p-coumaric acid as substrate

[0103] In order to further increase the yield of bakuchiol, based on the recombinant Yarrowia lipolytica S1 obtained in Example 1, recombinant strains S4-S8 that can stably and highly produce bakuchiol were constructed.

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

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

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

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

[0108] Integrating the gene IDI1 (nucleotide sequence shown in SEQ ID NO: 23) 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] ATGACGACGTCTTACAGCGACAAAATCAAGAGTATCAGCGTGAGCTCTGTGGCTCAGCAGTTTCCTGAGGTGGCGCCGATTGCGGACGTGTCCAAGGCTAGCCGGCCCAGCACGGAGTCGTCGGACTCGTCGGCCAAGCTATTTGATGGCCACGACGAGGAGCAGATCAAGCTGATGGACGAGATCTGTGTGGTGCTGGACTGGGACGACAAGCCGATTGGCGGCGCGTCCAAAAAGTGCTGTCATCTGATGGACAACATCAACGACGGACTGGTGCATCGGGCCTTTTCCGTGTTCATGTTCAACGACCGCGGTGAGCTGCTTCTGCAGCAGCGGGCGGCGGAAAAAATCACCTTTGCCAACATGTGGACCAACACGTGCTGCTCGCATCCTCTGGCGGTGCCCAGCGAGATGGGCGGGCTGGATCTGGAGTCCCGGATCCAGGGCGCCAAAAACGCCGCGGTCCGGAAGCTTGAGCACGAGCTGGGAATCGACCCCAAGGCCGTTCCGGCAGACAAGTTCCATTTCCTCACCCGGATCCACTACGCCGCGCCCTCCTCGGGCCCCTGGGGCGAGCACGAGATTGACTACATTCTGTTTGTCCGGGGCGACCCCGAGCTCAAGGTGGTGGCCAACGAGGTCCGCGATACCGTGTGGGTGTCGCAGCAGGGACTCAAGGACATGATGGCCGATCCCAAGCTGGTTTTCACCCCTTGGTTCCGGCTCATTTGTGAGCAGGCGCTGTTTCCCTGGTGGGACCAGTTGGACAATCTGCCCGCGGGCGATGACGAGATTCGGCGGTGGATCAAGTAG。

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

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

[0125]

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

[0127] S8 was added at the initial bacterial concentration OD 600 Under the conditions shown in Table 2 below, inoculate into 50 mL YPD medium containing p-coumaric acid at different concentrations (as shown in Table 2), ferment at 30°C, 220 rpm, and ferment for 72 h. Take 5 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, discard the supernatant and collect the bacterial sludge. After extraction and concentration, liquid phase detection was performed, and the content of bakuchiol after fermentation for 72 h under different conditions is shown in Table 2.

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

[0129]

[0130] It can be seen from Table 2 that with the increase of p-coumaric acid concentration, the content of bakuchiol gradually increased, and finally the content of bakuchiol reached 547.5 mg / L.

[0131] Example 6 Construction of recombinant yeast for high production of bakuchiol using glucose as substrate

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

[0133] The genes MvaS (the nucleotide sequence shown in SEQ ID NO:17), MvaE (the nucleotide sequence shown in SEQ ID NO:18), ERG20* (the nucleotide sequence shown in SEQ ID NO:19), ERG8 (the nucleotide sequence shown in SEQ ID NO:20), MVD1 (the nucleotide sequence shown in SEQ ID NO:21), ERG12 (the nucleotide sequence shown in SEQ ID NO:22) and IDI1 (the nucleotide sequence shown in SEQ ID NO:23) were successively integrated into the recombinant strain S3 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 concentration of OD600 = 0.2, and fermented for 72 h at 30°C and 220 rpm. The recombinant strain S9 was able to synthesize 89.3 mg / L of bakuchiol, which shows that the recombinant strain S9 can still synthesize a large amount of bakuchiol using only glucose without the addition of tyrosine or p-coumaric acid.

[0135] Example 7 Construction of recombinant yeast using Saccharomyces cerevisiae to synthesize bakuchiol using different enzyme types

[0136] Using the homologous recombination system, the gene MvaS (nucleotide sequence shown in SEQ ID NO: 17) and the gene MvaE (nucleotide sequence shown in SEQ ID NO: 18) were integrated into the wild-type Saccharomyces cerevisiae CEN.PK2-1C (Test Biotech, TS989763) to construct the Erg021 strain, and the gene tDN28194 (nucleotide sequence 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 document 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 at the same time and cultured overnight. 600=0.2 was inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid and fermented at 30°C, 220 rpm, for 72 h. The recombinant strain Erg021-tDN28194 was able to synthesize 25.4 mg / L of bakuchiol, while Erg021-BAK36(T1) could only produce 0.1 mg / L of bakuchiol.

[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 Yl590ΔURA3 strain constructed in the above Example 1 to construct the Yl590ΔURA3-BAK36(T1) strain. The recombinant strain Yl590ΔURA3-BAK36(T1) and the recombinant Yarrowia lipolytica S1 in Example 1 were simultaneously inoculated into YPD liquid medium for overnight culture. Yl590ΔURA3-BAK36(T1) and S1 were cultured at an initial bacterial concentration of OD 600 =0.2 was inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid and fermented for 72 h at 30°C and 220 rpm. The recombinant strain S1 was able to synthesize 4.6 mg / L of bakuchiol, while Yl590ΔURA3-BAK36(T1) was only detected at 0.05 mg / L of bakuchiol after grinding and 100-fold concentration.

[0140] Comparative Example 2

[0141] The gene tPcPT7 (disclosed in the patent application document with publication number CN118909814) was introduced into the Yarrowia lipolytica Yl590ΔURA3 strain constructed in the above Example 1 to construct the Yl590ΔURA3-tPcPT7 strain. The recombinant strain Yl590ΔURA3-tPcPT7 and the recombinant Yarrowia lipolytica S1 in Example 1 were simultaneously inoculated into YPD liquid medium for overnight culture. Yl590ΔURA3-tPcPT7 and S1 were cultured at an initial bacterial concentration of OD 600 =0.2 was inoculated into 50 mL YPD medium containing 250 mg / L p-coumaric acid and fermented for 72 h at 30°C and 220 rpm. The recombinant strain S1 was able to synthesize 4.6 mg / L of bakuchiol, while Yl590ΔURA3-tPcPT7 could only synthesize 0.15 mg / L of bakuchiol.

[0142] Comparative Example 3

[0143] The different strains and different substrates in Table 3 below were fermented and cultured, and all were inoculated into 50 mL YPD medium containing different concentrations and types of substrates at a concentration of OD600 = 0.2, and fermented for 72 h at 30°C and 220 rpm. 5 mL of fermentation broth was taken, centrifuged at 1000 rpm for 2 min, and the supernatant was discarded to collect the bacterial mud. After extraction and concentration, liquid phase detection was performed, and the content of bakuchiol obtained by different strains after 72 h of fermentation is 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 invention achieve the following technical effects: fermentation and culture of the recombinant Yarrowia lipolytica obtained by introducing the isopentenyl transferase encoding gene and other enzymes related to the synthesis pathway of bakuchiol 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 description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant yeast for producing bakuchiol, characterized in that: The recombinant yeast is any one of the following yeasts containing isopentenyl transferase: Yarrowia lipolytica or Saccharomyces cerevisiae; Wherein, the gene encoding the isopentenyl transferase is derived from Psoralea corylifolia Linn; The amino acid sequence of the isopentenyl transferase is shown in SEQ ID NO:

1.

2. The recombinant yeast according to claim 1, characterized in that The recombinant yeast further comprises tyrosine ammonia lyase, and the tyrosine ammonia lyase is derived from Flavobacterium johnsoniae.

3. The recombinant yeast according to claim 2, characterized in that The amino acid sequence of the tyrosine ammonia lyase is shown in SEQ ID NO:

9.

4. The recombinant yeast according to claim 2, characterized in that The recombinant yeast further comprises any one or more of the following enzymes: 3-deoxy-D-arabinoheptulose-7-phosphate synthase or chorismate mutase.

5. The recombinant yeast according to claim 4, characterized in that The amino acid sequence of the 3-deoxy-D-arabinoheptulose-7-phosphate synthase is shown in SEQ ID NO:10, and the amino acid sequence of the chorismate mutase is shown in SEQ ID NO:

11.

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

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

8.

8. A method for constructing a recombinant yeast according to any one of claims 1 to 7, characterized in that: The construction method comprises: Introducing a gene encoding isopentenyl transferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast; Wherein, the gene encoding the isopentenyl transferase is derived from Psoralea corylifolia Linn.

9. The construction method according to claim 8, characterized in that: The construction method comprises: introducing the genes encoding the isopentenyl transferase and the tyrosine ammonia lyase into the Yarrowia lipolytica or the Saccharomyces cerevisiae to obtain the recombinant yeast.

10. The construction method according to claim 8, characterized in that: The construction method comprises: introducing genes encoding the isopentenyl transferase and tyrosine ammonia lyase and genes encoding any one or more of the following enzymes into the Yarrowia lipolytica or the Saccharomyces cerevisiae to obtain the recombinant yeast: 3-deoxy-D-arabinoheptulose-7-phosphate synthase or chorismate mutase.

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

12. A method for preparing bakuchiol, characterized in that: The preparation method comprises: Fermenting and culturing the recombinant yeast for producing bakuchiol according to any one of claims 1 to 7 to obtain a fermentation broth; Centrifuging the fermentation liquid to obtain precipitated bacterial sludge; The precipitated bacterial mud is extracted, and the obtained solution is the bakuchiol.

13. The preparation method according to claim 12, characterized in that: The fermentation culture comprises: Placing the recombinant yeast in a YPD medium and a substrate for fermentation culture; Wherein, the substrate is selected from at least one of the following: p-coumaric acid, tyrosine or glucose.

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