Recombinant yeast for producing bakuchiol, construction method therefor, and use thereof

Abstract

Provided are a recombinant yeast for producing bakuchiol, a construction method therefor, and use thereof. The recombinant yeast is Yarrowia lipolytica containing an isopentenyl transferase. A gene encoding the isopentenyl transferase is derived from Psoralea corylifolia Linn. The recombinant yeast can be used to biosynthesize bakuchiol.

Description

Recombinant yeast for producing psoralen, its construction method and applications

[0001] This application is based on and claims priority to Chinese application CN application number 202510349127.2 filed on March 24, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of biosynthesis technology, and more specifically, to a recombinant yeast for producing psoralen, its construction method, and its application. Background Technology

[0003] Bakuchiol is a heteroterpenoid compound extracted from the plant Psoralea corylifolia Linn. It has attracted widespread attention due to its various physiological functions, including anti-wrinkle, antioxidant, and anti-inflammatory effects. Furthermore, bakuchiol not only possesses functions similar to retinol but also exhibits lower irritation and higher photostability, making it considered the best alternative to retinol and drawing significant attention in the cosmetics industry. The number of cosmetic brands containing bakuchiol has increased from 30 to approximately 150 in 2020. Statistics show that the global anti-aging market is projected to reach $191.7 billion in 2021, with the Chinese market experiencing a higher average annual growth rate than the global market, further accelerating the demand for bakuchiol.

[0004] Currently, the main methods for producing psoralen are direct extraction from plants or chemical synthesis. Psoralen is present in low amounts in natural plants, and obtaining it from plants requires consuming large quantities of psoralen plants, which can damage the ecological environment and lead to a severe shortage of plant resources. On the other hand, the chemical synthesis of psoralen has problems such as complex synthesis routes, low yields, large solvent consumption, and being uneconomical and environmentally unfriendly.

[0005] 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 increasing attention due to its low cost, high efficiency, and environmental friendliness. However, the technology for biosynthesizing psoralen is still immature. Therefore, finding an efficient method for the biosynthesis of psoralen is of great significance for its commercialization. Summary of the Invention

[0006] The main objective of this invention is to provide a recombinant yeast for producing psoralen, its construction method, and its application, so as to solve the problem of low synthesis efficiency of psoralen in the prior art.

[0007] To achieve the above objective, according to a first aspect of the present invention, a recombinant yeast for producing psoralea corylifolia is provided, wherein the recombinant yeast is Yarrowia lipolytica or Saccharomyces cerevisiae containing isopentenyltransferase; wherein the gene encoding isopentenyltransferase is derived from Psoralea corylifolia Linn.

[0008] Furthermore, the amino acid sequence of isopentenyltransferase tDN28194 is shown in SEQ ID NO:1.

[0009] Furthermore, the recombinant yeast also contains tyrosine ammonia lyase, which is derived from Flavobacterium johnsoniae.

[0010] Furthermore, the recombinant yeast also contains any one or more of the following enzymes: 3-deoxy-D-arabinohepulose-7-phosphate synthase or branched acid mutase.

[0011] Furthermore, the recombinant yeast also contains any one or more of the following enzymes: mevalonate-coenzyme A synthase, mevalonate-coenzyme A reductase, farnesyl pyrophosphate synthase, mevalonate-5-phosphate kinase, mevalonate diphosphate decarboxylase, mevalonate kinase, or isopentenyl pyrophosphate isomerase.

[0012] Furthermore, the amino acid sequence of tyrosine ammonia lyase is shown in SEQ ID NO:9.

[0013] Furthermore, the amino acid sequence of 3-deoxy-D-arabinohepenosyl-7-phosphate synthase is shown in SEQ ID NO:10, and the amino acid sequence of branched acid mutase is shown in SEQ ID NO:11.

[0014] Furthermore, the amino acid sequence of mevalonate-coenzyme A synthase is shown in SEQ ID NO:2; the amino acid sequence of mevalonate-coenzyme A 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.

[0015] To achieve the above objective, according to a second aspect of the present invention, a method for constructing the above-mentioned recombinant yeast is provided, the method comprising: introducing a gene encoding isopentenyltransferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast; wherein the gene encoding isopentenyltransferase is derived from Psoralea corylifolia Linn.

[0016] Furthermore, the construction method includes: introducing genes encoding isopentenyltransferase and tyrosine aminolyase into Yersinia lipophila or Saccharomyces cerevisiae to obtain recombinant yeast.

[0017] Further, the construction method includes: introducing genes encoding isopentenyltransferase and tyrosine aminolyase, as well as genes encoding any one or more of the following enzymes, into Yersinia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast: 3-deoxy-D-arabinohepulose-7-phosphate synthase or branched acid mutase.

[0018] Furthermore, the construction method also includes: introducing a gene encoding any one or more of the following enzymes into Yersinia 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.

[0019] To achieve the above objectives, according to a third aspect of the present invention, a method for preparing psoralen is provided, the method comprising: fermenting and culturing the above-mentioned recombinant yeast for producing psoralen to obtain a fermentation broth; centrifuging the fermentation broth to obtain a precipitated bacterial sludge; extracting the precipitated bacterial sludge; and obtaining a solution which is psoralen.

[0020] Further, the fermentation culture includes: placing the recombinant yeast in YPD medium and substrate for fermentation culture; wherein the substrate is selected from at least one of the following: p-coumaric acid, tyrosine, or glucose.

[0021] By applying the technical solution of this invention, the recombinant yeast of this application can be used to prepare psoralen in large quantities through biosynthesis in a relatively efficient and rapid manner, providing technical support for the subsequent industrialization of psoralen. Attached Figure Description

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

[0023] Figure 1 shows a schematic diagram of the relevant pathways for the synthesis of psoralen in this application;

[0024] Figure 2 shows the HPLC detection chromatogram of psoralen produced by recombinant Yersinia lipolytica in Example 1;

[0025] Figure 3 shows the LC-MS detection of psoralen produced by recombinant Yersinia lipolytica in Example 1;

[0026] In Figure 1, the abbreviations are as follows: HMG-CoA: 3-hydroxy-3-methylglutarate monoacyl-CoA, MvaE: mevalonate-CoA reductase, MvaS: mevalonate-CoA synthase, HMGR: HMG-CoA reductase, MVA: dimethylolvalerate, 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: isoprene pyrophosphate isomerase, DMAPP: dimethylallyl pyrophosphate. Acid, ERG20*: Farnesyl pyrophosphate synthase, GPP: Geraniyl pyrophosphate, E4P: Erythrose-4-phosphate, PEP: Phosphoenolpyruvate, ARO4: 3-deoxy-D-arabinohepulose-7-phosphate synthase, ARO1: Pentafunctional aromatic protein, ARO2: Ctenyl acid synthase, DHAP: 3-deoxy-D-arabinohepulose-7-phosphate, CHA: Ctenyl acid, ARO7: Ctenyl acid mutase, ARO8: Aromatic aminotransferase I, ARO9: Aromatic aminotransferase II, PPA: Prephenyl acid, FjTAL: Tyrosine amino lyase. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] As mentioned in the background section, existing methods for preparing psoralen are mostly chemical or natural extraction methods, resulting in low yields and complex operations. To achieve a higher yield of psoralen more efficiently, this application aims to provide a method for preparing psoralen using recombinant yeast.

[0029] In a first typical embodiment of this application, a recombinant yeast for producing psoralen is provided. This recombinant yeast is either *Yarrowia lipolytica* or *Saccharomyces cerevisiae*, both containing isopentenyltransferase; wherein the gene encoding the isopentenyltransferase is derived from *Psoralea corylifolia* Linn. The aforementioned genetically engineered bacteria exhibit the advantages of rapid growth, high activity, and low byproduct production. Using these genetically engineered bacteria to synthesize psoralen helps improve the production efficiency of psoralen. *Yarrowia lipolytica* is a single-celled eukaryote and, compared to *Saccharomyces cerevisiae*, has a highly efficient acetyl-CoA supply capacity, making it applicable to the preparation of various acetyl-CoA derivatives and terpenoid compounds. Since psoralen is mainly extracted from *Psoralea corylifolia* plants, currently only isopentenyltransferases derived from *Psoralea corylifolia* can catalyze the synthesis of psoralen from coumaric acid; isopentenyltransferases from other sources do not possess this activity. To further obtain psoralen with a clear structure, in a preferred embodiment, the amino acid sequence of isopentenyltransferase tDN28194 is shown in SEQ ID NO:1.

[0030] SEQ ID NO:1:

[0031] As shown in Figure 1, psoralen is obtained by catalyzing the precursors p-coumaric acid and geraniyl pyrophosphate via isopentenyltransferase tDN28194. Geraniyl pyrophosphate is derived from the mevalonate pathway, using acetyl-CoA, an intermediate product of glycolysis, as a precursor, and is synthesized under the sequential 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*. Another precursor, p-coumaric acid, originates from the tyrosine pathway. It 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-arabinohepenosose-7-phosphate synthase ARO4, branched acid mutase ARO7, and tyrosine ammonia lyase FjTAL.

[0032] Any enzyme that functions in this synthetic pathway is applicable to this application. In a preferred embodiment, the recombinant yeast further comprises tyrosine ammonia lyase TAL, derived from Flavobacterium johnsoniae. In a preferred embodiment, the recombinant yeast further comprises any one or more of the following enzymes: 3-deoxy-D-arabinohepenoyl-7-phosphate synthase ARO4 or branching acid mutase ARO7. In a preferred embodiment, the recombinant yeast further comprises any one or more of the following enzymes: mevalonate-coenzyme A synthase MvaS, mevalonate-coenzyme A 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 derived from Yarrowia lipolytica Yl590, and TAL is derived from Flavobacterium johnsoniae.

[0033] To further optimize the yield of psoralen, the specific sequences of the enzymes were further defined. In a preferred embodiment, the amino acid sequence of tyrosine ammonia lyase TAL is shown in SEQ ID NO:9. In another preferred embodiment, the amino acid sequence of 3-deoxy-D-arabinohepenosyl-7-phosphate synthase ARO4* is shown in SEQ ID NO:10, and the amino acid sequence of branched acid mutase ARO7* is shown in SEQ ID NO:11. In a preferred embodiment, the amino acid sequence of mevalonate-coenzyme A synthase MvaS is shown in SEQ ID NO:2; the amino acid sequence of mevalonate-coenzyme A 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; and the amino acid sequence of isopentenyl pyrophosphate isomerase IDI1 is shown in SEQ ID NO:8.

[0034] Among them, ERG20* is a mutant, with the mutation site being F at position 88 of the amino acid sequence of wild-type ERG20 (GenBank number CAG79180.1) mutated to W (F88W) and N at position 119 mutated to W (N119W). ARO4* is a mutant, with the mutation site being K at position 221 of the amino acid sequence of wild-type ARO4 (GenBank number CAG81841.1) mutated to L (K221L). ARO7* is a mutant, with the mutation site being G at position 139 of the amino acid sequence of wild-type ARO7 (GenBank number CAG79658.1) mutated to S (G139S).

[0035] Yeast containing the gene TAL, which encodes a tyrosine ammonia lyase, can synthesize psoralen using tyrosine as a substrate, meaning it can be prepared without the need for coumaric acid. Yeast containing the gene TAL, which encodes a tyrosine ammonia lyase, the gene ARO4*, which encodes 3-deoxy-D-arabinohepenoyl-7-phosphate synthase, and the gene ARO7*, which encodes a branched acid mutase, can synthesize psoralen using glucose as a substrate, meaning it can be prepared without the need for coumaric acid and tyrosine.

[0036] SEQ ID NO:2:

[0037] SEQ ID NO:3:

[0038] SEQ ID NO:4:

[0039] SEQ ID NO:5:

[0040] SEQ ID NO:6:

[0041] SEQ ID NO:7:

[0042] SEQ ID NO:8:

[0043] SEQ ID NO:9:

[0044] SEQ ID NO:10:

[0045] SEQ ID NO:11:

[0046] In a second typical embodiment of this application, a method for constructing the above-mentioned recombinant yeast is provided. The method includes: introducing a gene encoding isopentenyltransferase into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain recombinant yeast; wherein the gene encoding isopentenyltransferase is derived from Psoralea corylifolia Linn.

[0047] URA3 encodes orotidine-5′-phosphate decarboxylase, an enzyme that catalyzes a key reaction in the synthesis of pyrimidine nucleotides in yeast RNA. After URA3 is knocked out, yeast cannot grow if uridine or uracil is not added to the culture medium. This can be used to screen for plasmids carrying the URA3 gene, but it has no effect on the fermentation production of psoralen by *Yersinia lipolytica*. In a preferred embodiment, the *Yersinia lipolytica* described above is a *Yersinia lipolytica* strain with the URA3 gene knocked out.

[0048] This application utilizes homologous recombination technology to introduce genes of relevant enzymes into yeast. In a preferred embodiment, the construction method includes: introducing genes encoding isopentenyltransferase and tyrosine ammonia lyase into *Yersinia lipolytica* or *Saccharomyces cerevisiae* to obtain recombinant yeast. In a preferred embodiment, the construction method includes: introducing genes encoding isopentenyltransferase and tyrosine ammonia lyase, as well as genes encoding any one or more of the following enzymes, into *Yersinia lipolytica* or *Saccharomyces cerevisiae* to obtain recombinant yeast: 3-deoxy-D-arabinohepenosaccharide-7-phosphate synthase or branched acid mutase. In a preferred embodiment, the construction method further includes: introducing genes encoding any one or more of the following enzymes into *Yersinia 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.

[0049] Any gene capable of transcription and translation to obtain the amino acids of the above-mentioned enzymes is applicable to this application. In a preferred embodiment, the amino acid sequence of isopentenyltransferase tDN28194 is shown in SEQ ID NO:1; the amino acid sequence of mevalonate-coenzyme A synthase MvaS is shown in SEQ ID NO:2; the amino acid sequence of mevalonate-coenzyme A 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; and the amino acid sequence of tyrosine ammonia lyase TAL is shown in SEQ ID NO:8. The amino acid sequence of 3-deoxy-D-arabinohepenoyl-7-phosphate synthase ARO4* is shown in SEQ ID NO:10; the amino acid sequence of branched acid mutase ARO7* is shown in SEQ ID NO:11.

[0050] In a third typical embodiment of this application, a method for preparing psoralen is provided. The method includes: fermenting and culturing the recombinant yeast used for producing psoralen as described above to obtain a fermentation broth; centrifuging the fermentation broth to obtain a precipitated bacterial sludge; extracting the precipitated bacterial sludge; and obtaining a solution that is psoralen.

[0051] By conventionally fermenting the recombinant yeast, a large amount of fermentation broth product can be obtained. Subsequently, the psoralen in the bacterial sludge can be extracted in a relatively simple way to obtain a large amount of psoralen.

[0052] 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 YPD medium and substrate for fermentation culture; wherein the substrate is selected from at least one of the following: p-coumaric acid, tyrosine, or glucose. Specifically, recombinant yeast infused with the gene TAL encoding tyrosine ammonia lyase can synthesize bakuchiol using tyrosine as a substrate. Recombinant yeast infused with the gene TAL encoding tyrosine ammonia lyase, the gene ARO4* encoding 3-deoxy-D-arabinohepenoyl-7-phosphate synthase, and the gene ARO7* encoding branched acid mutase can synthesize bakuchiol using glucose as a substrate.

[0053] In order to synthesize psoralen more efficiently, in a preferred embodiment, the concentration of the recombinant yeast culture (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.

[0054] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0055] The types and components of the culture medium used in the embodiments of this application are as follows:

[0056] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder.

[0057] YPD medium: tryptone 20g / L, yeast extract 10g / L, glucose 20g / L.

[0058] SD-Ura medium (purchased from Solarbio).

[0059] SD-His medium (purchased from Solarbio).

[0060] The *Yeastraea lipolytica* used in this application embodiment is *Yeastraea lipolytica* Yl590, whose accession number is CGMCC NO.30855, accession date is June 4, 2024, and accession address is No. 3, Datun Road, Chaoyang District, Beijing. It has been disclosed in patent publication number CN118879517B. The *Saccharomyces cerevisiae* used is *Saccharomyces cerevisiae* CEN.PK2-1C (Taisto Biotechnology Co., Ltd., TS989763).

[0061] Detection method:

[0062] 1. Extraction and concentration of bakuchiol:

[0063] 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 homogenizer-96L (purchased from Shanghai Jingxin Industrial Development Co., Ltd.). Set the parameters to 60 Hz and 120 s, and repeat the homogenization process twice to break up the bacterial cells. Then add 500 μL of ethyl acetate, shake for 15 min to extract psoralen, transfer the ethyl acetate organic phase to a new centrifuge tube, concentrate under vacuum, and dissolve the product in 100 μL of anhydrous ethanol for subsequent liquid chromatography and mass spectrometry analysis.

[0064] 2. Liquid chromatography detection of bakuchiol:

[0065] The liquid chromatography method used was as follows: column: Titank C18 3μm 3×50mm P / N: FMB-5559-YONU; column temperature: 40℃; 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 gradient elution conditions were: 0-4 min, 30%-90% B; 4-5 min, 90%-30% B; 5-6 min, 30% B.

[0066] 3. Mass spectrometry detection of psoralen: Mass spectrometry conditions were positive ion mode, electrospray ionization (ESI) source, capillary voltage 3.0 kV, capillary temperature 500 °C, cone voltage 30 V, and molecular weight scan range 0–500. The instrument used was a Waters SQD-2.

[0067] Example 1

[0068] 1. Isopentenyltransferase gene was obtained through screening.

[0069] This application screened 60 other sources of isopentenyltransferases, including those from Psoralea corylifolia Linn, Ulmus davidiana var. japonica, and Piper Longum, but only the isopentenyltransferase from Psoralea corylifolia Linn was found to catalyze the synthesis of psoralen.

[0070] Transcriptome data from *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 Genewise Biotechnology Co., Ltd. A gene encoding isopentenyltransferase capable of catalyzing the synthesis of psoralen was identified and named DN28194. The full-length isopentenyltransferase DN28194 is 409 amino acids long, as shown in SEQ ID NO:12. Because the presence of its N-terminal signal peptide may affect the correct folding of the protein in yeast, leading to reduced catalytic activity, the N-terminal signal peptide was truncated to obtain the amino acid sequence of isopentenyltransferase tDN28194, as shown in SEQ ID NO:1.

[0071] SEQ ID NO:12:

[0072] 2. Constructing recombinant yeast

[0073] The URA3 gene in *Yersinia lipolytica* Yl590 was knocked out using homologous recombination technology, and the cells were inoculated into YPD liquid medium and cultured overnight. The next day, 1% of the cells were inoculated into 50 mL of YPD liquid medium, and competent yeast cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II. The isopentenyltransferase encoding gene tDN28194 (nucleotide sequence as shown in SEQ ID NO:13) was transformed into Yl-590ΔURA3 competent cells using homologous recombination technology, and the cells were screened on SD-Ura3 medium to obtain recombinant *Yersinia lipolytica* S1 producing psoralen.

[0074] SEQ ID NO:13:

[0075] Yl590ΔURA3 and recombinant Yl590S1 were inoculated into YPD medium and cultured overnight. The Yl590 and S1 strains were then cultured at their initial OD500 concentration. 600 =0.2% was inoculated into 50 mL of YPD medium containing 250 mg / L p-coumaric acid, and fermented at 30℃ and 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 results are shown in Figure 2, and the mass spectrometry results are shown in Figure 3.

[0076] After 72 hours of fermentation, no psoralen was detected in Yersinia lipolytica Yl590ΔURA3, while recombinant Yersinia lipolytica S1 was able to synthesize 4.6 mg / L of psoralen.

[0077] Example 2: Construction of a recombinant yeast that synthesizes psoralen using tyrosine as a substrate

[0078] To synthesize psoralen using tyrosine as a substrate, the gene TAL (as shown in the nucleotide sequence of SEQ ID NO:14) was integrated into recombinant strain S1 to obtain recombinant strain S2. Recombinant strains S1 and S2 were inoculated into YPD medium and cultured overnight. S1 and S2 were then cultured at an initial bacterial concentration of OD0.05. 600 =0.2% was inoculated into 50 mL of YPD medium containing 200 mg / L tyrosine, and fermented at 30°C and 220 rpm for 72 h.

[0079] SEQ ID NO:14:

[0080] Recombinant strain S2 was able to synthesize 3.1 mg / L of psoralen, while recombinant strain S1 was unable to synthesize psoralen. This indicates that recombinant strain S2, even with only tyrosine added, can still synthesize psoralen using only tyrosine.

[0081] Example 3: Construction of recombinant yeast for de novo synthesis of psoralen.

[0082] To achieve de novo synthesis of psoralen, the genes ARO4* (nucleotide sequence shown in SEQ ID NO:15) and ARO7* (nucleotide sequence shown in SEQ ID NO:16) were integrated into recombinant strain S2 to obtain recombinant strain S3. Recombinant strain S3 was inoculated into YPD medium and cultured overnight. S3 was then cultured at an initial bacterial concentration of OD0.05. 600 =0.2% was inoculated into 50 mL of YPD medium and fermented at 30°C and 220 rpm for 72 h.

[0083] SEQ ID NO:15:

[0084] SEQ ID NO:16:

[0085] The recombinant strain S3 was able to synthesize 2.3 mg / L of psoralen, which shows that the recombinant strain S3 can still synthesize psoralen using only glucose without the addition of tyrosine or p-coumaric acid.

[0086] Example 4: Construction of a recombinant yeast with high psoralen production using p-coumaric acid as a substrate.

[0087] To further increase the yield of psoralen, recombinant strains S4-S8, which can stably produce high yields of psoralen, were constructed based on the recombinant Yersinia lipolytica S1 obtained in Example 1.

[0088] 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 recombinant strain S1 to obtain recombinant strain S4;

[0089] The gene ERG20* (nucleotide sequence as shown in SEQ ID NO:19) was integrated into recombinant strain S4 to obtain recombinant strain S5;

[0090] The recombinant strain S6 was obtained by integrating 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) into the recombinant strain S5.

[0091] The gene ERG12 (nucleotide sequence as shown in SEQ ID NO:22) was integrated into recombinant strain S6 to obtain recombinant strain S7;

[0092] The gene IDI1 (such as the nucleotide sequence shown in SEQ ID NO:23) was integrated into recombinant strain S7 to obtain recombinant strain S8;

[0093] SEQ ID NO:17:

[0094] SEQ ID NO:18:

[0095] SEQ ID NO:19:

[0096] SEQ ID NO:20:

[0097] SEQ ID NO:21:

[0098] SEQ ID NO:22:

[0099] SEQ ID NO:23:

[0100] Recombinant strains S4-S8 were inoculated into YPD medium and cultured overnight. S4-S8 were then inoculated into 50 mL of YPD medium containing 250 mg / L p-coumaric acid at an initial bacterial concentration of OD600 = 0.2 and fermented at 30℃ and 220 rpm for 72 h. 5 mL of the fermentation broth was collected, centrifuged at 12000 rpm for 2 min, and the supernatant was discarded to collect the bacterial sludge. After extraction and concentration, liquid chromatography analysis was performed. The psoralen content of the recombinant strains S4-S8 after 72 h of fermentation is shown in Table 1.

[0101] Table 1: Fermentation results of different yeast engineered strains constructed in Example 4

[0102] Example 5: Effect of different concentrations of p-coumaric acid on psoralen synthesis

[0103] S8 was set at the initial bacterial concentration OD600 Under the conditions shown in Table 2, the culture was inoculated into 50 mL of YPD medium containing different concentrations of p-coumaric acid (as shown in Table 2), and fermented at 30℃ and 220 rpm for 72 h. 5 mL of the 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 analysis was performed. The contents of psoralen after 72 h of fermentation under different conditions are shown in Table 2.

[0104] Table 2: Yields of psoralen synthesized from p-coumaric acid at different concentrations

[0105] As shown in Table 2, the content of psoralen gradually increased with the increase of coumaric acid concentration, and finally the content of psoralen reached 547.5 mg / L.

[0106] Example 6: Construction of a recombinant yeast with high yield of psoralen using glucose as a substrate

[0107] To improve the ability of the strain to synthesize psoralen de novo, a recombinant strain S9 capable of stable and high-yield psoralen was constructed based on the recombinant Yersinia lipolytica S3 obtained in Example 3.

[0108] The genes MvaS (nucleotide sequence shown in SEQ ID NO:17), MvaE (nucleotide sequence shown in SEQ ID NO:18), ERG20* (nucleotide sequence shown in SEQ ID NO:19), ERG8 (nucleotide sequence shown in SEQ ID NO:20), MVD1 (nucleotide sequence shown in SEQ ID NO:21), ERG12 (nucleotide sequence shown in SEQ ID NO:22), and IDI1 (nucleotide sequence shown in SEQ ID NO:23) were sequentially integrated into recombinant strain S3 to obtain recombinant strain S9;

[0109] The recombinant strain S9 was inoculated into YPD medium and cultured overnight. Then, S9 was inoculated into 50 mL of YPD medium at an initial bacterial concentration of OD600 = 0.2 and fermented at 30°C and 220 rpm for 72 h. The recombinant strain S9 was able to synthesize 89.3 mg / L of psoralen, indicating that this recombinant strain S9 can synthesize a relatively large amount of psoralen using only glucose without the addition of tyrosine or p-coumaric acid.

[0110] Example 7: Constructing a recombinant yeast strain using different enzymes to synthesize bakuchiol using Saccharomyces cerevisiae.

[0111] Using a homologous recombination system, the genes MvaS (nucleotide sequence shown in SEQ ID NO:17) and MvaE (nucleotide sequence shown in SEQ ID NO:18) were integrated into wild-type Saccharomyces cerevisiae CEN.PK2-1C (Taisto Biotechnology Co., Ltd., TS989763) to construct the Erg021 strain. The gene tDN28194 (nucleotide sequence shown in SEQ ID NO:13) was then introduced into the Erg021 strain to construct the recombinant strain Erg021-tDN28194. The gene BAK36(T1) (disclosed in patent application publication number WO2023168043A1) was then introduced into the Erg021 strain to construct the recombinant strain Erg021-BAK36(T1).

[0112] Recombinant strains Erg021-tDN28194 and Erg021-BAK36(T1) were simultaneously inoculated into YPD liquid medium and cultured overnight. Erg021-tDN28194 and Erg021-BAK36(T1) were then cultured at the initial bacterial concentration OD0.05. 600 =0.2% was inoculated into 50 mL of YPD medium containing 250 mg / L p-coumaric acid, and fermented at 30℃ and 220 rpm for 72 h. The recombinant strain Erg021-tDN28194 was able to synthesize 25.4 mg / L of psoralen, while Erg021-BAK36(T1) could only produce 0.1 mg / L of psoralen.

[0113] Comparative Example 1

[0114] The gene BAK36(T1) (disclosed in patent application publication number WO2023168043A1) was introduced into the *Yeastra lipolytica* strain Yl590ΔURA3 constructed in Example 1 above, to construct the Yl590ΔURA3-BAK36(T1) strain. The recombinant strain Yl590ΔURA3-BAK36(T1) and the recombinant *Yeastra lipolytica* S1 from Example 1 were simultaneously inoculated into YPD liquid medium and cultured overnight. Yl590ΔURA3-BAK36(T1) and S1 were cultured at an initial bacterial concentration OD0.05. 600 =0.2g was inoculated into 50mL of YPD medium containing 250mg / L p-coumaric acid, and fermented at 30℃ and 220rpm for 72h. The recombinant strain S1 was able to synthesize 4.6mg / L of psoralen, while Yl590ΔURA3-BAK36(T1), after being ground, crushed and concentrated 100 times, was only detected to contain 0.05mg / L of psoralen.

[0115] Comparative Example 2

[0116] The gene tPcPT7 (disclosed in patent application CN118909814) was introduced into the *Yeastra lipolytica* strain Yl590ΔURA3 constructed in Example 1 above, to construct the Yl590ΔURA3-tPcPT7 strain. The recombinant strain Yl590ΔURA3-tPcPT7 and the recombinant *Yeastra lipolytica* S1 from Example 1 were simultaneously inoculated into YPD liquid medium and cultured overnight. Yl590ΔURA3-tPcPT7 and S1 were cultured at an initial bacterial concentration OD0.05. 600 =0.2% was inoculated into 50 mL of YPD medium containing 250 mg / L p-coumaric acid, and fermented at 30℃ and 220 rpm for 72 h. The recombinant strain S1 was able to synthesize 4.6 mg / L of psoralen, while Yl590ΔURA3-tPcPT7 could only synthesize 0.15 mg / L of psoralen.

[0117] Comparative Example 3

[0118] Different strains and substrates listed in Table 3 were fermented. All cultures were inoculated into 50 mL of YPD medium containing different concentrations and types of substrates at an OD600 concentration of 0.2. Fermentation was carried out at 30℃ and 220 rpm for 72 h. 5 mL of the fermentation broth was centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the bacterial sludge was collected. After extraction and concentration, liquid chromatography analysis was performed. The psoralen content of different strains after 72 h of fermentation is shown in Table 3.

[0119] Table 3:

[0120] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: by using the recombinant Yersinia lipolytica obtained by introducing the isopentenyltransferase encoding gene and other enzymes related to the synthesis pathway of psoralen, a large amount of psoralen can be synthesized efficiently through fermentation culture, which lays an important foundation for the subsequent commercial production of psoralen by Yersinia lipolytica and has significant application value.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. 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; The gene encoding the isopentenyltransferase is derived from Psoralea corylifolia Linn. The amino acid sequence of the isopentenyltransferase is shown in SEQ ID NO:

1.

2. The recombinant yeast according to claim 1, characterized in that, The recombinant yeast also contains tyrosine ammonia lyase 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 also contains any one or more of the following enzymes: 3-deoxy-D-arabinohepulose-7-phosphate synthase or branched acid mutase.

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

11.

6. The recombinant yeast according to claim 4, characterized in that, The recombinant yeast also contains any one or more of the following enzymes: mevalonate-coenzyme A synthase, mevalonate-coenzyme A 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 coenzyme A synthase is shown in SEQ ID NO:2; The amino acid sequence of the mevalonate coenzyme A 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-phosphokinase 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 recombinant yeast according to any one of claims 1-7, characterized in that, The construction method includes: The gene encoding isopentenyltransferase was introduced into Yarrowia lipolytica or Saccharomyces cerevisiae to obtain the recombinant yeast. The gene encoding the isopentenyltransferase is derived from Psoralea corylifolia Linn.

9. The construction method according to claim 8, characterized in that, The construction method includes: introducing the genes encoding the isopentenyltransferase and tyrosine aminolyase into the Yeast lipolyticus or the Saccharomyces cerevisiae to obtain the recombinant yeast.

10. The construction method according to claim 8, characterized in that, The construction method includes: introducing the genes encoding the isopentenyltransferase and tyrosine aminolyase, as well as the genes encoding any one or more of the following enzymes, into the *Yersinia lipolytica* or the *Saccharomyces cerevisiae* to obtain the recombinant yeast: 3-deoxy-D-arabinohepenosose-7-phosphate synthase or branched acid mutase.

11. The construction method according to claim 10, characterized in that, The construction method further includes: introducing a gene encoding any one or more of the following enzymes into the *Yersinia lipolytica* or the *Saccharomyces cerevisiae* to obtain the 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.

12. A method for preparing psoralen, characterized in that, The preparation method includes: The recombinant yeast for producing psoralen according to any one of claims 1-7 was used for fermentation culture to obtain a fermentation broth; The fermentation broth was centrifuged to obtain precipitated bacterial sludge; The precipitated bacterial sludge is extracted, and the resulting solution is psoralen.

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

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