Isopentenyl transferase and application thereof in synthesis of bakuchiol

Abstract

The invention discloses an isopentenyl transferase and an application of the isopentenyl transferase in synthesis of bakuchiol. Specifically, recombinant saccharomyces cerevisiae for expressing the isopentenyl transferase is constructed, the isopentenyl transferase CcPT6 from fructus psoraleae is obtained through screening, the isopentenyl transferase CcPT6 can catalyze an isopentenylation reaction of a coumaric acid substrate to generate bakuchiol, and the activity of the isopentenyl transferase CcPT6 is higher than that of a reported sequence BAK36. The CcPT7 is obtained by truncating 54 amino acids at the N end of the CcPT6, the catalytic activity is improved by 74%, and the activity of the CcPT7 is higher than that of a reported BAK36 mutant. In addition, the invention also provides a recombinant saccharomyces cerevisiae and yarrowia lipolytica engineering strain for integrally expressing the isopentenyl transferase CcPT6 or CcPT7, so that the artificial biosynthesis of the bakuchiol is realized. The discovery of the isopentenyl transferase and the construction of the recombinant engineering strain have important meanings for promoting the biosynthesis of the bakuchiol.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to an isopentenyltransferase and its application in the synthesis of psoralen. Background Technology

[0002] Psoralen is one of the main components of the legume herb *Psoralea corylifolia* Linn., belonging to the terpenoid class of compounds. Since its discovery and isolation in 1966, psoralen and its chemically modified analogues have been recognized for their diverse biological properties, such as anticancer, antioxidant, antibacterial, and liver-protective effects. It can also act as an estrogen, an inhibitor of human carboxylesterase 2 (hCE2), and an immunosuppressive agent. Furthermore, its structural framework has attracted widespread attention in the skincare industry due to its retinol-like functions. Compared to retinol, psoralen has few or no side effects, thus making it a perfect alternative. This evidence suggests that psoralen has the potential for further development, application, and research.

[0003] Although previous attempts have been made to chemically synthesize psoralen, the resulting synthetic schemes have been difficult and inefficient. Most psoralen currently used is extracted from plants through laborious and expensive extraction procedures. Psoralen is synthesized in nature via combinatorial modules based on isopentenyl and amino acids. In 1984, Banerji and Chintalwar, using different labeled precursors on mature Psoralen plants, discovered that the aromatic ring of psoralen originates from the phenylpropane pathway, while the monoterpene side chain originates from the mevalonic acid pathway, with equal label distribution in the isopentenyl pyrophosphate and dimethylallyl pyrophosphate moieties. However, the isopentenyl transferases that catalyze the aromatic ring and monoterpene precursors remain unknown to date.

[0004] Isopentenyltransferases are widely found in the biological world, and plants are rich in isopentenyltransferases. Currently, there are no reports on the synthesis of psoralen from isopentenyltransferases. Therefore, there is an urgent need in this field to develop a method for the natural synthesis of psoralen. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an isopentenyltransferase derived from psoralen. This isopentenyltransferase produces psoralen via a mechanism involving geranyl pyrophosphate (GPP) and p-coumaric acid. Compared to existing technologies, this increases the production yield of psoralen. This invention also provides a nucleic acid encoding the isopentenyltransferase, transgenic cells for producing psoralen, and a method for producing psoralen.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an isolated polypeptide, the polypeptide comprising:

[0008] (a) The amino acid sequence shown in SEQ ID NO:6; or

[0009] (b) A derivative polypeptide of (a), wherein the derivative polypeptide is selected from any of the following polypeptides:

[0010] (b1) A polypeptide formed by adding a tag sequence, signal sequence or secretion signal sequence to SEQ ID NO:6, which catalyzes the production of psoralen and exhibits isopentenyltransferase activity;

[0011] (b2) A polypeptide whose amino acid sequence has ≥85% homology with the amino acid sequence shown in SEQ ID NO:6 (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) and which catalyzes the production of psoralen, exhibiting isopentenyltransferase activity;

[0012] (b3) A polypeptide formed by substituting, deleting or adding one or more amino acid residues of the polypeptide with the amino acid sequence shown in SEQ ID NO:6, which has the ability to catalyze the production of psoralen and exhibits isopentenyltransferase activity.

[0013] In one or more embodiments, the polypeptide is used for in vitro isopentenyl transfer to catalyze the production of psoralen.

[0014] In one specific embodiment, the polypeptide is selected from the amino acid sequence shown in SEQ ID NO:6.

[0015] In a second aspect, the present invention provides an engineered enzyme, the enzyme comprising:

[0016] (a) The amino acid sequence of the N-terminus of the polypeptide described in the first aspect, truncated by 54 amino acids, as shown in SEQ ID NO:22; or

[0017] (b) A derivative polypeptide of (a), wherein the derivative polypeptide is selected from any of the following polypeptides:

[0018] (b1) A polypeptide formed by adding a tag sequence, signal sequence or secretion signal sequence to the polypeptide of (a) and exhibiting isopentenyltransferase activity, which catalyzes the production of psoralen.

[0019] (b2) A polypeptide whose amino acid sequence is ≥85% homology with the amino acid sequence of (a) (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) and which catalyzes the production of psoralen, exhibiting isopentenyltransferase activity;

[0020] (b3) A polypeptide formed by substituting, deleting or adding one or more amino acid residues of the polypeptide in (a), which catalyzes the production of psoralen and exhibits isopentenyltransferase activity.

[0021] In one specific embodiment, the engineered enzyme is selected from the amino acid sequence shown in SEQ ID NO:22.

[0022] This invention utilizes the screened and verified isopentenyltransferase and CcPT6 gene, along with its N-terminal truncated active sequence CcPT7, to express psoralen in Saccharomyces cerevisiae, which can synthesize psoralen and exhibit isopentenyltransferase activity.

[0023] Thirdly, the present invention provides an isolated polynucleotide selected from any one of (A)-(G):

[0024] (A) A nucleotide sequence encoding the polypeptide described in the first aspect or the engineered enzyme described in the second aspect;

[0025] (B) A nucleotide sequence encoding the polypeptide shown in SEQ ID NO:6 or the engineered enzyme or its derivative polypeptide shown in SEQ ID NO:22;

[0026] (C) The nucleotide sequence as shown in SEQ ID NO:12 or 61;

[0027] (D) A nucleotide sequence having at least 85% (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) identity with the sequence shown in SEQ ID NO:12 or 61;

[0028] (E) A nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-10) nucleotides to the 5' end and / or 3' end of the sequence shown in SEQ ID NO:12 or 61;

[0029] The complementary sequence of any of the nucleotide sequences described in (F)(A)-(E);

[0030] Fragments of 20-50 bases in length of the sequences described in (G)(A)-(F);

[0031] Furthermore, the encoded polypeptide catalyzes the production of psoralen, exhibiting isopentenyltransferase activity.

[0032] In one or more embodiments, the polynucleotide sequence is selected from SEQ ID NO:12, 61 or their complementary sequence.

[0033] Fourthly, the present invention provides a nucleic acid construct comprising the polynucleotides described in the third aspect, or expressing the isolated polypeptides described in the first or second aspect. Preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

[0034] Fifthly, the present invention provides a host cell, said host cell being selected from any one of the following (1)-(3):

[0035] (1) Expressing the polypeptide or derivative polypeptide described in the first or second aspect;

[0036] (2) Contains the polynucleotide sequence described in the third aspect; and / or

[0037] (3) Contains the nucleic acid constructs described in the fourth aspect.

[0038] In one or more embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0039] In one or more embodiments, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0040] In one or more embodiments, the host cell is a eukaryotic cell selected from Saccharomyces cerevisiae, Yersinia lipolytica or other yeast species, filamentous fungi, algae, and amoebae.

[0041] In one or more embodiments, the host cell is a Saccharomyces cerevisiae cell, such as Saccharomyces cerevisiae strain W303, expressing the isopentenyltransferase shown in SEQ ID NO. 12 or 61 or containing a recombinant plasmid linking the gene shown in SEQ ID NO. 12 or 61.

[0042] In one specific embodiment, the gene shown in SEQ ID NO.12 or 61 is ligated to the PGK promoter and then to the CYC1 terminator sequence in the Y22-PGK-CYC1 vector, and the recombinant plasmid is then transferred into Saccharomyces cerevisiae W303 to construct recombinant Saccharomyces cerevisiae.

[0043] In one or more embodiments, the host cell is a psoralen cell.

[0044] In one or more embodiments, the host cell is a prokaryotic cell selected from Escherichia coli (E. coll), Acinetobacter, Pseudomonas, Streptomyces and Mycobacterium.

[0045] Sixthly, the present invention provides a method for producing bakuchiol, comprising:

[0046] 1) Culture the host cells described in aspect 5 in a culture medium containing: (a) p-coumaric acid and optionally (b) geranyyl pyrophosphate (GPP), or,

[0047] 2) When the host cells described in the fifth aspect are cultured in the culture medium, coumaric acid and gerany pyrophosphate GPP can be produced endogenously by the host cells or transgenic cells, and no exogenous addition to the cell culture medium is required.

[0048] In one or more embodiments, the method involves inoculating the recombinant Saccharomyces cerevisiae into a culture medium and adding the substrate p-coumaric acid every 10–14 h after inoculation, and culturing for at least 72 h.

[0049] In a seventh aspect, the present invention provides the use of the isolated polypeptide described in the first aspect, the engineered enzyme described in the second aspect, the isolated polynucleotide described in the third aspect, or the host cell described in the fifth aspect, for catalyzing the following reaction, or for preparing a catalytic agent for catalyzing the following reaction: transferring an isopentenyl group from geranyl pyrophosphate GPP to p-coumaric acid to form psoralen.

[0050] In one or more embodiments, the isolated polypeptide is used to catalyze the following reaction, or to prepare a catalyst for catalyzing the following reaction:

[0051]

[0052] Eighthly, the present invention provides a method for producing 3-hydroxypsoralen, comprising:

[0053] 1) Culture the host cells described in aspect 5 in a culture medium containing (a) caffeic acid and (b) geranyyl pyrophosphate (GPP); or,

[0054] 2) When the host cells described in the fifth aspect are cultured in the culture medium, caffeic acid and gerany pyrophosphate GPP can be produced endogenously by the host cells or transgenic cells and do not require exogenous addition to the cell culture medium.

[0055] In one or more embodiments, the method involves inoculating the recombinant Saccharomyces cerevisiae into a culture medium and adding the substrate caffeic acid every 10–14 h after inoculation, and culturing for at least 72 h.

[0056] In a ninth aspect, the present invention provides the use of the isolated polypeptide described in the first aspect, the engineered enzyme described in the second aspect, the isolated polynucleotide described in the third aspect, or the host cell described in the fifth aspect, for catalyzing the following reaction, or for preparing a catalytic agent for catalyzing the following reaction: transferring isopentenyl groups from GPP to caffeic acid to form 3-hydroxypsoralen.

[0057] In one or more embodiments, the isolated polypeptide is used to catalyze the following reaction, or to prepare a catalyst for catalyzing the following reaction:

[0058]

[0059] In a tenth aspect, the present invention provides the use of the polypeptide isolated in the first aspect, the engineered enzyme in the second aspect, the polynucleotide isolated in the third aspect, or the host cell in the fifth aspect in the preparation of cosmetics or agricultural and medical drugs containing psoralen.

[0060] The beneficial effects of this invention are:

[0061] The isopentenyltransferase CcPT6 and its N-terminal truncated active sequence CcPT7, screened and validated in this invention, can synthesize psoralen when expressed in *Saccharomyces cerevisiae*. Their activity is higher than that of the sequence BAK36 reported in US patent application US20230340448A1, with CcPT7 exhibiting a 74% increase in catalytic activity, exceeding that of the BAK36 mutant reported in US20230340448A1. This is of great significance for advancing the biotechnological synthesis of psoralen. Furthermore, the isopentenyltransferase CcPT6 screened and validated in this invention can catalyze caffeic acid substrates to produce 3-hydroxypsoralen, which is of great significance for advancing the biotechnological synthesis of psoralen derivatives. Attached Figure Description

[0062] Figure 1 To verify the catalytic activity of isopentenyltransferase CcPT6, high-performance liquid chromatography (HPLC) results were obtained; the peak elution time of psoralen standard was 22.7 min.

[0063] Figure 2 The results of liquid chromatography-mass spectrometry were used to verify the catalytic activity of isopentenyltransferase CcPT6 on the substrate coumaric acid.

[0064] Figure 3 The results show the activity comparison between the N-terminal truncated active sequence (CcPT7) of isopentenyltransferase CcPT6 and the reported sequences (BAK36 and BAK36-7 site mutant) of US20230340448A1.

[0065] Figure 4The results of liquid chromatography-mass spectrometry (LC-MS) show the catalytic activity of isopentenyltransferase CcPT6 on the substrate caffeic acid. The peak elution time of 3-hydroxypsoralen standard was 21.8 min.

[0066] Figure 5 This is a schematic diagram of the biosynthetic pathway of psoralen in Yeast. Detailed Implementation

[0067] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0068] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. % in the following examples refers to mass percentages.

[0069] The following are the culture media, reagents, and methods used in the embodiments of the present invention:

[0070] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (solid medium with 1.5% agar powder added).

[0071] CM medium: YNB w / o AA (0.67%), glucose (2g / L), Dropout powder (0.083%), wherein the Dropout powder contains (mg / L): threonine 150, tyrosine 30, valine 150, lysine 30, glutamic acid 100, serine 150, aspartic acid 100, methionine 20, phenylalanine 50, isoleucine 30, arginine 20; other nutrients (mg / L): adenine 50, uracil 50, histidine 100, leucine 100, tryptophan 100. The liquid medium is adjusted to pH 5.6, and the solid medium is adjusted to pH 6.5 by adding 1.5% agar powder.

[0072] YPD medium: 1% yeast extract, 2% peptone, 2% glucose, solid medium, with 1.5% agar powder added.

[0073] PCR reaction system:

[0074]

[0075] PCR reaction procedure:

[0076]

[0077] Gibson assembly system

[0078]

[0079] Methods for detecting bakuchiol:

[0080] HPLC analysis:

[0081] Instrument: Shimadzu 1200 high performance liquid chromatograph

[0082] Chromatographic column: Waters C-18 reversed-phase column (4.6×250mm), UV detector, detection wavelength 290nm.

[0083] Mobile phases: Phase A is 0.1% formic acid; Phase B is acetonitrile.

[0084] Initial concentration A: 97% B: 3%

[0085] Flow rate: 0.6 mL / min

[0086] Column temperature: 30℃

[0087] Detector: PDA detector

[0088] Gradient elution program: (concentration is a percentage of phase B)

[0089]

[0090] MS analysis:

[0091] Mass spectrometer: Bruker-microOTOF-II

[0092] ESI ion source, negative ion mode

[0093] Nucleus-to-mass ratio (m / z): 50-1000

[0094] Nitrogen flow rate: 6.0 liters / minute

[0095] Temperature: 180℃

[0096] Atomizer pressure: 1 bar

[0097] Probe voltage: 14.5KV.

[0098] Example 1: Construction of recombinant vectors for CcPT1, CcPT2, CcPT3, CcPT4, CcPT5, CcPT6, BAK36, and BAK36-7 sitemutant genes.

[0099] Through third-generation genome sequencing and transcriptome sequencing analysis of the legume *Psoralea corylifolia*, six potential candidate isopentenyltransferase sequences were predicted and named CcPT1, CcPT2, CcPT3, CcPT4, CcPT5, and CcPT6. Two sequences, BAK36 and its mutant BAK36-7, were obtained through a US patent application [US20230340448A1_Bioproduction of engineered enzymes and psoralen]. The nucleotide sequences of these eight genes were optimized using codons from the *Saccharomyces cerevisiae* host and constructed on the Y22 vector after the PGK promoter and before the CYC1 terminator sequence (synthesized by Nanjing GenScript Biotech Co., Ltd.). The amino acid sequences of the six sequences derived from psoralen are shown in the sequence listings SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6. The codon-optimized nucleotide sequences are shown in the sequence listings SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12. The amino acid sequences of the two sequences obtained in US20230340448A1 are shown in the sequence listings SEQ ID NO.23 and SEQ ID NO.24.

[0100] Example 2: Construction of Y33-ScERG20 F96W、N127W Recombinant vector

[0101] According to the literature Ignea C, Pontini M, Maffei ME, Makris AM, Kampranis SC. Engineering monoterpene production in yeast using a synthetic dominant negative geranyldiphos phate synthase. ACS Synth Biol. 2014 May 16; 3(5):298-306., the F96W and N127W double mutants of GPP synthase Sc ERG20 from Saccharomyces cerevisiae can effectively increase GPP production.

[0102] Using the genome of Saccharomyces cerevisiae S288C (GenBank: GCA_902192305.1) as a template, specific amplification was performed using ScERG20-F (Gibson) (SEQ ID NO.13) and ScERG20-R (Gibson) (SEQ ID NO.14) as primers to obtain the ScERG20 gene fragment, which encodes the endogenous GPP synthase in Saccharomyces cerevisiae. The amplified fragment was then purified by gel extraction.

[0103] The ScERG20 gene fragment and the artificially synthesized bidirectional promoter ADH1+TDH3 ​​were assembled into the artificially synthesized vector YCplac33 (Genbank ID: X75456.1) using Gibson to obtain the recombinant vector Y33-ScERG20.

[0104] Using the recombinant vector Y33-ScERG20 as a template, specific amplification was performed using F96W-F (SEQ ID NO.15) and Sc-Amp-Gibson R (SEQ ID NO.16) to obtain the ScERG20-F96W-UP gene fragment, and the obtained amplified fragment was purified by gel cutting.

[0105] Using the recombinant vector Y33-ScERG20 as a template, specific amplification was performed using F96W-R (SEQ ID NO.18) and Sc-Amp-Gibson F (SEQ ID NO.17) to obtain the ScERG20-F96W-DN gene fragment, and the obtained amplified fragment was purified by gel cutting.

[0106] The ScERG20-F96W-UP and ScERG20-F96W-DN gene fragments were assembled using Gibson to obtain the recombinant vector Y33-ScERG20F96W.

[0107] Using the recombinant vector Y33-ScERG20F96W as a template, specific amplification was performed using N127W-F (SEQ ID NO.19) and primer Sc-Amp-Gibson-R to obtain the ScERG20-F96W+N127W-UP gene fragment, and the obtained amplified fragment was purified by gel cutting.

[0108] Using the recombinant vector Y33-ScERG20F96W as a template, specific amplification was performed using N127W-R (SEQ ID NO.20) and Sc-Amp-Gibson-F to obtain the ScERG20-F96W+N127W-DN gene fragment, and the obtained amplified fragment was purified by gel cutting.

[0109] The ScERG20-F96W+N127W-UP and ScERG20-F96W+N127W-DN gene fragments were assembled using Gibson to obtain the recombinant vector Y33-ScERG20. F96W、N127W ScERG20 F96W、N127W The nucleotide sequence is shown in the sequence listing SEQ ID NO.21.

[0110] Example 3: Construction of recombinant brewer's yeast

[0111] Using conventional lithium acetate conversion methods, the recombinant vectors Y22-CcPT1, Y22-CcPT2, Y22-CcPT3, Y22-CcPT4, Y22-CcPT5, and Y22-CcPT6 described in Example 1 and the recombinant vector Y33-ScERG20 described in Example 2 were respectively converted. F96W、N27W The strain was transformed into *Saccharomyces cerevisiae* W303-1B. In the control strain, the Y22 vector without the foreign gene and the Y33 vector without the foreign gene were transformed into the control strain. Single clones that could grow on CM medium plates with tryptophan and uracil auxotrophic ...

[0112] Example 4: Fermentation Production of Bakuchiol

[0113] Single clones were picked from the plate and placed into test tubes containing 4 mL of sterilized tryptophan and uracil auxotrophic CM medium. The engineered strains of Saccharomyces cerevisiae SC01 (control strain), SC02, SC03, SC04, SC05, SC06 and SC07 were cultured overnight at 30℃ and 200 rpm.

[0114] The seed culture was inoculated into an Erlenmeyer flask containing 50 mL of tryptophan and uracil auxotrophic medium at a ratio of 1:50. After fermentation at 30 °C and 200 rpm for 12-14 h, p-coumaric acid substrate was added to a final concentration of 0.5 mM, and fermentation continued for 72 h.

[0115] Example 5: Extraction and Identification of Reaction Products

[0116] After fermentation, an equal volume of ethyl acetate was added to the fermentation broth in the shake flask. Extraction was performed on a shaker for 1 hour, followed by centrifugation at 5000 rpm for 15 minutes to separate the aqueous and organic phases. The upper organic phase (ethyl acetate) was collected and rotary evaporated. Finally, 200 μL of methanol was added to reconstitute the organic phase, and the solution was transferred to a 2 mL EP tube. Centrifugation was performed at 12000 rpm for 10 minutes. 150 μL of the solution was transferred to a glass vial for high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis. Compared to the control strain SC01, the fermentation sample of strain SC07 showed a new peak at 22.55 minutes, and the elution time was consistent with that of the psoralen standard. Mass spectrometry analysis confirmed the peak as psoralen. (See attached image) Figure 1 , 2 ).

[0117] Example 6: Comparison of psoralen production yields by CcPT6, CcPT7, and BAK36, BAK37-7 site mutants

[0118] In the early stages of the experiment, an active CcPT6 sequence was obtained through screening. The 54 amino acids of its N-terminal chloroplast transport peptide were truncated to obtain CcPT7. The amino acid sequence of CcPT7 is shown in SEQ ID NO. 22, and its nucleotide sequence is shown in SEQ ID NO. 61. Y22-CcPT6, Y22-CcPT7, and Y22-BAK36 and Y22-BAK37-7 site mutants were then coupled with the recombinant vector Y33-ERG20 described in Example 2. F96W、N27W In the Saccharomyces cerevisiae W303-1B strain, the negative control strain was transformed with the Y22 vector without the foreign gene and the Y33 vector without the foreign gene. Single clones that could grow on CM medium plates with tryptophan and uracil auxotrophic ...

[0119] Single clones were picked from the plate and placed into test tubes containing 4 mL of sterilized tryptophan and uracil auxotrophic CM medium. The engineered strains of Saccharomyces cerevisiae SC01 (control strain), SC07, SC08, SC09 and SC010 were cultured overnight at 30℃ and 200 rpm.

[0120] The seed culture was inoculated at a ratio of 1:50 into Erlenmeyer flasks containing 50 mL of tryptophan and uracil auxotrophic CM medium. Fermentation was carried out at 30°C and 200 rpm for 12-14 h. Then, 0.5 mM p-coumaric acid was added, and fermentation continued for another 72 h. Liquid chromatography analysis showed that the SC08 strain carrying Y22-CcPT7 produced the highest psoralen content. Compared to the SC07 strain, its psoralen yield increased by 74%; compared to the highest active sequence reported in patent (US20230340448), the psoralen yield increased by 42%. Figure 3 ).

[0121] Example 7: Substrate spectral analysis of CcPT6

[0122] Single clones were picked from the plate and placed into test tubes containing 4 mL of sterilized tryptophan and uracil auxotrophic CM medium, and cultured overnight at 30°C and 200 rpm for the engineered strains of Saccharomyces cerevisiae SC01 (control strain) and SC07.

[0123] The seed culture was inoculated at a ratio of 1:50 into Erlenmeyer flasks containing 50 mL of tryptophan and uracil auxotrophic CM medium. Fermentation was carried out at 30℃ and 200 rpm for 12-14 h. Then, a final concentration of 1.0 mM of the substrate p-coumaric acid / p-hydroxyphenylpropionic acid / cinnamic acid / p-methylcinnamic acid / 3-hydroxycinnamic acid / caffeic acid / ferulic acid / dihydrocaffeic acid / 3,4-dimethoxyphenylpropionic acid / coumaric acid was added. Fermentation continued for 72 h, followed by extraction and liquid chromatography analysis. The results showed that CcPT6 catalyzed the isopentenylation of the substrate caffeic acid to produce 3-hydroxypsoralen (…). Figure 4 ).

[0124] Example 8: Construction of a recombinant *Yersinia lipophila* strain expressing CcPT6

[0125] The starting strain of Yarrowia lipolytica was W29Δku70, which integrates the Cas9 protein at the KU70 site. It was prepared according to the method described in Holkenbrink C, Dam MI, Kildegaard KR, et al. EasyCloneYALI: CRISPR / Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica[J]. Biotechnology Journal, 2018, 13(9): 1700-543.

[0126] The following gene sequences were constructed into the pUC57 vector (synthesized and constructed by Nanjing GenScript): alfalfa-derived tyrosine ammonia-lyase CluTAL (amino acid and nucleotide sequences are shown in SEQ ID NO. 25 and SEQ ID NO. 26), Rhodotorula glutinis-derived phenylalanine ammonia-lyase RgPAL (amino acid and nucleotide sequences are shown in SEQ ID NO. 27 and SEQ ID NO. 28), EbC4H (amino acid and nucleotide sequences are shown in SEQ ID NO. 29 and SEQ ID NO. 30) and P450 reductase ATR2 gene (amino acid and nucleotide sequences are shown in SEQ ID NO. 31 and SEQ ID NO. 32), and CcPT6 gene (nucleotide sequence is shown in SEQ ID NO. 35) after optimization of the Yersinia lipolytica host codons.

[0127] To construct the integrating strain, a single gRNA vector and homologous donor plasmid for gene knock-in were constructed using the CRISPR / Cas9 system. The original gRNA knockout plasmid vector was pCfB6627, purchased from Addgene (https: / / www.addgene.org / ), catalog number #106159. G5, G18, and G3 knockout vectors were constructed by targeting different integration sites (IntE-1, E13, IntC-3) with the original gRNA plasmid pCfB6627.

[0128] Homologous donor plasmids, i.e., integration vectors, were constructed using pMD-19T as the backbone to create integration vectors p5-IntE-1, p18-E13, and p3-IntC-3 with different integration sites. The integration site sequences are referenced in the literature (Holkenbrink C, Dam MI, Kildegaard KR, et al. EasyCloneYALI: CRISPR / Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica[J]. Biotechnology Journal, 2018, 13(9): 1700543.). The specific construction methods of the three vectors are described in (I).

[0129] (I) Construction of p5, p18 and p3 integration plasmid vectors

[0130] (1) Using the genome of Yersinia lipolyticis W29 as a template, the endogenous strong promoters GPD and TEFIN of Yersinia lipolyticis were amplified by primer pairs pGPD-F (SEQ ID NO.36) / pGPD-R (SEQ ID NO.37) and pTEFIN-F (SEQ ID NO.38) / pTEFIN-R (SEQ ID NO.39), respectively. The fusion bidirectional promoter-1 fragment was then amplified by overlap PCR. The bidirectional promoter-2 fragment was amplified by primer pair tPEX20-pGPD-F (SEQ ID NO.40) / pTEFIN-R as a template.

[0131] (2) The p5, p18 and p3 vectors stored in our laboratory were linearized using primer pairs p-T1-tPEX20-F (SEQ ID NO.41) / p-T1-tLIP2-F (SEQ ID NO.42) to obtain the linearized p5, p18 and p3 vector backbones respectively.

[0132] (3) The CluTAL gene was amplified using primer pair pTEFIN-CluTAL-F (SEQ ID NO.43) / tlip2-CluTAL-R (SEQ ID NO.44); the RgPAL gene was amplified using primer pair pGPD-RgPAL-F (SEQ ID NO.45) / tPEX20-RgPAL-R (SEQ ID NO.46); the ATR2 gene was amplified using primer pair pGPD-ATR2-F (SEQ ID NO.47) / XRP2-ATR2-R (SEQ ID NO.48); the CcPT6 gene was amplified using pTEFIN-CcPT6-F (SEQ ID NO.49) / tlip2-CcPT6L-R (SEQ ID NO.50); ​​and the CcPT6 gene was amplified using primer pair pTEFIN-CYB5-F (SEQ ID NO.51) / tlip2-CYB5-R (SEQ ID NO.44) as a template. The endogenous gene CYB5 of Yersinia lipolytica (amino acid and nucleotide sequences are shown in SEQ ID NO.33 and SEQ ID NO.34) was amplified using primer pair tXPR2-F (SEQ ID NO.53) / tXPR2-R (SEQ ID NO.54) to obtain the endogenous terminator XPR2 of Yersinia lipolytica; the fusion fragment XPR2-pGPD-EbC4H was obtained by overlap PCR amplification.

[0133] (4) The linearized p3 backbone fragment, bidirectional promoter-2 fragment, and CcPT6 gene fragment were assembled using the Gibson assembly method to obtain the recombinant vector P3-CcPT6; the linearized p18 backbone fragment, bidirectional promoter-1 fragment, CluTAL gene fragment, and RgPAL gene fragment were assembled using the Gibson assembly method to obtain the recombinant vector P18-CluTAL-RgPAL. The linearized p5 backbone fragment, bidirectional promoter-1 fragment, CYB5 gene fragment, ATR2 gene fragment, and XPR2-pGPD-EbC4H fragment were assembled using the Gibson assembly method to obtain the recombinant vector P5-CYB5-ATR2-EbC4H. The three recombinant vectors were transformed into DMT competent cells, and positive clones were screened on LB plates containing 100 mg / ml ampicillin using an ampicillin tag. After colony PCR, Sanger sequencing, etc., the correct recombinant plasmids were finally obtained.

[0134] (II) Construction of recombinant Yersinia lipolytica for the production of bakuchiol

[0135] (1) Using the p18-CluTAL-RgPAL recombinant plasmid as a template, the linearized target gene fragment T18-CluTAL-RgPAL (containing the CluTAL gene and the RgPAL gene) was obtained using primers YL-P18-F (SEQ ID NO.55) / YL-P18-R (SEQ ID NO.56); using the p5-CYB5-ATR2-EbC4H recombinant plasmid as a template, the linearized target gene fragment T5-CYB5-ATR2-EbC4H (containing the CYB5 gene, ATR2 gene, and EbC4H gene) was obtained using primers YL-P5-F (SEQ ID NO.57) / YL-P5-R (SEQ ID NO.58); using the p3-CcPT6 recombinant plasmid as a template, the linearized target gene fragment T18-CluTAL-RgPAL (containing the CluTAL gene and the RgPAL gene) was obtained using primers YL-P18-F (SEQ ID NO.55) / YL-P18-R (SEQ ID NO.56). NO.60) Obtain the linearized target gene fragment T3-CcPT6 (containing the CcPT6 gene);

[0136] (2) Using conventional lithium acetate conversion method, T18-CluTAL-RgPAL and its corresponding knockout plasmid were converted into the E13 site of the original strain W29.

[0137] (3) Positive transformants were preferably screened on YPD plates containing hygromycin B (250 mg / L) or norstilbenone (400 mg / L). After yeast transformation, the transformed yeast was plated onto YPD plates and incubated at 30°C for 1-2 days. Then, a single clone was picked and placed into a deep-well plate containing 2.5 mL of YNB and incubated at 30°C and 220 rpm for 24 h. This produced the engineered strain YL-P1.

[0138] (4) Re-inoculate YL-P1 onto a new YPD plate containing hygromycin B and incubate for 1 day to obtain a more successful gRNA marker deletion. Pick a single colony from the culture dish and inoculate it into a 24-well plate containing 2.5 mL of YPD medium.

[0139] (5) T5-CYB5-ATR2-EbC4H and its corresponding knockout plasmid were integrated into the IntE-1 site of YL-P1 by lithium acetate conversion to obtain recombinant strain YL-P2.

[0140] (6) T3-CcPT6 and its corresponding knockout plasmid were integrated into the IntC-3 site of YL-P2 by lithium acetate conversion to obtain recombinant strain YL-P3.

[0141] (III) Fermentation detection of recombinant lipolytic yeast for the production of psoralen.

[0142] The YL-P3 obtained in (II) was inoculated into 2.5 mL of YPD medium for seed activation for 48 h. It was then inoculated into a sterilized shake flask containing YPD medium at a ratio of 1:30. After fermentation at 30 °C and 220 rpm for 4 days, HPLC analysis was performed, and the target product psoralen was quantified as 0.29 mg / L.

[0143] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An isolated polypeptide, characterized in that, The polypeptide comprises: (a) an amino acid sequence as set forth in SEQ ID NO: 6; or (b) a derivative polypeptide of (a) selected from any one of the following polypeptides: (b1) a polypeptide formed by adding a tag sequence, a signal sequence or a secretion signal sequence to SEQ ID NO: 6, and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; (b2) a polypeptide having a homology of > 85% (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) in amino acid sequence to the amino acid sequence as set forth in SEQ ID NO: 6, and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; (b3) a polypeptide formed by substitution, deletion or addition of one or more amino acid residues to the polypeptide of the amino acid sequence as set forth in SEQ ID NO: 6, and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; Preferably, the polypeptide is used for in vitro prenyltransfer, catalyzing the production of bakuchiol. Further preferably, the polypeptide is selected from the amino acid sequence as set forth in SEQ ID NO:

6.

2. An engineered enzyme characterized in that, The enzyme comprises: (a) an amino acid sequence of the polypeptide of claim 1 after N-terminal truncation of 54 amino acids, and the amino acid sequence is as set forth in SEQ ID NO: 22; or (b) a derivative polypeptide of (a) selected from any one of the following polypeptides: (b1) a polypeptide formed by adding a tag sequence, a signal sequence or a secretion signal sequence to the polypeptide of (a), and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; (b2) a polypeptide having a homology of > 85% (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) in amino acid sequence to the polypeptide of (a), and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; (b3) a polypeptide formed by substitution, deletion or addition of one or more amino acid residues to the polypeptide of (a), and having catalytic activity for the production of bakuchiol, exhibiting prenyltransferase activity; Preferably, the polypeptide is used for in vitro prenyltransfer, catalyzing the production of bakuchiol. Further preferably, the engineered enzyme is selected from the amino acid sequence as set forth in SEQ ID NO:

22.

3. An isolated polynucleotide selected from any one of the following (A)-(G): (A) a nucleotide sequence encoding the polypeptide of claim 1 or the engineered enzyme of claim 2; (B) a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 6 or the engineered enzyme as set forth in SEQ ID NO: 22 or a derivative polypeptide thereof; (C) a nucleotide sequence as set forth in SEQ ID NO: 12 or 61. (D) a nucleotide sequence that is at least 85% (preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%) identical to the sequence set forth in SEQ ID NO: 12 or 61; (E) a nucleotide sequence formed by truncating or adding 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5' end and / or 3' end of the sequence set forth in SEQ ID NO: 12 or 61; (F) a complement of the nucleotide sequence of any one of (A)-(E); (G) a fragment of 20-50 bases of any one of (A)-(F); and the polypeptide encoded thereby has catalytic production of bakuchiol, exhibiting prenyltransferase activity. Preferably, the polynucleotide sequence is selected from SEQ ID NO: 12, 61 or a complement thereof.

4. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide of claim 3, or expresses the polypeptide of claim 1 or the engineered enzyme of claim 2. Preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

5. A host cell, characterized in that, The host cell is selected from any one of (1)-(3) below: (1) expressing the polypeptide of claim 1 or the engineered enzyme of claim 2; (2) containing the polynucleotide sequence of claim 3; and / or (3) containing the nucleic acid construct of claim 4; The host cell is a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell. Further preferably, the yeast cell is selected from Saccharomyces cerevisiae, Yarrowia lipolytica or other yeast species, filamentous fungi, algae and amoebae. More preferably, the host cell is a Saccharomyces cerevisiae cell, such as Saccharomyces cerevisiae strain W303, expressing the prenyltransferase set forth in SEQ ID NO. 12 or 61 or containing a recombinant plasmid having the gene set forth in SEQ ID NO. 12 or 61 linked thereto. In a specific embodiment, the gene set forth in SEQ ID NO. 12 or 61 is linked to the PGK promoter in the Y22-PGK-CYC1 vector followed by the CYC1 terminator sequence, and the recombinant plasmid is then transformed into Saccharomyces cerevisiae W303 to construct a recombinant Saccharomyces cerevisiae. Preferably, the host cell is a Psoralea cell. Preferably, the host cell is a prokaryotic cell, selected from E. coll, Acinetobacter, Pseudomonas, Streptomyces and Mycobacterium.

6. A method for producing bakuchiol, characterized in that, The method comprises: 1) culturing the host cell of claim 5 in a culture medium containing (a) p-coumaric acid and (b) geranyl pyrophosphate (GPP); or 2) culturing the host cell of claim 5 in a culture medium, wherein the p-coumaric acid and geranyl pyrophosphate GPP can be endogenously produced by the host cell or transgenic cell and do not need to be exogenously added to the cell culture medium; Preferably, the method is to inoculate the recombinant Saccharomyces cerevisiae into a culture medium and add the substrate p-coumaric acid every 10-14 h after inoculation, and culture for at least 72 h.

7. Use of the isolated polypeptide of claim 1, the engineered enzyme of claim 2, the isolated polynucleotide of claim 3, or the host cell of claim 5, to catalyze the reaction of transferring a prenyl group from geranyl pyrophosphate (GPP) to p-coumaric acid to form a bakuchiol, or to make a catalytic preparation that catalyzes the reaction of transferring a prenyl group from geranyl pyrophosphate (GPP) to p-coumaric acid to form a bakuchiol. Preferably, the isolated polypeptide or engineered enzyme or host cell is used to catalyze one or more of the following reactions, or to make a catalytic preparation that catalyzes one or more of the following reactions:

8. A method for producing 3-hydroxy-4-methylesculetol, characterized by, including: 1) culturing the host cell of claim 5 in a culture medium comprising (a) caffeic acid and (b) geranyl pyrophosphate (GPP); or, 2) culturing the host cell of claim 5 in a culture medium, caffeic acid and geranyl pyrophosphate (GPP) can be endogenously produced by the host cell or transgenic cell and do not need to be exogenously added to the cell culture medium; Preferably, the method is culturing the recombinant S. cerevisiae in a culture medium and adding the substrate caffeic acid every 10-14 h after inoculation, for at least 72 h.

9. Use of the isolated polypeptide of claim 1, the engineered enzyme of claim 2, the isolated polynucleotide of claim 3, or the host cell of claim 5, to catalyze the reaction of transferring a prenyl group from geranyl pyrophosphate (GPP) to caffeic acid to form 3-hydroxybakuchiol, or to make a catalytic preparation that catalyzes the reaction of transferring a prenyl group from geranyl pyrophosphate (GPP) to caffeic acid to form 3-hydroxybakuchiol. Preferably, the isolated polypeptide or engineered enzyme or host cell is used to catalyze the reaction, or to make a catalytic preparation that catalyzes the reaction:

10. Use of the isolated polypeptide of claim 1, the engineered enzyme of claim 2, the isolated polynucleotide of claim 3, or the host cell of claim 5, in the manufacture of a cosmetic or an agricultural or medicinal drug containing bakuchiol.

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