Application of integration site as target spot in production and / or preparation of icaritin

Abstract

The invention relates to the field of bioengineering, in particular to application of integration sites as targets in production and / or preparation of icaritin, and the integration sites comprise one or more of a YPRC [delta] 15 site, a YORW [delta] 22 site, a HO site, a YNRC [delta] 9 site, a PDC5 site, a YCRW [delta] 12 site and a YMRW [delta] 15 site. The saccharomyces cerevisiae is used as a chassis cell, two-step conversion from kaempferol to icaritin is focused on, a novel method for enhancing synthesis of icaritin from the perspective of pathway engineering and enzyme engineering is provided, and an advanced strategy is provided for biosynthesis of flavonoid compounds and natural products.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to the application of integration sites as targets in the production and / or preparation of icariin. Background Art

[0002] Natural products are an important component of medicines and food supplements, and they play an indispensable role in the pharmaceutical and food industries. Among them, flavonoids are aromatic natural compounds with a variety of health-promoting properties, such as anti-inflammatory, antiviral and anti-cancer. As an important flavonoid compound, icariin is the main active ingredient of the traditional Chinese medicine Epimedium, and has a wide range of biological activities and medicinal values. For example, it protects the cardiovascular system, improves immunity and enhances bone formation. As an internationally renowned traditional Chinese medicine, epimedium flavonoids have historically been used as a drug for kidney tonification, aphrodisiac, anti-rheumatic and osteoporosis treatment. In addition, it can inhibit the occurrence and growth of hepatocellular carcinoma and induce apoptosis of acute myeloid leukemia cells. On January 10, 2022, the State Food and Drug Administration approved the conditional listing of icariin soft capsules (Acoradine) for the treatment of advanced hepatocellular carcinoma, which is another major breakthrough in the modernization of traditional Chinese medicine after artemisinin. With the continuous exploration of the biological activity and medical value of icariin, market demand and prices are also rising.

[0003] At present, there are two traditional methods for the source of icariin, including plant extraction and chemical synthesis, both of which have great limitations and shortcomings. The unfriendliness and unsustainability of the environment hinder the commercial application of icariin. First, icariin is an endangered species, and the wild plant resources are limited and have been overexploited in recent years. Secondly, the artificial cultivation of icariin is time-consuming and labor-intensive, and requires harsh environmental conditions. Third, the icariin content in plants is low and difficult to obtain, and the subsequent separation and purification work is costly and challenging. Chemical synthesis usually requires strict reaction conditions and the use of toxic preparations, which runs counter to the green production model. Complex synthetic routes limit the efficient production of icariin. As a natural product with broad application prospects, synthetic biology and metabolic engineering provide an eco-friendly and sustainable method for the production of natural products.

[0004] As an important model organism, Saccharomyces cerevisiae has been used as a chassis strain for the biosynthesis of a variety of natural products, such as naringenin, kaempferol, and icariin, due to its genetic operability, strong robustness, and excellent fermentation performance. Zhou Zhihua's research group collaborated with Wang Ying's team to establish a new icariin biosynthetic pathway in an engineered Saccharomyces cerevisiae strain by introducing 11 heterologous genes. In order to overcome the problem of pH incompatibility mediated by heterologous enzymes and eukaryotic cytoplasm, the researchers located the methyltransferase in Saccharomyces cerevisiae in the mitochondria and complexed it with Escherichia coli, achieving the biosynthesis of icariin with glucose as a carbon source, with yields of 7.2 mg / L and 19.7 mg / L, respectively. However, the process requires two different types of chassis biological relays, which increases the complexity and difficulty of operation, and increases the production cost. For the first time, Li Chun's team cracked the effective reaction sequence of the four-step post-modification of kaempferol to icariin, and successfully built an artificial biosynthetic pathway for the de novo synthesis of icariin, with a yield of 130 μg / L. Summary of the invention

[0005] In view of this, the present invention provides the use of integration sites as targets in the production and / or preparation of icariin. Compared with the existing icariin production methods, the present invention is green and sustainable, with mild conditions, less pollution, and no need for special equipment; at the same time, as a recognized safe strain, the cerevisiae strain has better biosafety and is easier to implement subsequent optimization processes; the present invention combines protein engineering with icariin, increases the yield of icariin, and optimizes substrate preference. The icariin yield obtained by the present invention is the highest yield reported in cerevisiae.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of the integration site as a target in the production and / or preparation of icariin;

[0008] The integration sites include: one or more of a YPRCΔ15 site, a YORWΔ22 site, a HO site, a YNRCΔ9 site, a PDC5 site, a YCRWΔ12 site and a YMRWΔ15 site.

[0009] The present invention also provides an enzyme composition, comprising: endogenous enzymes and / or exogenous enzymes;

[0010] The endogenous enzymes include: one or more of ScCK enzyme, ERG13 enzyme, ERG12 enzyme, ERG8 enzyme, ERG19 enzyme, IDI enzyme and HMGR enzyme;

[0011] The exogenous enzymes include: one or more of EkF8PT or its mutants from Epimedium koreanum, EpPT8 from Epimedium pubescens, LaPT2 from Lupinus albus, AtIPK from Arabidopsis thaliana, MpOMT4 or its mutants from Mentha x piperita, MET13 from Saccharomyces cerevisiae and Mthfr from Arabidopsis thaliana.

[0012] In some embodiments of the present invention, the HMGR in the above enzyme composition has:

[0013] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0014] (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or

[0015] (3) an amino acid sequence that is at least 80% identical to the amino acid sequence of (1) or (2); and / or

[0016] The EkF8PT mutant has:

[0017] (4) the amino acid sequence shown in SEQ ID NO: 2; or

[0018] (5) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or

[0019] (6) an amino acid sequence that is at least 80% identical to the amino acid sequence of (4) or (5); and / or

[0020] The mutant of MpOMT4 has:

[0021] (7) the amino acid sequence shown in SEQ ID NO: 3; or

[0022] (8) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (7), and having the same or similar function as the amino acid sequence shown in (7); or

[0023] (9) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (7) or (8).

[0024] In some embodiments of the present invention, the sequence of SEQ ID NO:1 above is: MAADQLVKTEVTKKSFTAPVQKASTPVLTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKNKEVAALVIHGKLPLYALEKKLGDTTRAVAVRRKALSILAEAPVLASDRLPYKNYDYDRVFGACCENVIGYMPLPVGVIGPLVIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTVLTKDGMTRGPVVRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTCLAGDLLFMRFRTTTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIEGRGKSVVAEATIPGDVVRKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVESSNCITLMKEVDGDLRISVSMPSIEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLAGELSLCAALAAGHLVQSHMTHNRKPAEPTKPNNLDATDINRLKDGSVTCIKS*。

[0025] In some embodiments of the present invention, the sequence of SEQ ID NO:2 above is: MTHENELLFKDKSPTREKGRLSATSSENAPSSFSTKLDMFIKFVRPYATIGIIGNTICMCILPVQTMADLSPRFFIGVAQAIASMVLMNLFNVAVNQVYDVELDKVNKPYLPLASGGVSMTSATLFTILTAALSIALGYFSSPALFYGSIAFFLSASAYSVNFPLLRWKHNALGAIISLMLWGISLQTGVFFHIQQYVLGKPMVLTKSFIYAIIFQSLFSIVAATLKDLPDVEGDKANGSTNLTILIGKEKVFWGSTSLMLATYIGTAAFGATLPVLENKLIIMIAHSAIAVFLWLQAKQTNLADDASTQSYYLLMWKLCNIEYLLIPFVG*。

[0026] In some embodiments of the present invention, the sequence of SEQ ID NO:3 is as follows: MVADEEVRVRAEAWNNAFGYIKPTAVATAVELGLPDILENHDGPMSLLELSAATDCPAEPLHRLMRFLVFHGIFKKTAKPPLSNEAVYYARTALSRLFTRDELGDFMLLQTGPLSQHPAGLTASSLRTGKPQFIRSVNGEDSWTDPVNGYHMKVFSDAMAAHARETTAAIVRYCPAAFEGIGTVVDVGGRHGVALEKLVAAFPWVRGISFDLPEIVAKAPPRPGIEFVGGSFFESVPKGDLVLLMWILHDWSDESCIEIMKKCKEAIPTSGKVMIVDAIVDEDGEGDDFAGARLSLDLIMMALLARGKERTYREWEYLLREAGFTKFVVKNINTVEFVIEAYP*。

[0027] In some embodiments of the present invention, in the above combination, the MET13 derived from Saccharomyces cerevisiae and the Mthfr derived from Arabidopsis thaliana exist in the form of a fusion protein; the sequence of the fusion protein is shown in SEQ ID NO: 7: *.

[0028] The present invention also provides a nucleic acid molecule encoding the above enzyme composition, characterized in that the nucleic acid molecule encoding the HMGR enzyme has:

[0029] (10), the nucleotide sequence shown in SEQ ID NO: 4; or

[0030] (11) A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or

[0031] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence of (10) or (11); and / or

[0032] The nucleic acid molecule encoding the mutant of EkF8PT has:

[0033] (13), the nucleotide sequence shown in SEQ ID NO: 5; or

[0034] (14) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13), and having the same or similar function as the nucleotide sequence shown in (13); or

[0035] (15) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13) or (14); and / or

[0036] The nucleic acid molecule encoding the mutant of MpOMT4 has:

[0037] (16), the nucleotide sequence shown in SEQ ID NO: 6; or

[0038] (17) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (16), and having the same or similar function as the nucleotide sequence shown in (16); or

[0039] (18) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (16) or (17).

[0040]

[0041]

[0042]

[0043]

[0044] The present invention also provides an expression cassette, comprising the above nucleic acid molecule.

[0045] In some embodiments of the present invention, the expression cassette is expressed at the integration site in the above application.

[0046] In some embodiments of the present invention, the expression cassette includes one or more of expression cassettes 1 to 10;

[0047] The expression cassette 1 comprises: GPM1 -IDI-T TEF2 -P TPI1 -tHMGR-T CPS1 ;

[0048] The expression cassette 2 comprises: TDH3 -EkF8PT-T PGK1 ;

[0049] The expression cassette 3 comprises: TDH3 -EpPT8-T PGK1 ;

[0050] The expression cassette 4 comprises: TDH3 -LaPT2-T PGK1 ;

[0051] The expression cassette 5 comprises: TEF1 -ScCK-T ADH1 -P GPM1 -IDI-T TEF2 -P TDH3 -AtIPK-T PGK ;

[0052] The expression cassette 6 comprises: GPM1 -ERG13-T TEF2 -P TPI1 -ERG12-T CPS1 -P TDH3 -ERG8-T PGK1 -P PGK1 -ERG19-T HXT7 ;

[0053] The expression cassette 7 comprises: TDH3 -t61EkF8PT-T PGK1 ;

[0054] The expression cassette 8 comprises: TDH3 -MpOMT4-T PGK ;

[0055] The expression cassette 9 comprises: TEF1-MET13-Mthfr-T ADH1 -P TDH3 -MpOMT4 V303L -T PGK1 .

[0056] In some embodiments of the present invention, the expression cassette 1 is expressed at the YPRCΔ15 site; and / or

[0057] The expression cassette 5 is expressed at the YORWΔ22 site; and / or

[0058] The expression cassette 6 is expressed at the HO site; and / or

[0059] The expression cassette 7 is expressed at the YNRCΔ9 site and the PDC5 site; and / or

[0060] The expression cassette 8 is expressed at the YCRWΔ12 site; and / or

[0061] The above expression cassette 9 is expressed at the YMRWΔ15 site.

[0062] The present invention also provides a recombinant plasmid, comprising: the above expression cassette.

[0063] The present invention also provides a strain for transforming and / or transfecting the above recombinant plasmid;

[0064] The chassis strains for transformation and / or transfection include: Saccharomyces cerevisiae.

[0065] In some embodiments of the present invention, in the above strains, the Saccharomyces cerevisiae is CEN.PK.2-1D.

[0066] The present invention also provides the use of the enzyme composition, the nucleic acid molecule, the expression cassette, the recombinant plasmid and / or the strain in the production and / or preparation of icariin.

[0067] The present invention also provides a method for producing and / or preparing icariin, wherein the above-mentioned strain is inoculated into a substrate and fermented to obtain the icariin;

[0068] The substrate includes: kaempferol.

[0069] Beneficial effects of the present invention:

[0070] (1) Compared with other chassis microorganisms, Saccharomyces cerevisiae has biosafety and good robustness, a clear genetic background, and mature molecular manipulation technology, which facilitates the introduction of exogenous pathways and metabolic regulation;

[0071] (2) Saccharomyces cerevisiae is easy to culture at high density, which can effectively alleviate the problem of substrate and product inhibition and improve the expression level and catalytic efficiency of inefficient enzymes;

[0072] (3) Compared with the published icariin biosynthesis methods, this study utilized different key enzymes and optimized the known synthetic pathways through donor and cofactor enhancement, protein engineering, and optimization.

[0073] (4) Compared with the published methods for the biosynthesis of icariin, the advanced strategy proposed in this study enhanced the production of icariin from multiple angles and multiple sites, providing more reference significance and research targets for the biosynthesis of icariin and other natural products.

[0074] (5) The purpose of the present invention is to fill the gap in the prior art and provide an advanced strategy for strengthening the two rate-limiting steps downstream of icariin biosynthesis, while providing a reference for the biosynthesis of other natural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0076] Figure 1 The titers of icariin of strains yLN002, yLN003, and yLN004 after 72 h of fermentation are shown;

[0077] Figure 2 Shown are schematic diagrams of RMSD within 100ns of molecular dynamics simulation of enzyme MpOMT4 and mutant MpOMT4V303, and schematic diagrams of the binding cavities of enzyme MpOMT4 and mutant MpOMT4V303 with substrate 8-isopentylkaempferol respectively; where: the numbers in the lower left corner of the figure below represent the binding free energies of the two. DETAILED DESCRIPTION

[0078] The invention discloses the application of integration sites as targets in the production and / or preparation of icariin.

[0079] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0080] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0081] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0082] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0083] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0084] The gene involved in the present invention is:

[0085] IDI: YPL117C, SGD: S000006038;

[0086] HMGR: YML075C, SGD: S000004540;

[0087] EkF8PT:QXN66318.1;

[0088] EpPT8:UYI00165.1;

[0089] LaPT2: AWK21939.1;

[0090] ScCK: CP046092.1;

[0091] AtIPK: NP_173986.2;

[0092] ERG13: SGD:S000004595;

[0093] ERG12: SGD:S000004821;

[0094] ERG8: SGD:S000004833;

[0095] ERG9: SGD:S000001233;

[0096] MpOMT4: AAR09602.1;

[0097] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0098] The present invention will be further described below in conjunction with embodiments:

[0099] Example 1 Construction of a Saccharomyces cerevisiae strain capable of screening for isopentenyl transferase

[0100] The strain yLN001 was obtained by integrating endogenous IDI (isopentenyl diphosphate δ-isomerase) and tHMGR (truncated hydroxymethylglutaryl-CoA reductase) into the YPRCΔ15 site of the CEN.PK.2-1D chassis strain.

[0101] Experimental methods:

[0102] PCR amplification of yeast promoter P GPM1 and P TPI1 , terminator T TEF2 and T CPS1 , endogenous IDI and tHMGR genes, and the three were connected into module P by Overlap PCR method. GPM1 -IDI-T TEF2 -P TPI1 -tHMGR-T CPS1 , sequence verification; PCR obtained about 400 bp sequences of Saccharomyces cerevisiae YPRCΔ15, named YPRCΔ15-L and YPRCΔ15-R, respectively, and linked to the promoter P GPM1 and terminator T CPS1 Overlap the sequences to obtain YPRCΔ15-LP GPM1 and YPRCΔ15-RT CPS1 As the connecting homology arm. According to the YPRCΔ15 sequence, a gRNA plasmid (containing the gene encoding the cas9 protein) was constructed. The gRNA plasmid, YPRCΔ15-L-P GPM1 and YPRCΔ15-RT CPS1 Homology arms, module P GPM1 -IDI-T TEF2 -P TPI1 -tHMGR-T CPS1 The gene was introduced into the YPRCΔ15 locus of Saccharomyces cerevisiae by the lithium acetate transformation method, and the SC screening plate was coated. The obtained transformants were streaked for pure culture and the yeast genome was extracted for PCR verification and sequencing. The verified correct transformant was named yLN001.

[0103] Example 2 Verification of a strain capable of converting kaempferol into 8-isopentylkaempferol

[0104] Using strain yLN001 as the chassis, strains yLN002, yLN003 and yLN004 were obtained by expressing EkF8PT from Epimedium koreanum, EpPT8 from Epimedium pubescens and LaPT2 from Lupinus albus through free plasmids, which were able to convert kaempferol into 8-isopentylkaempferol. After 72h of fermentation, the yields were 3.05 mg / L, 2.60 mg / L and 2.82 mg / L, respectively.

[0105] Experimental methods:

[0106] PCR amplification of yeast promoter P TDH3 and terminator T PGK1 , overlapped with the EkF8PT gene to obtain module P TDH3 -EkF8PT-T PGK1 The pRS413 plasmid was digested with SacI and SpeI restriction endonucleases to linearize the vector pRS413 and the module P TDH3 -EkF8PT-T PGK1 Gibson assembly was performed, heat shock transformation was performed into E. coli DH5α competent cells, Amp resistance screening was performed, transformants were picked for preliminary verification by colony PCR, the correct strains were inoculated into LB liquid medium for activation, plasmids were extracted and sequenced for verification, and the correct transformants were maintained. The verified correct plasmid was named pRS413-P TDH3 -EkF8PT-T PGK1 .

[0107] Plasmid pRS413-P was obtained by the same method. TDH3 -EpPT8-T PGK1 and pRS413-P TDH3 -LaPT2-T PGK1 .

[0108] The plasmid pRS413-P was transformed into TDH3 -EpPT8-T PGK1 and pRS413-P TDH3 -LaPT2-T PGK1 They were transformed into the yLN001 strain respectively to obtain strains yLN002, yLN003 and yLN004.

[0109] Experimental results:

[0110] After 72 h of fermentation, with 100 mg / L kaempferol as substrate, strains yLN002, yLN003, and yLN004 produced 3.05 mg / L, 2.60 mg / L, and 2.82 mg / L icariin, respectively. Figure 1 shown.

[0111] Example 3 Construction of a Saccharomyces cerevisiae strain with enhanced isoprenylation

[0112] Based on strain yLN001, ScCK (endogenous choline kinase), AtIPK (isopentenyl phosphate kinase from Arabidopsis thaliana) and IDI were integrated at the YORWΔ22 site, ERG13 (hydroxymethylpentyl-CoA reductase), ERG12 (mevalonate kinase), ERG8 (phosphomevalonate kinase) and ERG19 (mevalonate pyrophosphate decarboxylase) were integrated at the HO site, and t61EkF8PT with a 61-amino acid truncation was integrated at the YNRCΔ9 and PDC5 sites to obtain strain yLN030.

[0113] Experimental methods:

[0114] Referring to the method in Example 1, the modules P TEF1 -ScCK-T ADH1 -P GPM1 -IDI-T TEF2 -P TDH3 -AtIPK-T PGK The YORWΔ22 locus of Saccharomyces cerevisiae was introduced by lithium acetate transformation method, SC screening plates were coated, and the transformants were streaked and pure cultured. The yeast genome was extracted for PCR verification and sequencing. The same method was used to integrate the module P at the HO locus. GPM1 -ERG13-T TEF2 -P TPI1 -ERG12-T CPS1 -P TDH3 -ERG8-T PGK1 -P PGK1 -ERG19-T HXT7 , P was integrated into the YNRCΔ9 and PDC5 loci TDH3 -t61EkF8PT-T PGK1 , to overexpress t61EkF8PT with 61 amino acids truncated, and the correctly sequenced transformant was named yLN030 after plasmid loss through subculture.

[0115] Example 4 Verification of a strain capable of converting 8-isoprenylated kaempferol into icariin

[0116] Based on strain yLN030, MpOMT4 (4'-O methyltransferase from Mentha x piperita) was integrated at the YCRWΔ12 site to obtain strain yLN031, which can convert kaempferol into icariin. After 72h of fermentation, the yield of icariin was 4.77 mg / L, and the yield of byproduct kaempferol (4'methylation product of kaempferol) was 31.6 mg / L.

[0117] Experimental methods:

[0118] Referring to the method in Example 1, the modules P TDH3 -MpOMT4-T PGK The gene was introduced into the YCRWΔ12 locus of Saccharomyces cerevisiae by the lithium acetate transformation method, and the SC screening plate was coated. The obtained transformants were streaked for pure culture and the yeast genome was extracted for PCR verification and sequencing.

[0119] Experimental results:

[0120] After 72 h of fermentation, strain yLN031 produced 4.77 mg / L icariin using 100 mg / L kaempferol as substrate.

[0121] Example 5 Construction of Saccharomyces cerevisiae strains with enhanced SAM supply and overexpression of modified O-methyltransferase

[0122] Based on strain yLN031, MpOMT4 was integrated into the YMRWΔ15 locus V303L The strain yLN036 was obtained by combining MET13-Mthfr (a fusion of MET13 from Saccharomyces cerevisiae and Mthfr from Arabidopsis thaliana), which was able to convert kaempferol to icariin. After 72 h of fermentation, the icariin yield was 14.4 mg / L, and the molar ratio of icariin to byproduct kaempferol was 1.07.

[0123] Experimental methods:

[0124] Referring to the method in Example 1, the modules P TEF1 -MET13-Mthfr-T ADH1 -P TDH3 -MpOMT4 V303L -T PGK1 The gene was introduced into the YMRWΔ15 locus of Saccharomyces cerevisiae by the lithium acetate transformation method, and the SC screening plate was coated. The obtained transformants were streaked for pure culture and the yeast genome was extracted for PCR verification and sequencing.

[0125] Experimental results:

[0126] After 72h fermentation, strain yLN036 produced 14.4 mg / L icariin and 10.9 mg / L byproduct kaempferol with 100 mg / L kaempferol as substrate, and the molar ratio of icariin to byproduct kaempferol was 1.07. Molecular docking and dynamics simulation were used to calculate the root mean square deviation of MpOMT4 and 8-isopentenyl kaempferol, which tended to be stable, indicating that the structure was stable after mutation. The binding free energy between the wild type and mutant of MpOMT4 and 8-isopentenyl kaempferol was calculated by MM / GBSA method, which was -65.4606 and -66.7538 kca / mol, respectively, indicating that the mutation led to a higher affinity between the enzyme and the substrate. From the protein surface model, it can be seen that the mutation changed the shape of the binding pocket, reduced the steric hindrance, and made the enzyme bind more tightly to the substrate.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of integration sites as targets in the production and / or preparation of icariin; The integration sites include: One or more of the YPRCΔ15 site, the YORWΔ22 site, the HO site, the YNRCΔ9 site, the PDC5 site, the YCRWΔ12 site and the YMRWΔ15 site.

2. An enzyme composition, characterized in that include: Endogenous enzymes and / or exogenous enzymes; The endogenous enzymes include: one or more of ScCK enzyme, ERG13 enzyme, ERG12 enzyme, ERG8 enzyme, ERG19 enzyme, IDI enzyme and HMGR enzyme; The exogenous enzymes include: one or more of EkF8PT or its mutants from Epimedium koreanum, EpPT8 from Epimedium pubescens, LaPT2 from Lupinus albus, AtIPK from Arabidopsis thaliana, MpOMT4 or its mutants from Mentha x piperita, MET13 from Saccharomyces cerevisiae and Mthfr from Arabidopsis thaliana.

3. The enzyme composition according to claim 2, characterized in that The HMGR has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (1), and having the same or similar function as the amino acid sequence shown in (1); or (3) an amino acid sequence that is at least 80% identical to the amino acid sequence of (1) or (2); and / or The EkF8PT mutant has: (4) the amino acid sequence shown in SEQ ID NO: 2; or (5) An amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (4), and having the same or similar function as the amino acid sequence shown in (4); or (6) an amino acid sequence that is at least 80% identical to the amino acid sequence of (4) or (5); and / or The mutant of MpOMT4 has: (7) the amino acid sequence shown in SEQ ID NO: 3; or (8) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (7), and having the same or similar function as the amino acid sequence shown in (7); or (9) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (7) or (8).

4. A nucleic acid molecule encoding an enzyme composition according to claim 2 or 3, characterized in that The nucleic acid molecule encoding the HMGR enzyme has: (10), the nucleotide sequence shown in SEQ ID NO: 4; or (11) A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence of (10) or (11); and / or The nucleic acid molecule encoding the mutant of EkF8PT has: (13), the nucleotide sequence shown in SEQ ID NO: 5; or (14) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13), and having the same or similar function as the nucleotide sequence shown in (13); or (15) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13) or (14); and / or The nucleic acid molecule encoding the mutant of MpOMT4 has: (16), the nucleotide sequence shown in SEQ ID NO: 6; or (17) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (16), and having the same or similar function as the nucleotide sequence shown in (16); or (18) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (16) or (17).

5. An expression cassette, characterized in that Comprising the nucleic acid molecule as described in claim 4.

6. The expression cassette according to claim 5, characterized in that The expression cassette is expressed at the integration site in the use of claim 1.

7. A recombinant plasmid, characterized in that include: The expression cassette of claim 5 or 6.

8. A strain characterized in that Transforming and / or transfecting the recombinant plasmid according to claim 7; The chassis strains for transformation and / or transfection include: Saccharomyces cerevisiae.

9. Use of the enzyme composition according to claim 2 or 3, the nucleic acid molecule according to claim 4, the expression cassette according to claim 5 or 6, the recombinant plasmid according to claim 7 and / or the strain according to claim 8 in the production and / or preparation of icariin.

10. A method for producing and / or preparing icariin, characterized in that: Inoculating the strain as claimed in claim 8 into a substrate and fermenting to obtain the icariin; The substrate includes: kaempferol.

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