Enzymes for synthesis of glabridin

Abstract

The invention relates to the field of biological engineering and technology, in particular to an enzyme for synthesizing glabridin, a biological synthesis method of glabridin and an engineering-modified biological host. The invention discloses a series of glabridin synthesizing enzymes (4 '-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, Vissisoketoreductase, isoprenyltransferase, pterocarpin reductase, oxidative cyclase and demethylase) from glycyrrhiza glabra for the first time, and the glabridin synthesizing enzymes have corresponding activities in various biological hosts. The heterologous biosynthesis of the glabridin is realized for the first time, and the yield of the glabridin can reach 60% or above.

Description

Technical Field

[0001] The invention relates to the field of bioengineering and technology, and in particular to an enzyme for synthesizing glabridin, a biosynthetic method of glabridin and an engineered biological host. Background Art

[0002] Glabridin is a flavonoid compound extracted from licorice. It has a variety of physiological activities, including anti-inflammatory, antioxidant, antibacterial and tyrosinase inhibition. Therefore, glabridin is widely used in the field of whitening and skin care, and is often used in various cosmetics. In addition, glabridin also shows certain anti-cancer activity and has potential application value in anti-allergy, antiviral and cardiovascular system protection. Due to its natural origin and multiple pharmacological properties, glabridin has gradually become a research hotspot in the fields of drug development, medical beauty and functional food.

[0003] At present, the extraction of glabridin mainly relies on the extraction of licorice from the plant Glycyrrhiza glabra ( Glycyrrhiza glabra L. ) for separation and purification, but the content of glabridin is low, accounting for about 0.09% of glabridin, and it needs to go through multiple steps such as drying, grinding, and crushing, and combined with ultrasound, microwave and other means to assist extraction. The process is complex, the extraction efficiency is low, and the cost is high. In addition, licorice has a well-developed root system, which can prevent wind and sand. It is mainly distributed in Central Asia and Northwest China and has important ecological application value. The acquisition mode of root extraction has seriously damaged the vegetation distribution of licorice and the local ecological environment. The licorice used at this stage mainly comes from imports and artificial cultivation, but artificially cultivated licorice has disadvantages such as long growth cycle, occupation of arable land area and susceptibility to climatic conditions, which seriously limits the production of glabridin. The current method of obtaining glabridin is unsustainable and contrary to the construction of a green and environmentally friendly ecological environment. It is necessary to create a new acquisition method to change the source of raw materials to solve the current problems.

[0004] Therefore, an efficient and environmentally friendly method for synthesizing glycyrrhizin is urgently needed in the relevant technology. Summary of the invention

[0005] The present invention solves one of the problems of the related art from at least the following aspects.

[0006] To this end, the first aspect of the present invention provides an enzyme for synthesizing glabridin, selected from one or more of the following: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; A pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:15.

[0007] The second aspect of the present invention provides a method for the biosynthesis of glycyrrhizin, comprising: providing a biological host; causing the biological host to express one or more enzymes selected from the group consisting of: 4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, visconol reductase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase and demethylase to obtain an engineered biological host; and culturing the engineered biological host to obtain the glycyrrhizin.

[0008] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is selected from one or more of SEQ ID NOs: 1 and 2.

[0009] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is selected from one or more of SEQ ID NOs: 3 and 4.

[0010] In some embodiments, the amino acid sequence of the isoflavone reductase is selected from one or more of SEQ ID NOs: 5 and 6.

[0011] In some embodiments, the amino acid sequence of the visconazole reductase is selected from one or more of SEQ ID NOs: 7 and 8.

[0012] In some embodiments, the amino acid sequence of the prenyl transferase is selected from one or more of SEQ ID NOs: 9, 10, and 16.

[0013] In some embodiments, the amino acid sequence of the pterostilbene reductase is selected from one or more of SEQ ID NOs: 11 and 12.

[0014] In some embodiments, the amino acid sequence of the oxidative cyclase is selected from SEQ ID NO:13.

[0015] In some embodiments, the amino acid sequence of the demethylase is selected from one or more of SEQ ID NOs: 14 and 15.

[0016] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is shown in SEQ ID NO:2.

[0017] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is shown in SEQ ID NO:4.

[0018] In some embodiments, the amino acid sequence of the isoflavone reductase is shown in SEQ ID NO:6.

[0019] In some embodiments, the amino acid sequence of the visconazole reductase is shown in SEQ ID NO:8.

[0020] In some embodiments, the amino acid sequence of the isoprenyl transferase is as shown in SEQ ID NO:10.

[0021] In some embodiments, the amino acid sequence of the pterostilbene reductase is as shown in SEQ ID NO:11 or 12.

[0022] In some embodiments, the amino acid sequence of the oxidative cyclase is shown in SEQ ID NO:13.

[0023] In some embodiments, the amino acid sequence of the demethylase is shown in SEQ ID NO:15.

[0024] In some embodiments, the biological host is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeast, filamentous fungi, and tobacco.

[0025] In some embodiments, the Gram-positive bacteria include Bacillus subtilis ( Bacillus subtilis ) and Corynebacterium glutamicum ( Corynebacterium glutamicum ), the Gram-negative bacteria include Escherichia coli ( Escherichia coli ), the actinomycetes include Streptomyces coelicolor ( Streptomyces coelicolour ), the yeast includes Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ) and Pichia pastoris ( Shepherd's pie ), the filamentous fungi include black mold ( Aspergillus niger ), the tobacco includes Nicotiana benthamiana ( Nicotiana benthamiana ).

[0026] In some embodiments, the glabridin is biosynthesized with a substrate selected from the group consisting of daidzein, formononetin, visconolone, and meditartin.

[0027] In some embodiments, the biological host is Nicotiana benthamiana, and the engineered biological host heterologously expresses: an isoprenyl transferase with an amino acid sequence as shown in SEQ ID NO: 10, a pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO: 11, an oxidative cyclase with an amino acid sequence as shown in SEQ ID NO: 13, and a demethylase with an amino acid sequence as shown in SEQ ID NO: 15, and the substrate for the biosynthesis of the glabridin is medipterostilbene.

[0028] In some embodiments, the biological host is yeast, and the engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:1, isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:3, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:5, visconol reductase with an amino acid sequence as shown in SEQ ID NO:7, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO:16, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO:11, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13, and demethylase with an amino acid sequence as shown in SEQ ID NO:14, and the substrate for the biosynthesis of the glabridin is daidzein.

[0029] In some embodiments, the biological host is yeast, and the engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:2, isoflavone 2''-hydrogenase with an amino acid sequence as shown in SEQ ID NO:4, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:6, visconol reductase with an amino acid sequence as shown in SEQ ID NO:8, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO:10, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO:12, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13, and demethylase with an amino acid sequence as shown in SEQ ID NO:15, and the substrate for the biosynthesis of the glabridin is daidzein.

[0030] A third aspect of the present invention provides an engineered biological host, wherein the engineered biological host expresses one or more enzymes selected from the group consisting of: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; A pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:15.

[0031] In a fourth aspect, an embodiment of the present invention provides the use of a 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO: 1 in the synthesis of formononetin or glabridin.

[0032] In a fifth aspect, an embodiment of the present invention provides use of an isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO: 3 in the synthesis of 2'-hydroxyforononetin or glabridin.

[0033] In a sixth aspect, an embodiment of the present invention provides the use of an isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 5 in the synthesis of visconazole or glabridin.

[0034] The seventh aspect of the present invention provides a use of a visconol reductase having an amino acid sequence as shown in SEQ ID NO: 7 in the synthesis of medipterostilbene or glabridin.

[0035] In an eighth aspect, an embodiment of the present invention provides the use of a prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 9 or 16 in the synthesis of glabridin.

[0036] A ninth aspect of the present invention provides a method for synthesizing glabridin by using a demethylase having an amino acid sequence as shown in SEQ ID NO: 14.

[0037] Compared with the related art, the embodiments of the present invention achieve at least the following beneficial effects: The present invention realizes the full biosynthesis of glabridin by mining a series of enzymes related to the biosynthesis of glabridin from the plant Glycyrrhiza glabrifolia, expressing oxygen methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, visconol reductase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase, and demethylase in vitro. In particular, the present invention realizes the heterologous biosynthesis of glabridin for the first time by screening highly efficient and specific methyltransferase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase, and demethylase, using daidzein and the like as raw materials, and through biocatalysis, and the yield of glabridin can reach more than 60%.

[0038] The complete glabridin biosynthetic pathway and method constructed in the embodiment of the present invention is simple, rapid and environmentally friendly, and is a potential method for realizing large-scale industrial production of glabridin. It is expected to overturn the existing plant extraction method of glabridin, which has a long cycle, low efficiency and long-term occupation of arable land, by digging roots to extract the glabridin. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0040] The present invention is made based on the following knowledge of the inventors: The existing plant extraction method of glabridin, which has a long cycle, low efficiency, and long-term occupation of arable land, is unsustainable and contrary to the construction of a green and environmentally friendly ecological environment. It is necessary to create a new method to change the source of raw materials to solve the current problems. The chemical synthesis method has the disadvantages of low yield, chiral separation of products, harsh reaction process, and complex operation. In the related technology, the research progress on the biosynthesis of glabridin is limited to its precursors 4'-O-methyl pre-glabridin and medipterostilbene.

[0041] The inventors of the present application have discovered a series of enzymes related to the biosynthesis of glabridin from the plant Glycyrrhiza glabra, and have not only realized the in vitro biosynthesis of glabridin for the first time, but have also realized the full biosynthesis of glabridin with daidzein as a substrate by expressing oxygen methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, visconol reductase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase, and demethylase in vitro. In particular, the present invention has realized the heterologous biosynthesis of glabridin for the first time by screening highly efficient and specific methyltransferase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase, and demethylase, using daidzein and the like as raw materials, and through biocatalysis, and the yield of glabridin can reach more than 60%.

[0042] The complete glabridin biosynthetic pathway and method constructed in the embodiment of the present invention is simple, rapid and environmentally friendly, and is a potential method for realizing large-scale industrial production of glabridin. It is expected to overturn the existing plant extraction method of glabridin, which has a long cycle, low efficiency and long-term occupation of arable land, by digging roots to extract the glabridin.

[0043] The first aspect of the present invention provides an enzyme for synthesizing glabridin, selected from one or more of the following: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; A pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:15.

[0044] The second aspect of the present invention provides a method for the biosynthesis of glycyrrhizin, comprising: providing a biological host; causing the biological host to express one or more enzymes selected from the group consisting of: 4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, visconol reductase, isoprenyl transferase, pterostilbene reductase, oxidative cyclase and demethylase to obtain an engineered biological host; and culturing the engineered biological host to obtain the glycyrrhizin.

[0045] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is selected from one or more of SEQ ID NOs: 1 and 2.

[0046] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is selected from one or more of SEQ ID NOs: 3 and 4.

[0047] In some embodiments, the amino acid sequence of the isoflavone reductase is selected from one or more of SEQ ID NOs: 5 and 6.

[0048] In some embodiments, the amino acid sequence of the visconazole reductase is selected from one or more of SEQ ID NOs: 7 and 8.

[0049] In some embodiments, the amino acid sequence of the prenyl transferase is selected from one or more of SEQ ID NOs: 9, 10, and 16.

[0050] In some embodiments, the amino acid sequence of the pterostilbene reductase is selected from one or more of SEQ ID NOs: 11 and 12.

[0051] In some embodiments, the amino acid sequence of the oxidative cyclase is selected from SEQ ID NO:13.

[0052] In some embodiments, the amino acid sequence of the demethylase is selected from one or more of SEQ ID NOs: 14 and 15.

[0053] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is shown in SEQ ID NO:2.

[0054] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is shown in SEQ ID NO:4.

[0055] In some embodiments, the amino acid sequence of the isoflavone reductase is shown in SEQ ID NO:6.

[0056] In some embodiments, the amino acid sequence of the visconazole reductase is shown in SEQ ID NO:8.

[0057] In some embodiments, the amino acid sequence of the isoprenyl transferase is as shown in SEQ ID NO:10.

[0058] In some embodiments, the amino acid sequence of the pterostilbene reductase is as shown in SEQ ID NO:11 or 12.

[0059] In some embodiments, the amino acid sequence of the oxidative cyclase is shown in SEQ ID NO:13.

[0060] In some embodiments, the amino acid sequence of the demethylase is shown in SEQ ID NO:15.

[0061] In some embodiments, the biological host is selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeast, filamentous fungi, and tobacco.

[0062] In some embodiments, the Gram-positive bacteria include Bacillus subtilis ( Bacillus subtilis ) and Corynebacterium glutamicum ( Corynebacterium glutamicum ), the Gram-negative bacteria include Escherichia coli ( Escherichia coli ), the actinomycetes include Streptomyces coelicolor ( Streptomyces coelicolour ), the yeast includes Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ) and Pichia pastoris ( Shepherd's pie ), the filamentous fungi include black mold ( Aspergillus niger ), the tobacco includes Nicotiana benthamiana ( Nicotiana benthamiana ).

[0063] In some embodiments, the glabridin is biosynthesized with a substrate selected from the group consisting of daidzein, formononetin, visconolone, and meditartin.

[0064] In some embodiments, the biological host is Nicotiana benthamiana, and the engineered biological host heterologously expresses: an isoprenyl transferase with an amino acid sequence as shown in SEQ ID NO: 10, a pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO: 11, an oxidative cyclase with an amino acid sequence as shown in SEQ ID NO: 13, and a demethylase with an amino acid sequence as shown in SEQ ID NO: 15, and the substrate for the biosynthesis of the glabridin is medipterostilbene.

[0065] In some embodiments, the biological host is yeast, and the engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:1, isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:3, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:5, visconol reductase with an amino acid sequence as shown in SEQ ID NO:7, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO:16, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO:11, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13, and demethylase with an amino acid sequence as shown in SEQ ID NO:14, and the substrate for the biosynthesis of the glabridin is daidzein.

[0066] In some embodiments, the biological host is yeast, and the engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:2, isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:4, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:6, visconol reductase with an amino acid sequence as shown in SEQ ID NO:8, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO:10, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO:12, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13, and demethylase with an amino acid sequence as shown in SEQ ID NO:15, and the substrate for the biosynthesis of the glabridin is daidzein.

[0067] A third aspect of the present invention provides an engineered biological host, wherein the engineered biological host expresses one or more enzymes selected from the group consisting of: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; A pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:15.

[0068] In a fourth aspect, an embodiment of the present invention provides the use of a 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO: 1 in the synthesis of formononetin or glabridin.

[0069] In a fifth aspect, an embodiment of the present invention provides use of an isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO: 3 in the synthesis of 2'-hydroxyforononetin or glabridin.

[0070] In a sixth aspect, an embodiment of the present invention provides the use of an isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 5 in the synthesis of visconazole or glabridin.

[0071] The seventh aspect of the present invention provides a use of a visconol reductase having an amino acid sequence as shown in SEQ ID NO: 7 in the synthesis of medipterostilbene or glabridin.

[0072] In an eighth aspect, an embodiment of the present invention provides the use of a prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 9 or 16 in the synthesis of glabridin.

[0073] A ninth aspect of the present invention provides a method for synthesizing glabridin by using a demethylase having an amino acid sequence as shown in SEQ ID NO: 14.

[0074] It can be understood that the uses of the embodiments of the fourth to ninth aspects of the present invention include but are not limited to intracellular catalysis, extracellular catalysis, enzyme catalysis, or cell-free catalysis.

[0075] The biological hosts applicable to the uses of the fourth to ninth aspects of the present invention are the same as those of the second aspect, and will not be described in detail here.

[0076] the term As used herein, the "engineered bacteria" or "engineered plants" or "modified biological hosts" refer to fungal cell lines or plants that use genetic engineering methods to efficiently express exogenous genes.

[0077] As used herein, the "starting strain" and "chassis strain" or "biological host" refer to the original strain or plant used for transformation.

[0078] As used herein, the "gene circuit" refers to a gene expression system that contains all the necessary elements required to express a target polypeptide, and generally includes the following elements: a promoter, an encoded reporter gene, and a terminator.

[0079] The terms “comprising” or “including” described in the present invention are open-ended terms. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0080] The term “include” or “comprising” described in the present invention is an open description, which contains the specified components or steps described and other specified components or steps that will not be substantially affected.

[0081] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0082] The correspondence between the abbreviations used in this application and their full names is shown in Table 1 below.

[0083] Table 1

[0084] Source of experimental materials Nicotiana benthamiana Nicotiana benthamiana ) The wild type (WT) is recorded in “Yuan J, Yu Z, Li Y,Shah SHA, Xiao D, Hou X, Li Y. Ectopic expression of BrIQD35 promotes droughtstress tolerance in Nicotiana benthamiana. Plant Biol (Stuttg). 2022 Aug;24(5):887-896.”, which is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0085] Streptomyces coelicolor S. coelicolorA3(2), recorded in “Hopwood DA (1999) Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145(9):2183–2202.”, is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0086] Yarrowia lipolytica ATCC 201249, described in “Du HX, Xiao WH, Wang Y, Zhou X, Zhang Y, Liu D, et al. (2016) Engineering Yarrowia lipolytica for Campesterol Overproduction. PLoS ONE 11(1): e0146773. " is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0087] The present invention will be further described below by the specific embodiment of heterologous biosynthesis of glabridin, the purpose of which is only for illustration and is not intended to limit the scope of the present invention. Without departing from the scope of the claims, those skilled in the art may modify the various aspects of the present invention, but these modifications also belong to the protection scope of the present invention. For example, the cell expression system and the vector plasmid used in the present embodiment may be replaced with other expression systems and vectors commonly used in the art, which can be understood and realized by other persons skilled in the art.

[0088] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0089] Example 1: Expression and functional verification of 4'-O-methyltransferase 4'-O-methyltransferase (GgOMT, amino acid sequence SEQ ID NO:2) from Glycyrrhiza glabra was selected, and gene synthesis was performed according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:17. The gene was amplified by PCR and connected to the Agrobacterium expression vector pBI121 using Gibson assembly. The recombinant plasmid was introduced into the Agrobacterium competent cell EHA105 by electrotransformation. The Agrobacterium clone transformed with the expression plasmid was selected and placed in 1 ml LB medium containing the corresponding antibiotics, and cultured at 28°C with a shaker at 250 rpm for 24 hours. Add 100 μl 0.5 M MES and 2 μl 100 mM AS to 5 ml LB medium containing corresponding antibiotics, and then inoculate 50 μl Agrobacterium culture solution, and culture at 28°C in a shaker at 250 rpm until OD600 = 1.0. Collect the bacteria by centrifugation at 4000 rpm for 10 minutes, resuspend them with 10 mM MgCl2 to OD600 = 1.0, add 100 mM AS at a ratio of 2 μl per ml of bacterial solution, and let it stand for more than 3 hours.

[0090] Take Nicotiana benthamiana ( Nicotiana benthamiana ), use a syringe to suck up the bacterial solution, remove the needle, press the front of the leaf with your finger, and infiltrate the bacterial solution from the back of the leaf. At the same time, inject the reaction substrate 1mM daidzein solution into the leaf. After 24-48 hours, take a sample to test the product.

[0091] The leaves were cut into pieces and then broken by bead beating at -20 °C. The crushed precipitate and the separated supernatant were extracted with equal volumes of ethyl acetate, and the obtained organic phase was evaporated to dryness and reconstituted with methanol. The production of formononetin was detected by ultra-performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0092] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that formononetin was produced.

[0093]

[0094] Example 2: Expression and functional verification of isoflavone 2'-hydrogenase Isoflavone 2'-hydrogenase (GgI2'H, amino acid sequence SEQ ID NO:4) from Glycyrrhiza glabra was selected, and gene synthesis was performed according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:18. The gene was amplified by PCR and connected to the Escherichia coli expression vector pET28a using Gibson assembly. The recombinant plasmid was introduced into the competent Escherichia coli BL21 cell by electroporation. The clone transformed with the expression plasmid was selected and placed in 1 ml LB medium containing the corresponding antibiotics, and cultured at 37°C and 220 rpm for 12 hours. 0.1 ml of bacterial solution was inoculated into 5 ml LB medium containing the corresponding antibiotics. After culturing for 3 hours, the OD value was measured by ultraviolet spectrophotometer. When OD600=0.6-0.8, the shaking temperature was lowered to 16°C, and 1 mM IPTG was added to the bacterial solution for induction. After 10 hours, the reaction substrate 1mM formononetin was added, the shaking temperature was adjusted to 30°C, and the product was sampled and detected after 24-48 hours of reaction.

[0095] The culture solution after the reaction was centrifuged, the supernatant was taken, extracted with an equal volume of ethyl acetate, the obtained organic phase was evaporated to dryness and reconstituted with methanol. The content of the product 2'-hydroxyforononetin was detected by ultra-performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0096] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that 0.96 mM of 2'-hydroxyforononetin could be obtained.

[0097]

[0098] Example 3: Expression and functional verification of isoflavone reductase The isoflavone reductase (GgIFR, amino acid sequence SEQ ID NO:6) from Glycyrrhiza glabra was selected, and the gene was synthesized according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:19. The gene was amplified by PCR and connected to the Saccharomyces cerevisiae expression vector pYES2 by Gibson assembly. The recombinant plasmid was introduced into the competent Escherichia coli DH5α cells by electrotransformation. The clones transformed with the expression plasmid were picked and placed in 1 ml LB medium containing the corresponding antibiotics, and cultured at 37℃ and 220 rpm for 12 hours. The bacteria were obtained by centrifugation and the plasmid was extracted. The plasmid was transformed into Saccharomyces cerevisiae INVSC cells, and the positive clones obtained after transformation were inoculated into SD-Ura medium and cultured at 30℃ and 250 rpm for 16 hours. Then 20 g / L galactose was added for induction, and 1mM 2'-hydroxy-formononetin as the reaction substrate was added at the same time. The reaction was shaken at 30℃ and 220 rpm for 24-48 hours, and the product was sampled and detected.

[0099] The culture solution after the reaction is centrifuged, the supernatant is taken, extracted with an equal volume of ethyl acetate, the obtained organic phase is evaporated to dryness and reconstituted with methanol. The content of the product visconazole is detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0100] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that 0.88 mM of visnezyl could be obtained.

[0101]

[0102] Example 4: Expression and functional verification of visnetolide reductase The visconazole reductase (GgVR, amino acid sequence SEQ ID NO:8) from Glycyrrhiza glabra was selected, and the gene was synthesized according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:20. The gene was amplified by PCR and connected to the Saccharomyces cerevisiae expression vector pAK203 using Gibson assembly. The recombinant plasmid was introduced into the competent Escherichia coli cell DH5α by electroporation. The clones transformed with the expression plasmid were picked and placed in 1 ml LB medium containing the corresponding antibiotics, and cultured at 37°C with a shaker at 220 rpm for 12 hours. The bacteria were centrifuged and the plasmid was extracted. The plasmid was transformed into Streptomyces coelicolor cells through protoplasts, and the positive clones obtained after transformation were inoculated into the culture medium and cultured at 30°C with a shaker at 250 rpm for 24 hours. Then, thiostrepton was added for induction, and 1mM visconazole as the reaction substrate was added at the same time. After the reaction was carried out at 30°C with a shaker at 220 rpm for 24-48 hours, the product was sampled and tested.

[0103] The culture solution after the reaction is centrifuged, the supernatant is taken, extracted with an equal volume of ethyl acetate, the obtained organic phase is evaporated to dryness and reconstituted with methanol. The content of the product visconazole is detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0104] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that 0.9 mM of meditartin could be obtained.

[0105]

[0106] Example 5: Expression and functional verification of prenyltransferase, pterostilbene reductase, oxidative cyclase, and demethylase Glycyrrhiza glabra isoprenyl transferase (GgPT, amino acid sequence SEQ ID NO: 10), glycyrrhiza glabra pterostilbene reductase (GgPTR, amino acid sequence SEQ ID NO: 11), glycyrrhiza glabra oxidative cyclase (GgOC, amino acid sequence SEQ ID NO: 13) and glycyrrhiza glabra demethylase (GgDMT, amino acid sequence SEQ ID NO: 15) were selected, and genes were synthesized according to the amino acid sequences. The corresponding nucleotide sequences are SEQ ID NO: 21, 22, 23, and 24. The genes were PCR amplified and connected to the Agrobacterium expression vector pBI121 using Gibson assembly. The recombinant plasmid was introduced into the Agrobacterium competent cell EHA105 by electrotransformation. The Agrobacterium clone transformed with the expression plasmid was picked and placed in 1 ml LB medium containing the corresponding antibiotics and cultured at 28°C with a shaker at 250 rpm for 24 hours. Add 100 μl 0.5 M MES and 2 μl 100 mM AS to 5 ml LB medium containing corresponding antibiotics, and then inoculate 50 μl Agrobacterium culture solution, and culture at 250 rpm at 28°C until OD600 = 1.0. Collect the bacteria by centrifugation at 4000 rpm for 10 minutes, resuspend them with 10 mM MgCl2 to OD600 = 1.0, add 100 mM AS at a ratio of 2 μl per ml of bacterial solution, and let it stand for more than 3 hours.

[0107] Take Nicotiana benthamiana ( Nicotiana benthamiana ), use a syringe to suck up the bacterial solution, remove the needle, press the front of the leaf with your finger, and infiltrate the bacterial solution from the back of the leaf. At the same time, inject the reaction substrate 1mM medipterostilbene solution into the leaf. After 24-48 hours, take a sample to test the product.

[0108] The leaves were cut into pieces and then broken by bead beating at -20 °C. The crushed precipitate and the separated supernatant were extracted with equal volumes of ethyl acetate, and the obtained organic phase was evaporated to dryness and reconstituted with methanol. The formation of glabridin was detected by ultra-performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0109] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that glabridin was produced.

[0110]

[0111] Example 6: Construction and verification of a pathway for synthesizing glabridin using daidzein as a substrate 4'-O-methyltransferase (P1OMT9, amino acid sequence SEQ ID NO: 1) from kudzu vine, isoflavone 2'-hydrogenase (GeCYP81E1, amino acid sequence SEQ ID NO: 3) from glycyrrhiza uralensis, isoflavone reductase (MsIFR, amino acid sequence SEQ ID NO: 5) from alfalfa, visnezone reductase (MsVR, amino acid sequence SEQ ID NO: 7) from alfalfa, isoprenyltransferase (tPcM4DT, amino acid sequence SEQ ID NO: 16) from pterostilbene (GgPTR, amino acid sequence SEQ ID NO: 11) from glycyrrhiza glabra, oxidative cyclase (GgOC, amino acid sequence SEQ ID NO: 13) from glycyrrhiza glabra, and demethylase (NhDMT, amino acid sequence SEQ ID NO: 14) from Fusarium solani were selected, and gene synthesis was performed according to the amino acid sequences. The corresponding nucleotide sequences are SEQ ID NO: NO: 25, 26, 27, 28, 29, 30, 31, 32. The genes were amplified by PCR and connected into expression cassettes using OE-PCR and Gibson assembly, and constructed into the commonly used vector plasmid pYL15 (GenBank accession: KU378202) of Yarrowia lipolytica. The expression cassette sequence is: P TDH -PlOMT9-T VMA -P FBA -GmCYP81E1-T CYC -P TEF -MsIFR-T CDC -P TEF -MsVR-T ATP -P ENO -tPcM4DT - T ADH -P TDH -Gg PTR -T RPL -P HSP12 -Gg OC -T SOD1 -P HSP26 -Nh DMT -T FBP-URA3. The recombinant plasmid was introduced into the competent E. coli cell DH5α by transformation. The clones transformed with the expression plasmid were picked and placed in 1 ml LB medium containing the corresponding antibiotics, and cultured at 37℃ and 220 rpm for 12 hours. The bacteria were obtained by centrifugation and the plasmid was extracted. The plasmid was transformed into Yarrowia lipolytica ATCC201249, and the positive clones obtained after transformation were inoculated into SD-Ura medium and cultured at 30℃ and 250 rpm for 16 hours. The reaction substrate 5mM soybean aglycone was added, and the reaction was carried out at 30℃ and 220 rpm for 96 hours, and then the product was sampled and tested.

[0112] The culture after the reaction was broken by bead beating, extracted with an equal volume of ethyl acetate, and the obtained organic phase was evaporated to dryness and reconstituted with methanol. The content of the product glabridin was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0113] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that 1.8 mM glabridin could be obtained.

[0114] Example 7: Construction and verification of a pathway for synthesizing glabridin using daidzein as a substrate 4'-O-methyltransferase (GgOMT, amino acid sequence SEQ ID NO: 2) from Glycyrrhiza glabra, isoflavone 2'-hydrogenase (GgI2'H, amino acid sequence SEQ ID NO: 4) from Glycyrrhiza glabra, isoflavone reductase (GgIFR, amino acid sequence SEQ ID NO: 6) from Glycyrrhiza glabra, visconol reductase (GgVR, amino acid sequence SEQ ID NO: 8) from Glycyrrhiza glabra, isoprenyltransferase (GgPT, amino acid sequence SEQ ID NO: 10) from Glycyrrhiza glabra, pterostilbene reductase (GgPTR, amino acid sequence SEQ ID NO: 12) from Glycyrrhiza glabra, oxidative cyclase (GgOC, amino acid sequence SEQ ID NO: 13) from Glycyrrhiza glabra, and demethylase (GgDMT, amino acid sequence SEQ ID NO: 15) from Glycyrrhiza glabra were selected, and genes were synthesized according to the amino acid sequences. The corresponding nucleotide sequences are SEQ ID NO: NO: 33, 34, 19, 35, 36, 37, 38, 39. After PCR amplification, the genes were connected into expression cassette HOL-P using OE-PCR and Gibson assembly. TDH3 -GgOMT-T VMA1 -P FBA1 -GgI2'HT CYC1 -P TEF1 -GgIFR-T CDC19 -P TEF2 -GgVR-T ATP1 -P ENO2 -GgPT - T ADH1 -P TDH1 -Gg PTR -T RPL1 -P HSP12 -Gg OC -T SOD1 -P HSP26 -Gg DMT -T FBP1 -URA3-HOR and connected to the vector pUC19. The recombinant plasmid was introduced into the competent E. coli cell DH5α by transformation. The clone transformed with the expression plasmid was selected and placed in 1 ml LB medium containing the corresponding antibiotics. The clone was cultured at 37℃ and 220 rpm for 12 hours. The bacterial cells were obtained by centrifugation and the plasmid was extracted. TDH3 -GgOMT-T VMA1 -P FBA1 -GgI2'H -T CYC1 -P TEF1 -GgIFR-T CDC19 , ②T CDC19 -P TEF2-GgVR-T ATP1 -P ENO2 -GgPT - T ADH1 -P TDH1 -Gg PTR -T RPL1 -P HSP12 ③P HSP12 -Gg OC -T SOD1 -P HSP26 -Gg DMT -T FBP1 -URA3-HOR were amplified by PCR and then transformed into Saccharomyces cerevisiae BY4741. The positive clones obtained after transformation were inoculated into SD-Ura medium and cultured at 30℃ and 250 rpm for 16 hours. 1mM daidzein was added as the reaction substrate, and the reaction was carried out at 30℃ and 220 rpm for 96 hours before sampling and product detection.

[0115] The culture after the reaction was broken by bead beating, extracted with an equal volume of ethyl acetate, and the obtained organic phase was evaporated to dryness and reconstituted with methanol. The content of the product glabridin was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0116] The fermentation products of the engineered strains were quantitatively determined by UPLC (Agilent 1260). A Poroshell 120 EC-C18 column with a particle size of 2.7 μm and 3.0×100 mm was used. The detection wavelength was 254 nm, the column oven was 35 °C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution process was: 0 min, 20% B; 0–4.5 min, 20%–35% B; 4.5–6 min, 35%–50% B; 6–12 min, 50%–65% B; 12–18 min, 65%–85% B; 18–22.5 min, 85%–95% B; 22.5–24 min, 95%–20% B; and 24–30 min, 20% B. The flow rate was 0.2 mL / min and the injection volume was 1 μL. The external standard method was used for qualitative and quantitative determination. The test results showed that 0.6 mM glabridin could be obtained.

[0117] The enzymes, biological hosts, substrates and products used in the above examples are shown in Table 3. As can be seen from Table 3, the present invention discloses for the first time a series of enzymes for synthesizing Glycyrrhiza glabra derived from Glycyrrhiza glabra, which have corresponding activities in a variety of biological hosts, and realizes the heterologous biosynthesis of glabridin for the first time, and the yield of glabridin can reach more than 60%.

[0118] Table 3

[0119] Sequence information: >SEQ ID NO: 1 (PlOMT9) MGSNNGRKASEIFQGQALLYRHMFAFVDSMCLKSIVELGIPNIIHKHGQPITLSELMSILQVPPAKVGHVQSLMRYLAHHGFFERLRIHEKDAYALTAASELLVKGTEPYLAPMVECMLDPTLSASFHQMKKWVYEEDLSVFDISLGSNLWDFLTKNPAYNEIFNEAMASDSQMSNLALRDCKLVFEGLESIVDVGGGTGTTAKIICEAFPNLKCIVFDRPQVVENLLENNNLTYVGGDMFKSIPKANAVLLKWILHDWTDKDCRKILENCKEAISNNSKRGKIIIIDIVINEKQDEHKVTELKLLFDVAMACVLNGKERNEEEWKKLFMEVGLQDYKISPLTGYLSLIEIYP* >SEQ ID NO: 2 (GgOMT) MDFSTNGSEESELYHAQIHLYKHVYNFVSSMALKSAMELGIADVIHNHGKPITLPQLASALKLHPSKVGVLYRFLRLLTHNGFFAKTTVPSQNGKEGEEEETAYALTPPSKLLVKGKPTCLASIVRGALHPSSLDMWRSSEKWFNEDKELTLFESATGESFWDFLNKDSESGTLSMFQEAMAADSQMFKLALKECRHVFEGLESLVDVGGGTGGVTKLIHEEFPHLKCTVFDQPQVVGNLSGNENLKFVGGDMFKSIPPADAVLLKWVLHDWNDELSLKILKNSKEAISGKGKEGKVIIIDISIDEASGDRELTELQLDYDLVMLTMFNGKEREKKEWEKLISDAGFSSYKITPICGFKSLIEVFP* >SEQ ID NO: 3 (GeCYP81E1) MEILSLLSYSVFYLALFFIFNIVIRARKFKNLPPGPPSLPIIGNLHHLKRPLHRTFKGLSEKYGHVFSLWFGSRLVVVVSSASEFQQCFTKNDVVLANRPRFLSGKYIFYNYTTLGSTSYGEHWRNLRRITALDVLSNHRINSFSGIRRDETQRLITRLADDSSTNFAEMELSSRLYDMTFNNIMRMISGKRYYGEDCDTSDLQEASQFRDMVSELLQLSGANNKTDFMPLLRFLDFENLEKRLKDISGKTDAFLRGLIEEHRTKKERANTMIDHLLNLQDSQPEYYTDQIIKGLALAMLLAGTDSSAVTLEWSMSNLLNHPEVLKKVKDELDTHVGQDRLVDESDLPKLTYLKNVINETLRLYTPAPLLLPHSTSDECNIGGYKVPQDTIVLINAAIHRDPELWTEATTFKPERFEKKGELEKLIAFGMGRRACPGEGLAIRAISMTLALLIQCFDWKLINGDKIDLAERDGFTLTKLVPLKAMCKSRPVINKVFKQ* >SEQ ID NO: 4 (GgI2'H) MEILSLLSYSVFYVALFFICNLLFQARKFKNLPPGPPSLPIIGNLHHLKRPLHRTFKGLSEKYGHVISLWFGSRLVVVVSSLSVFQECFTKNDIVLADRPFLSGKYIFYNYTTVGSSSYGEHWRNLRRRITSLDVLSTHRINSFSGVRRDETQRLIQKLAEESSTDFAEIELTSKFYDMTFNNIMRMMISGKRYYGDDCDMMDMEAKQFRAMVSELLQLSGANNKTDFMPVLRLVDFENLEKRLKKISSKTDTFLRGLIQEHRNKKQHTNTMVDHLLSLQESQPEYYTDQIIKGLALGMLLAGTDSAVTLEWALSCLLNHPEALKKARDELETHVGQDRLLEESDLTKLPYLKNIIYETLRLYTPAPLLLPHSSSDECIIGGFKVPRETIILINAWSIHRDPQIWSEATSFKPERFEKEGELDKLIAFGLGRRACPGEALALRGISLTLGLLIQCFEWKLVGDKEIDMREESGFTLSRLIPLKAMCKARPVANKLVNTQAVSLGD* >SEQ ID NO: 5 (MsIFR) MATENKILILGPTGAIGRHIVWASIKAGNPTYALVRKTPGNVNKPKLITAANPETKEELIDNYQSLGVILLEGDINDHETLVKAIKQVDIVICAAGRLLIEDQVKIIKAIKEAGNVKKFFPSEFGLDVDRHDAVEPVRQVFEEKASIRRVIEAEGVPYT YLCCHAFTGYFLRNLAQLDATDPPRDKVVIlgDGNVKGAYVTEADDVGTFTIRAANDPNTLNKAVHIRLPKNYLTQNEVIALWEKKIGKTLEKTYVSEEKVLKDIQESSFPNHYLLALYHSQQIKGDAVYEIDPAKDIEASEAYPDVTYTTADEYLNQFV* >SEQ ID NO: 6 (GgIFR) MNMIEVGGYPGNPINTKQSNAQQHKPTSALTTYQNKKKKNTHPSFSNSPYFFNNIPMAAENKILILGPTGAIGRHIVWASVKAGNPTFALVRKTNGPVNGPVNKPKLITAANPESKEQLLESYQNAGVTLLEGDINDHESLVAIKQVDVVICATGRLLIDDQVKIIAAIKEAGNVKRFFPSEFGLDVDRHDSVEPVREVFEDKARIRRVIEAEGVPYTYLCCHAFTGYFLRNLAQLDATVPPRDKVVILGDGNVKGAYVTEADVGTYTIRAANDPRTLNKAVHIRLPANYLTANEVVSLWEKKIGKTLEKTYVPEEKVLKDIQESSFPHNYLLALYHSQQIKGDAVYEIDPAKDVEAYDLYSDVKYTTADEYLNQFV* >SEQ ID NO: 7 (MsVR) MAEGKGRVCVTGGTGFLGSWIIKSLENGYSVNTTIRADPERKRDVSFLTNLPGASEKLHFNADLSNPDSFAAAIEGCVGIFHTASPIDFAVSEPEEIVTKRTVDGALGILKACVNSKTVKRFIYTSSGSAVSFNGKDKDVLDESDWSDVDLLRSVKPFGWNYAVSKTLAEKAVLEFGEQNGIDVVTLILPFIVGRFVCPKLPDSIEKALVLVLGKKEQIGVTRFHMVHVDDVARAHIYLLENSVPGGRYNCSPFIVPIEEMSQLLSAKYPEYQILTVDELKEIKGARLPDLNTKKLVDAGFDFKYTIEDMFDDAIQCCKEKGYL* >SEQ ID NO: 8 (GgVR) MAEGKGRVCVTGGTGFLGSWLIKRLLEDGYGVNTTIRSDPERKRDISFLTNLPGASERLKIFNADLSDPESFGPAIEGCVGVFHTASPIDFAVSEPEEIVTKRTVDGTLGILKACKNSKTVKRVVYTSSGAAVSWGGTEKDVLDESDWSDVDMLRSVKPYSWSYAVSKTMTEKAVLEFGEQHGLDVVTLILPFIVGRFICPKIPDSVEKALVLVLGKREQIGVTRFHMVHVEDVARAYIFLLEHPNPKGRYNCSPFIVPIEEVSELLSAKYPEYQIPTVEELKEIKGAKLPDLKSKKLVDAGFEFKYTIEDMFDDAIQCCKEKGYL* >SEQ ID NO:9(PcM4DT) MDWGLAISSSSKAYSVTTGANLWRSKHVTNNIHYPSSCITKASQHKKKTQIEYNVLRFQRPSLGHGYNFSGGGGSTYQECNRKYAVKAVYDQPKDFELEASNPKNILDSAKKFLAAFYYFSYPYTMIGITLCAFCSSALAVEKLSDISLPFFIGVLQAVIPQLFIEIYLSGVNQLYDLEIDKINKPHLPMASGQFSFKTGVIMSAAFLALSFGFTWLTGSWPLIWNLVVIASSWTAYSIDVPFMRWKRYPLVAAMCMIATWGLALPISFFHHMQTFVLQRPIGFPRSLGFLVAFMTFYSMGIALSKDIPDVEGDKEHGINSFSVLLGQKRIFWICVSLFEMAFGVGLLGGVTSPHFWTKIITGLGNVILGSILWYQAKSFDLTDKASTGSFYMYIWKLLYAALLLMAFVR* >SEQ ID NO:10(GgPT) MSSLFGSNVFESISTYRSYAPKASQHKRKIQKEYNFLRSRQPSLKHIYKGIEGGSTQQECNRKYVVKTVPKPSFESDPRSIDQKDILECIKNFLDALYMFTTPSFFSSALYIISVSFIVVEKLSDISPLFFTGVLQAVVPNLFVHIYMTGLNQLCDVEIDKINKPYLPLVSGKISFATGVIVVASCLFLSLWLGWIVGSWPSTCVVISIAMIWTAYSINVPLLRWKKYPVLAAMCIFLSLAVVNPIGYFLHMQTFVFKRPASFSRPLIFVIAFKSFFSLAVALFKDVPDIEGDQTFGVQSFVARFGKKRVFWICISLIEMAYGFALLMGATSAFLWSKIVTVLGHAVLASFVLYRAKSIDLRSKTSITSFYMLIWKLMYMEYFLMPLVR* >SEQ ID NO: 11 (GgPTR) MAETTTSKSKILFIGGTGYIGKFIVEASVNAGHPTFVLIRDSTLSNPAKSPIIDKFKSLAVNLVFGDLYDHQSLVKAIKQVDVVISTVGHLQLGDQDKIISAIKESGNVKRFFPSEFGNDVDRTHAVEPAKSAFATKAKIRRTIEAEGIPYTYVSSNFFAGYFLPTLSQPGATAAPRDKVIILGDGNPKAVANKEEDIATYTIKAVDDPRTLNKILYIRPPANTLSFNELVSLWEKKIGKTLGRVYVPEEQLLKQIQESSPPINVILSIGHSVYVKGDHTNFEIPAFGVEASALYPDVKYTTVDEYLNQFV* >SEQ ID NO: 12 (GgPTR) MDSTAASKILVIGGTGYMGRFLVEASAKAGHPTFALVRHSTVTNPDKSSIIHSFNTLGVNLLLGDINDHQSLVKAIKQADVVISTVNHQYISDQYKIISAIKEAGNIKRFFPSEFGNDVVRTHGVDGAKALFDIKAKFRRTIEAEGIPHTYVVANFLTQHFLPTRSRLLAIAAPLDKVVILGDGNTKATFNTEEGVATFTIRAVDDPRTLNKILYIRPPANTLSYNDLVSLWEKKTGNTLERVYVPEEQVLRLIQESSYPLNMALSICHAAYVKGDHTNYEIEPSFGVEALELYPDVKFTTVDDYLEQNRDCTPFYLNQLIPITNGTKF* >SEQ ID NO:13(GgOC) MAFTYSTKKLALLSIITIFISSFPEPSTSVDLESGFLQCFSSGLGNSNSTAELVLTKNSSSYASLLQSSIRNLRFMGTSVPKPTLIVTPRDLFHIQTSIKCSVKQGLQIRVRSGGHDYEGLSYVSNDDVPFLIIDLTNLRSITIDIKDETAWVQSGATLGELYYAIAKKSNVHGFPAGSCPTVGVGGHFSGGGFGTIFRKYGLAADNVIDAQMVDVNGKILKNRTLMGEDLFWAIRGGGGSSFGVVTAWKVKLVHVPPKVTVFNIPKTLDQNASTLFHKWQIVADKLPGELFLHSVMGVSNANSISSSSGGGRTVLVSFTGLYLGTVENLLPLMQNNFAELGLQHNNCTEMSWIQSVLFFAGYDPINDSLEVLLQRNQTFGSFKAKSDYVMEPIPTSGLEGLWNMLLEENSSPTLILTPYGGRMSEISESETPFPHRNGSIYGIQYLVYWDSSEETPKHIAWMRRVYSYMVLYVSKFPRAAYLNYRDLDIGVNRGNTSYDYEEAKSWGLKYFKCNFERLAKVKAEVDPSNFFRHEQSIPLLF* >SEQ ID NO:14(NhDMT) MLVDTGLGLISELQAKLGWAVLLQIVPITIVAYNLLWFIYASFFSSLRKIPGPFLARISRVWEMKKTATGNIHEIMMDLHRRHGAIVRIGPRRYDFDTMEALKIIYRIGNALPKADYYKPFGLPSFPNLFDEQNPARHSAIKKQVASLYTMTALLSYEEGVDGQTAILKEQLQRFCDQKQVIDLPRFLQYYAFDVIGVITVGKSMGMMESNSDTNGACSALDGMWHYASMMAYIPNMHAWWLRLSSLLPIEVPIKGLTEYVERRIIQYRLKAAEFGDDAALKGENNFLAKLLLMEKKGTVTPVETQQAVGLNIGAGSDTTANALSTILYYLYTNPRTLHTLREELERYVKDGPISFQQSQSMPYLQAVIKEALRLHPGVGTQLTRVVPKGGLVIEGQFFPEGTEVGVNGWALYHNKAIFGNDASIFRPERWLEANENINIGGSFAFGAGSRSCIGKNISILEMSKAIPQIVRNFDIEINHGDMTWKNECWWFVKPEYKAMIKPRRCCLSRDESLV* >SEQ ID NO:15(GgDMT) MEHFYMSLLLLFVTLVSLSLFFLIFYHNKHNMNNNNNLPPGKMGYPVIGESLEFLSMGWKGHPEKFIFDRMVRYSSELIKTSILGVPTVIFCGPACNKFLFSNENKLVTAWWPDSVNKIFPTTSNSKEESKKMRKLLPQFLKPEALQRYVGIMDTLAQRHFASLWEEKTHVTVYPLAKRYTFMLACRLFMSVEDENHVAKFREPFHLLASGIISVPIDLPWTPFNRGIKASNFIRKELLKIIRQRKVDLAQGVASPTQDILSHMLLTCDDENGEFMTELNIADKILGLLIGGHDTASAACTFIVKYLAELPHIYDRVYQEQMEIANSKSPGELLNWDDINKMRYSWNVASEVMRVAPPLQGGFREAINDFVFNGFSIPKGWKLYWSANSTHKNPEYFPAPEKFDPTRFEGNGPAAYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEMLIPEEKIVVDPLPMPANDLPIRLYPHNT* >SEQ ID NO:16(tPcM4DT) ASQHKKKTQIEYNVLRFQRPSLGHGYNFSGGGGSTYQECNRKYAVKAVYDQPKDFELEASNPKNILDSAKKFLAAFYYFSYPYTMIGITLCAFCSSALAVEKLSDISLPFFIGVLQAVIPQLFIEIYLSGVNQLYDLEIDKINKPHLPMASGQFSFKTGVIMSAAFLALSFGFTWLTGSWPLIWNLVVIASSWTAYSIDVPFMRWKRYPLVAAMCMIATWGLALPISFFHHMQTFVLQRPIGFPRSLGFLVAFMTFYSMGIALSKDIPDVEGDKEHGINSFSVLLGQKRIFWICVSLFEMAFGVGLLGGVTSPHFWTKIITGLGNVILGSILWYQAKSFDLTDKASTGSFYMYIWKLLYAALLLMAFVR* >SEQ ID NO:17(GgOMT) >SEQ ID NO:18(GgI2’H) >SEQ ID NO:19(GgIFR) >SEQ ID NO:20(GgVR) ATGGCCGAGGGCAAGGGCCGGGTATGTGTCACGGGCGGCACCGGCTTCCTGGGATCGTGGCTGATCAAGCGTTTGCTCGAGGACGGCTACGGCGTCAACACCACGATCCGGAGTGACCCGGAGCGCAAGCGGGACATCAGCTTCCTCACCAACCTGCCCGGGGCGAGCGAGCGGCTGAAGATCTTCAACGCCGACCTGTCCGACCCCGAATCCTTCGGCCCCGCGATTGAAGGCTGCGTGGGTGTCTTCCACACCGCCTCCCCGATCGACTTCGCCGTCTCGGAGCCCGAGGAGATCGTGACCAAGCGCACCGTCGACGGGACGCTGGGCATCCTCAAGGCCTGCAAGAACTCCAAGACCGTCAAGCGGGTCGTGTACACGTCCAGCGGCGCCGCGGTCAGCTGGGGCGGGACCGAGAAGGACGTGCTCGACGAGAGCGACTGGTCGGATGTGGACATGCTGCGCTCGGTGAAGCCGTACTCCTGGTCCTACGCCGTGAGCAAGACGATGACCGAAAAGGCGGTGCTGGAGTTCGGCGAGCAGCACGGCCTGGACGTCGTCACCCTGATCCTGCCGTTCATCGTCGGCCGCTTCATCTGCCCCAAGATCCCCGACTCCGTCGAGAAGGCGCTGGTCCTGGTGCTCGGCAAGCGCGAGCAGATCGGCGTGACCCGGTTCCACATGGTCCACGTCGAGGACGTGGCCAGGGCCTACATCTTCCTGCTGGAGCACCCGAACCCGAAGGGGCGCTACAACTGCTCCCCCTTCATCGTGCCGATCGAGGAGGTCTCGGAGCTCCTCTCCGCGAAGTACCCAGAGTACCAGATCCCGACGGTGGAGGAACTCAAGGAGATCAAGGGTGCCAAGCTCCCGGACCTCAAATCGAAGAAGCTGGTCGACGCGGGGTTCGAGTTCAAGTACACCATCGAGGACATGTTCGACGACGCCATCCAGTGCTGCAAGGAGAAGGGATACCTGTAA >SEQ ID NO:21(GgPT) >SEQ ID NO:22(GgPTR) ATGGCTGAAACAACTACCTCAAAATCAAAGATTCTTTTCATTGGAGGAACTGGCTACATTGGGAAATTTATTGTTGAAGCTTCTGTTAATGCTGGTCATCCAACTTTTGTGTTAATAAGAGATTCAACTCTGAGCAATCCAGCGAAGTCTCCCATAATTGATAAATTCAAGAGTCTCGCAGTGAACCTCGTTTTTGGGGACTTGTATGATCACCAGAGTCTTGTGAAAGCAATCAAACAAGTTGATGTTGTGATCTCCACCGTTGGACATCTTCAGCTTGGAGATCAAGATAAGATAATATCAGCCATTAAAGAGTCTGGTAATGTTAAGAGGTTTTTCCCTTCTGAATTTGGGAATGATGTCGACCGTACACATGCTGTAGAGCCCGCTAAGTCTGCCTTTGCAACCAAGGCAAAAATCCGCAGGACAATAGAGGCCGAGGGAATTCCATACACTTACGTATCGTCCA ACTTCTTTGCTGGATATTTCTTGCCTACTCTATCTCAACCTGGTGCAACTGCCGCTCCAAGAGACAAGGTTATAATCTTAGGGGATGGAAACCCTAAAGCTGTCGCTAACAAGGAGGAAGATATTGCTACATATACAATCAAGGCGGTGGATGATCCAAGAACATTGAACAAAATACTTTATATTCGGCCGCCGGCAAATACTTTATCTTTCAATGAATTGGTGAGTCTGTGGGAAAAGAAAATTGGTAAAACTCTTGGCCGAGTTTATGTGCCAGAAGAGCAGCTCTTGAAGCAGATTCAAGAATCGAGTCCTCCAATTAATGTCATACTAAGCATCGGCCACTCAGTATATGTTAAAGGTGATCATACAAATTTTGAGATTGAGCCTGCATTTGGTGTCGAAGCATCTGCACTGTATCCTGACGTAAAGTACACGACGGTTGACGAATATTTGAATCAATTTGTTTGA >SEQ ID NO:23(GgOC) >SEQ ID NO:24(GgDMT) >SEQ ID NO:25(PlOMT9) >SEQ ID NO:26(GmCYP81E1) >SEQ ID NO: 27 (MsIFR) ATGGCTACCGAAAACAAGATCTTGATTTTGGGTCCAACTGGTGCCATCGGTAGACACATTGTCTGGGCTTCCATCAAGGCTGGTAACCCTACCTACGCCTTAGTCAGAAAGACCCCAGTAATGTTAACAAGCCAAAGTTGATCACTGCTGCTAACCCAGAAACCAAAGAAGAATTGATTGACAACTACCAATCTTTGGGTGTCATCTTGTTGGAAGGTGACATCAACGACCACGAAAC ATTGGTCAAGGCTATTAAGCAAGTCGATATTGTTATCTGTGCTGCCGGCCGTTTATTGATCGAAGACCAAGTCAAGATAATCAAAGCTATCAAGGAAGCAGGTAACGTCAAGAAGTTCTTCCCATCTGAATTCGGTTTAGACGTTGACAGACATGATGCTGTTGAACCAGTTAGACAAGTTTTTGAAGAAAAAGCTTCCATTAGAAGAGTTATTGAAGCTGAAGGTGTTCCATACACTT ATTTGTGTTGTCACGCTTTCACCGGTTACTTCTTGAGAAACTTGGCTCAATTGGACGCCACCGACCCTCCAAGAGACAAGGTTGTTATTTTAGGTGATGGTAACGTTAAGGGTGCTTACGTTACTGAAGCTGATGTTGGTACTTTTACTATCCGTGCTGCTAATGACCCAAACACTTTAAACAAAGCCGTCCATATCAGATTGCCAAAGAACTACTTGACTCAAAACGAAGTCATTGCT TTGTGGGAAAAGAAGATTGGTAAGACCTTGGAAAAGACTTACGTCTCAGAAGAACAAGTTTTGAAGGATATCCAAGAATCTTCTTTCCCACACACAACTATCTACTGGCTTTGTACCACTCTCAACAAATCAAGGGTGATGCTGTATACGAAATTGATCCAGCTAAGGACATTGAAGCCTCCGAGGCTTACCCAGACGTCACCTACACCACTGCCGATGAATACTTGAACCAATTCGTCTGA >SEQ ID NO:28(MsVR) ATGGCCGAAGGTAAAGGTAGAGTCTGTGTCACCGGTGGTACTGGCTTCTTGGGTTCCTGGATTATAAAGTCTTTATTAGAAAACGGTTACTCCGTCAACACCACTATCAGAGCAGATCCAGAGAGAAAGAGAGATGTCAGTTTCTTGACTAATTTGCCAGGTGCCTCTGAAAAATTGCACTTCTTCAACGCCGACTTGTCTAACCCAGATTCTTTTGCTGCTGCTATTGAAGGTTGTGTTGGTATCTTCCACACTGCTAGCCCAATCGACTTTGCTGTTTCGAACCAGAAGAAATCGTTACCAAGAGAACCGTTGACGGTGCTCTTGGAATCTTGAAAGCTTGTGTTAACTCTAAGACTGTCAAGCGTTTCATTTACACCTCTTCTGGTTCTGCTGTTTCTTTCAATGGTAAGGACAAGGATGTTTTGGACGAATCCGACTGGTCTGACGTCGACTTGTTGAGATCTGTCAAGCCATTCGGTTGGAACT ACGCTGTCTCCAAGACCCTAGCTGAAAAGGCTGTATTGGAATTCGGTGAACAAAACGGTATTGACGTTGTTACTTTGATTTTGCCTTTCATTGTCGGTCGTTTCGTCTGCCCTAAGTTGCCAGACTCCATTGAAAAGGCTTTGGTCCTAGTTTTGGGTAAGAAAGAACAAATTGGTGTTACCAGATTTCACATGGTTCACGTTGATGACGTCGCCAGAGCCCATATCTATTTATTGGAAAACTCCGTTCCAGGTGGTAGATACAACTGTTCTCCCATTCATCGTTCCAATTGAAGAAATGTCACAATTGTTGTCCGCTAAGTACCCAGAATACCAAATCTTGACTGTGGATGAATTGAAGGAAATCAAGGGTGCTAGATTACCAGATTTGAACACTAAGAAGTTAGTCGATGCTGGTTTCGACTTCAAGTACACAATCGAAGACATGTTCGATGATGCTATCCAATGTTGTAAGGAAAAGGGTTACTTGTGA >SEQ ID NO:29(tPcM4DT) >SEQ ID NO:30(GgPTR) ATGGCCGAAACCACCACTTCTAAGTCTAAAATATTGTTTATTGGTGGTACCGGTTACATTGGCAAGTTCATTGTTGAAGCCAGTGTCAATGCCGGTCACCCAACTTTCGTCTTGATTAGAGATAGCACTTTGTCTAACCCAGCGAAGTCCCCAATCATTGATAAATTCAAGTCTTTGGCTGTCAACTTGGTTTTCGGTGACTTGTACGACCACCAATCTTTGGTCAAGGCCATC AAACAAGTTGATGTAGTGATCTCCACAGTCGGTCATTTGCAATTGGGTGACCAAGACAAGATTATTTCCGCTATCAAGGAATCCGGTAATGTCAAGAGATTCTTCCCATCAGAATTCGGTAACGACGTTGACAGAACCCACGCTGTCGAACCAGCTAAATCCGCTTTCGCTACCAAGGCTAAGATCAGACGTACTATCGAAGCTGAAGGTATCCCATACACTTACGTTTCTTCCA ACTTTTTTGCTGGTTACTTCTTGCCAACCTTATCCCAACCAGGTGCTACTGCTGCTCCAAGAGACAAGGTTATCATCTTGGGTGACGGTAACCCAAAGGCTGTTGCTAACAAGGAAGAAGATATTGCTACTTACACTATCAAGGCAGTCGATGACCCACGTACCTTAAACAAGATTTTGTACATCAGACCACCAGCCAACACTCTATCTTTCAACGAATTGGTTTCCTTATGGGA AAAGAAGATCGGTAAGACCTTGGGTAGAGTCTATGTCCCAGAAGAACAACTGTTGAAGCAAATTCAAGAGTCTTCTCCACCTATCAACGTTATCTTATCTATTGGTCACTCTGTTTACGTCAAGGGTGACCACACCAACTTCGAAATTGAACCTGCTTTCGGTGTTGAAGCCTCTGCTTTGTATTCCAGATGTTAAGTACACCACTGTTGATGAATACTTGAACCAATTCGTTTGA >SEQ ID NO:31(GgOC) >SEQ ID NO:32(NhDMT) >SEQ ID NO:33(GgOMT) >SEQ ID NO:34(GgI2’H) >SEQ ID NO:35(GgVR) ATGGCTGAAGGTAAGGGTAGAGTCTGTGTCACCGGTGGTACTGGTTTCCTAGGTTCATGGTTAATCAAGCGTTTATTGGAAGACGGTTACGGCGTCAACACTACCATCAGATCCGACCCAGAAAGAAAGAGAGACATTTCTTTCTTGACTAACTTGCCAGGTGCCTCCGAAAGATTAAAAATCTTCAATGCTGACTTGTCCGACCCAGAATCCTTCGGACCAGCTATTGAAGGTTGTGTTGGTGTCTTCCACACTGCTTCCCCAATTGACTTCGCCGTCAGTGAACCAGAAGAAATTGTCACCAAGCGTACTGTTGATGGTACTTTGGGTATCTTGAAGGCTTGCAAGAACTCCAAGACTGTTAAGAGAGTTGTCTACACCTCTTCTGGTGCTGCTGTCTCTTGGGGTGGTACCGAAAAGGATGTCTTGGACGAATCTGACTGGTCCGATGTGGACATGTTGAGATCTGTCAAGCCATACTCTTGGTCTT ACGCTGTTTCCAAGACCATGACTGAAAAGGCCGTCTTGGAATTCGGTGAACAACACGGTTTGGATGTTGTTACATTGATTTTGCCATTCATTGTCGGTAGATTTATCTGTCCAAAGATCCCTGACTCTGTTGAGAAAGCTTTGGTCCTCGTTTTGGGTAAGAGAGAACAAATCGGTGTTACCAGATTCCACATGGTTCATGTTGAAGACGTTGCTAGAGCTTACATCTTTTTGCTTGAACACCCAAACCCAAAGGGGAGATACAACTGTTCTCCATTTATTGTTCCAATCGAAGAAGTTTCTGAATTGTTGTCTGCCAAATACCCAGAATACCAAATTCCAACCGTTGAAGAATTAAAAGAAATCAAGGGTGCTAAGTTGCCTGATTTGAAGTCTAAAAAGTTAGTTGATGCTGGTTTCGAATTCAAGTACACTATTGAAGATATGTTCGACGATGCCATCCAATGTTGTAAGGAAAAGGGTTATTTGTGA >SEQ ID NO:36(GgPT) >SEQ ID NO:37(GgPTR) ATGGACTCTACTGCTGCCTCCAAGATCTTGGTTATTGGTGGTACTGGTTACATGGGTCGTTTTCTGGTCGAAGCTTCTGCCAAGGCCGGTCATCCTACCTTTGCTTTAGTACGTCACAGTACTGTTACCAACCCAGACAAGTCCTCTATTATTCACTCTTTCAACACCCTAGGCGTCAATCTATTGTTGGGTGACATTAATGATCATCAATCTTTGGTCAAGGCCATCAAGCAAGCTGATGTTGTCATCTCTACCGTTAACCACCAATATATTTCTGACCAATACAAGATTATTAGCGCTATCAAGGAAGCAGGTAACATCAAACGTTTCTTCCCATCTGAATTCGGTAACGACGTTGTCAGAACTCACGGTGTCGACGGTGCTAAGGCTTTGTTCGATATCAAGGCTAAATTCAGAAGAACCATCGAAGCTGAAGGTATCCCTCACACTTACGTCGTTGCCAACTTCTTGACTCAACACTTCTTGCCAACTAGATCCAGATTATTGGCTATTGCTGCTCCATTAGATAAGGTTGTCATATTGGGTGATGGTAACACCAAGGCCACATTCAACACTGAAGAAGGTGTTGCTACCTTCACTATCAGAGCTGTCGACGACCCAAGAACTTTGAACAAGATCTTATACATTAGACCACCAGCTAACACTTTGTCCTACAACGATTTGGTTTCCTTGTGGGAAAAGAAGACCGGTAACACCTTGGAAAGAGTTTATGTTCCAGAAGAACAAGTTTTGAGATTGATCCAAGAATCTTCTTACCCATTGAACATGGCTTTGTCCATCTGTCACGCTGCTTACGTTAAGGGTGACCACACCAACTACGAAATTGAACCATCCTTCGGTGTTGAAGCCTTGGAATTGTACCCAGATGTCAAGTTCACCACTGTGGATGACTACTTGGAACAAAACAGAGACTGTACTCCATTCTACTTAAATCAATTGATTCCAATCACTAACGGTACCAAATTTTGA >SEQ ID NO:38(GgOC) >SEQ ID NO:39(GgDMT) The nucleotide sequences provided above are only used as examples for illustration, and any codon-optimized nucleic acid sequences obtained based on the amino acid sequences are considered to be within the protection scope of the present invention.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0121] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0122] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An enzyme for synthesizing glabridin, characterized in that Select one or more of the following: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; The pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:

15.

2. A method for biosynthesis of glabridin, characterized in that: include: Provide a biological host; causing the biological host to express one or more enzymes selected from the group consisting of: 4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, visconol reductase, prenyltransferase, pterostilbene reductase, oxidative cyclase and demethylase to obtain an engineered biological host; and The engineered biological host is cultured to obtain the glabridin.

3. The biosynthesis method of glabridin according to claim 2, characterized in that: The amino acid sequence of the 4'-O-methyltransferase is selected from one or more of SEQ ID NOs: 1 and 2; Optionally, the amino acid sequence of the isoflavone 2'-hydrogenase is selected from one or more of SEQ ID NOs: 3 and 4; Optionally, the amino acid sequence of the isoflavone reductase is selected from one or more of SEQ ID NOs: 5 and 6; Optionally, the amino acid sequence of the visconazole reductase is selected from one or more of SEQ ID NOs: 7 and 8; Optionally, the amino acid sequence of the prenyltransferase is selected from one or more of SEQ ID NOs: 9, 10 and 16; Optionally, the amino acid sequence of the pterostilbene reductase is selected from one or more of SEQ ID NOs: 11 and 12; Optionally, the amino acid sequence of the oxidative cyclase is selected from SEQ ID NO: 13; Optionally, the amino acid sequence of the demethylase is selected from one or more of SEQ ID NOs: 14 and 15.

4. The biosynthesis method of glabridin according to claim 2 or 3, characterized in that: The amino acid sequence of the 4'-O-methyltransferase is shown in SEQ ID NO: 2; Optionally, the amino acid sequence of the isoflavone 2'-hydrogenase is as shown in SEQ ID NO:4; Optionally, the amino acid sequence of the isoflavone reductase is shown in SEQ ID NO: 6; Optionally, the amino acid sequence of the visconazole reductase is as shown in SEQ ID NO: 8; Optionally, the amino acid sequence of the isoprenyl transferase is as shown in SEQ ID NO: 10; Optionally, the amino acid sequence of the pterostilbene reductase is as shown in SEQ ID NO: 11 or 12; Optionally, the amino acid sequence of the oxidative cyclase is shown in SEQ ID NO: 13; Optionally, the amino acid sequence of the demethylase is shown in SEQ ID NO:

15.

5. The biosynthesis method of glabridin according to claim 2, characterized in that: The biological host is selected from the group consisting of: Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeast, filamentous fungi and tobacco; Optionally, the Gram-positive bacteria include Bacillus subtilis ( Bacillus subtilis ) and Corynebacterium glutamicum ( Corynebacterium glutamicum ), the Gram-negative bacteria include Escherichia coli ( Escherichia coli ), the actinomycetes include Streptomyces coelicolor ( Streptomyces coelicolour ), the yeast includes Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ) and Pichia pastoris ( Pichia pastoris ), the filamentous fungi include black mold ( Aspergillus niger ), the tobacco includes Nicotiana benthamiana ( Nicotiana benthamiana ).

6. The biosynthesis method of glabridin according to claim 2, characterized in that: The glabridin is biosynthesized with a substrate selected from the group consisting of daidzein, formononetin, visconolone and meditartin.

7. The biosynthesis method of glabridin according to claim 2, characterized in that: The biological host is Nicotiana benthamiana, The engineered biological host heterologously expresses: an isoprenyl transferase with an amino acid sequence as shown in SEQ ID NO: 10, a pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO: 11, an oxidative cyclase with an amino acid sequence as shown in SEQ ID NO: 13, and a demethylase with an amino acid sequence as shown in SEQ ID NO: 15, The substrate for the biosynthesis of glabridin is medipterocarpin.

8. The biosynthesis method of glabridin according to claim 2, characterized in that: The biological host is yeast, The engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO: 1, isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO: 3, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO: 5, visconol reductase with an amino acid sequence as shown in SEQ ID NO: 7, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO: 16, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO: 11, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO: 13, and demethylase with an amino acid sequence as shown in SEQ ID NO: 14, The substrate for the biosynthesis of glabridin is daidzein.

9. The biosynthesis method of glabridin according to claim 2, characterized in that: The biological host is yeast, The engineered biological host heterologously expresses: 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:2, isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:4, isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:6, visconol reductase with an amino acid sequence as shown in SEQ ID NO:8, isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO:10, pterostilbene reductase with an amino acid sequence as shown in SEQ ID NO:12, oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13, and demethylase with an amino acid sequence as shown in SEQ ID NO:15, The substrate for the biosynthesis of glabridin is daidzein.

10. An engineered biological host, characterized in that: The engineered biological host expresses one or more enzymes selected from the group consisting of: A 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO:2; Isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO:4; An isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6; The amino acid sequence of visnetoside reductase is shown in SEQ ID NO:8; The isoprenyl transferase having an amino acid sequence as shown in SEQ ID NO: 10; A pterostilbene reductase having an amino acid sequence as shown in SEQ ID NO: 11 or 12; An oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13; The demethylase has an amino acid sequence as shown in SEQ ID NO:

15.

11. Use of a 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO: 1 in the synthesis of formononetin or glabridin.

12. Use of isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO: 3 in the synthesis of 2'-hydroxyforononetin or glabridin.

13. Use of the isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 5 in the synthesis of visconazole or glabridin.

14. Use of visconol reductase having an amino acid sequence as shown in SEQ ID NO: 7 in the synthesis of medipterocarpin or glabridin.

15. Use of the prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 9 or 16 in the synthesis of glabridin.

16. Use of a demethylase having an amino acid sequence as shown in SEQ ID NO: 14 in the synthesis of glabridin.

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