Enzyme for synthesizing glabridin

Abstract

The present invention relates to the field of bioengineering and technology, and particularly to enzymes for synthesizing glabridin, a method for the biosynthesis of glabridin, and an engineered biological host. The present invention discloses for the first time a series of enzymes derived from Glycyrrhiza glabra for synthesizing glabridin (4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, vestitone reductase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase), which have corresponding activities in a variety of biological hosts, and for the first time realizes the heterologous biosynthesis of glabridin, and the yield of glabridin can reach more than 60%.

Description

Technical Field

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

[0002] Glabridin is a flavonoid compound extracted from licorice. It has various physiological activities, including anti-inflammatory, antioxidant, antibacterial, and inhibiting tyrosinase activity. Therefore, glabridin has a wide range of applications in the field of whitening 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, anti-virus, and protecting the cardiovascular system. Due to its natural source and multiple pharmacological properties, glabridin has gradually become a research hotspot in the fields of drug development, medical aesthetics, and functional foods.

[0003] Currently, the extraction of glabridin mainly relies on the separation and purification from the roots of the plant Glycyrrhiza glabra Glycyrrhiza glabra L. ). However, the content of glabridin is low, accounting for about 0.09% of Glycyrrhiza glabra, and it needs to go through multiple steps such as drying, grinding, and crushing. At the same time, ultrasonic, microwave and other means are combined to assist extraction, with complex processes, low extraction efficiency, and high costs. In addition, the root system of licorice is well-developed, which can prevent wind and fix sand. It is mainly distributed in Central Asia and northwest China and has important ecological application value. The acquisition mode of digging roots for extraction seriously damages the vegetation distribution of licorice and the local ecological environment. At present, the licorice used mainly comes from imports and artificial cultivation, but artificially cultivated licorice has disadvantages such as a long growth cycle, occupying arable land area, and being easily affected by climate conditions, which severely limits the production of glabridin. The current methods for obtaining glabridin are not sustainable and are contrary to building a green and environmentally friendly ecological environment. There is a need to create a new acquisition method that changes the raw material source to solve the existing problems.

[0004] Therefore, there is an urgent need for an efficient and environmentally friendly method for synthesizing glabridin in the related art. Summary of the Invention

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

[0006] To this end, the first aspect embodiment of the present invention provides an enzyme for synthesizing glabridin, selected from one or more of the following:

[0007] 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:2;

[0008] Isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:4;

[0009] Isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:6;

[0010] vestitone reductase having an amino acid sequence as shown in SEQ ID NO:8;

[0011] isoprenyltransferase having an amino acid sequence as shown in SEQ ID NO:10;

[0012] pterocarpan reductase having an amino acid sequence as shown in SEQ ID NO:11 or 12;

[0013] oxidocyclase having an amino acid sequence as shown in SEQ ID NO:13;

[0014] demethylase having an amino acid sequence as shown in SEQ ID NO:15.

[0015] The second aspect embodiment of the present invention provides a method for biosynthesis of glabridin, 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, vestitone reductase, isoprenyltransferase, pterocarpan reductase, oxidocyclase and demethylase to obtain an engineered biological host; and culturing the engineered biological host to obtain the glabridin.

[0016] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is selected from one or more of SEQ ID NO:1 and 2.

[0017] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is selected from one or more of SEQ ID NO:3 and 4.

[0018] In some embodiments, the amino acid sequence of the isoflavone reductase is selected from one or more of SEQ ID NO:5 and 6.

[0019] In some embodiments, the amino acid sequence of the vestitone reductase is selected from one or more of SEQ ID NO:7 and 8.

[0020] In some embodiments, the amino acid sequence of the isoprenyltransferase is selected from one or more of SEQ ID NO:9, 10 and 16.

[0021] In some embodiments, the amino acid sequence of the pterocarpan reductase is selected from one or more of SEQ ID NO:11 and 12.

[0022] In some embodiments, the amino acid sequence of the oxidocyclase is selected from SEQ ID NO:13.

[0023] In some embodiments, the amino acid sequence of the demethylase is selected from one or more of SEQ ID NO: 14 and 15.

[0024] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is as shown in SEQ ID NO: 2.

[0025] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is as shown in SEQ ID NO: 4.

[0026] In some embodiments, the amino acid sequence of the isoflavone reductase is as shown in SEQ ID NO: 6.

[0027] In some embodiments, the amino acid sequence of the vestitone reductase is as shown in SEQ ID NO: 8.

[0028] In some embodiments, the amino acid sequence of the prenyltransferase is as shown in SEQ ID NO: 10.

[0029] In some embodiments, the amino acid sequence of the pterocarpan reductase is as shown in SEQ ID NO: 11 or 12.

[0030] In some embodiments, the amino acid sequence of the oxidative cyclase is as shown in SEQ ID NO: 13.

[0031] In some embodiments, the amino acid sequence of the demethylase is as shown in SEQ ID NO: 15.

[0032] 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.

[0033] 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 include Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), and Pichia pastoris ( Pichia pastoris ), the filamentous fungi include Aspergillus niger ( Aspergillus niger ), and the tobacco includes Nicotiana benthamiana ( Nicotiana benthamiana ).

[0034] In some embodiments, the glabridin is biosynthesized using a substrate selected from the group consisting of daidzein, formononetin, vestitone, and medicarpin.

[0035] In some embodiments, the biological host is Nicotiana benthamiana, and the engineered biological host heterologously expresses: prenyltransferase with an amino acid sequence as shown in SEQ ID NO: 10, pterocarpan 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: 15. The substrate for biosynthesis of the glabridin is medicarpin.

[0036] 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, vestitone reductase with an amino acid sequence as shown in SEQ ID NO: 7, prenyltransferase with an amino acid sequence as shown in SEQ ID NO: 16, pterocarpan 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 biosynthesis of the glabridin is daidzein.

[0037] 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, vestitone reductase with an amino acid sequence as shown in SEQ ID NO: 8, prenyltransferase with an amino acid sequence as shown in SEQ ID NO: 10, pterocarpan 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 biosynthesis of the glabridin is daidzein.

[0038] An embodiment of the 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:

[0039] 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO: 2;

[0040] isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO: 4;

[0041] Isoflavone reductase with an amino acid sequence as shown in SEQ ID NO: 6;

[0042] Vestitone reductase with an amino acid sequence as shown in SEQ ID NO: 8;

[0043] Isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO: 10;

[0044] Pterocarpan reductase with an amino acid sequence as shown in SEQ ID NO: 11 or 12;

[0045] Oxidocyclase with an amino acid sequence as shown in SEQ ID NO: 13;

[0046] Demethylase with an amino acid sequence as shown in SEQ ID NO: 15.

[0047] An embodiment of the fourth aspect of the present invention provides the use of 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO: 1 in the synthesis of formononetin or glabridin.

[0048] An embodiment of the fifth aspect of the present invention provides the use of isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO: 3 in the synthesis of 2'-hydroxyformononetin or glabridin.

[0049] An embodiment of the sixth aspect of the present invention provides the use of isoflavone reductase with an amino acid sequence as shown in SEQ ID NO: 5 in the synthesis of vestitone or glabridin.

[0050] An embodiment of the seventh aspect of the present invention provides the use of vestitone reductase with an amino acid sequence as shown in SEQ ID NO: 7 in the synthesis of medicarpin or glabridin.

[0051] An embodiment of the eighth aspect of the present invention provides the use of isoprenyltransferase with an amino acid sequence as shown in SEQ ID NO: 9 or 16 in the synthesis of glabridin.

[0052] An embodiment of the ninth aspect of the present invention provides the use of demethylase with an amino acid sequence as shown in SEQ ID NO: 14 in the synthesis of glabridin.

[0053] Compared with the related art, the embodiments of the present invention at least achieve the following beneficial effects:

[0054] The present invention has achieved the total biosynthesis of glabridin by mining a series of enzymes related to glabridin biosynthesis from the plant Glycyrrhiza glabra L. var. glabra. The in vitro expression of O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, vestitone reductase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase has realized the total biosynthesis of glabridin. In particular, by screening highly efficient and specific methyltransferase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase, and using daidzein and other raw materials, the heterologous biosynthesis of glabridin has been achieved for the first time through biocatalysis, and the yield of glabridin can reach more than 60%.

[0055] The complete glabridin biosynthesis pathway and method constructed in the examples of the present invention are simple, rapid, and environmentally friendly, and are a potential method for realizing the large-scale industrial production of glabridin, which is expected to subvert the existing plant extraction method for glabridin with a long cycle, low efficiency, and long-term occupation of cultivated land. Detailed implementation manners

[0056] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided examples are only for clarifying the present invention and do not limit the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0057] The present invention is based on the following understanding of the inventors:

[0058] The existing plant extraction method for glabridin with a long cycle, low efficiency, and long-term occupation of cultivated land 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 raw material source to solve the existing problems. The chemical synthesis method has defects such as low yield, the need for chiral resolution of products, harsh reaction conditions, and complex operations. In related technologies, the research progress on the biosynthesis of glabridin stays at its precursors such as 4'-O-methylpreglabrin and medicarpin.

[0059] The inventors of the present application have mined a series of enzymes related to glabridin biosynthesis from the plant Glycyrrhiza glabra L. var. glabra. Not only has the in vitro biosynthesis of glabridin been achieved for the first time, but also the total biosynthesis of glabridin using daidzein as a substrate has been realized by expressing O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, vestitone reductase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase in vitro. In particular, by screening highly efficient and specific methyltransferase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase, and using daidzein and other raw materials, the heterologous biosynthesis of glabridin has been achieved for the first time through biocatalysis, and the yield of glabridin can reach more than 60%.

[0060] The complete biosynthetic pathway and method constructed in the embodiments of the present invention are simple, rapid and environmentally friendly, and are a potential method for realizing the large-scale industrial production of glabridin, which is expected to subvert the existing plant extraction method of glabridin with long cycle, low efficiency and long-term occupation of cultivated land.

[0061] The embodiments of the first aspect of the present invention provide enzymes for synthesizing glabridin, selected from one or more of the following:

[0062] 4'-O-methyltransferase with the amino acid sequence shown in SEQ ID NO:2;

[0063] Isoflavone 2'-hydrogenase with the amino acid sequence shown in SEQ ID NO:4;

[0064] Isoflavone reductase with the amino acid sequence shown in SEQ ID NO:6;

[0065] Vestitone reductase with the amino acid sequence shown in SEQ ID NO:8;

[0066] Isoprenyltransferase with the amino acid sequence shown in SEQ ID NO:10;

[0067] Pterocarpan reductase with the amino acid sequence shown in SEQ ID NO:11 or 12;

[0068] Oxidocyclase with the amino acid sequence shown in SEQ ID NO:13;

[0069] Demethylase with the amino acid sequence shown in SEQ ID NO:15.

[0070] The embodiments of the second aspect of the present invention provide a method for the biosynthesis of glabridin, comprising: providing a biological host; expressing in the biological host one or more enzymes selected from the group consisting of 4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, vestitone reductase, isoprenyltransferase, pterocarpan reductase, oxidocyclase and demethylase to obtain an engineered biological host; and culturing the engineered biological host to obtain the glabridin.

[0071] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is selected from one or more of SEQ ID NO:1 and 2.

[0072] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is selected from one or more of SEQ ID NO:3 and 4.

[0073] In some embodiments, the amino acid sequence of the isoflavone reductase is selected from one or more of SEQ ID NO:5 and 6.

[0074] In some embodiments, the amino acid sequence of the vestitone reductase is selected from one or more of SEQ ID NO:7 and 8.

[0075] In some embodiments, the amino acid sequence of the prenyltransferase is selected from one or more of SEQ ID NO:9, 10 and 16.

[0076] In some embodiments, the amino acid sequence of the pterocarpan reductase is selected from one or more of SEQ ID NO:11 and 12.

[0077] In some embodiments, the amino acid sequence of the oxidative cyclase is selected from SEQ ID NO:13.

[0078] In some embodiments, the amino acid sequence of the demethylase is selected from one or more of SEQ ID NO:14 and 15.

[0079] In some embodiments, the amino acid sequence of the 4'-O-methyltransferase is as shown in SEQ ID NO:2.

[0080] In some embodiments, the amino acid sequence of the isoflavone 2'-hydrogenase is as shown in SEQ ID NO:4.

[0081] In some embodiments, the amino acid sequence of the isoflavone reductase is as shown in SEQ ID NO:6.

[0082] In some embodiments, the amino acid sequence of the vestitone reductase is as shown in SEQ ID NO:8.

[0083] In some embodiments, the amino acid sequence of the prenyltransferase is as shown in SEQ ID NO:10.

[0084] In some embodiments, the amino acid sequence of the pterocarpan reductase is as shown in SEQ ID NO:11 or 12.

[0085] In some embodiments, the amino acid sequence of the oxidative cyclase is as shown in SEQ ID NO:13.

[0086] In some embodiments, the amino acid sequence of the demethylase is as shown in SEQ ID NO:15.

[0087] 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.

[0088] 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 ( Pichia pastoris ), the filamentous fungi include Aspergillus niger ( Aspergillus niger ), and the tobacco includes Nicotiana benthamiana ( Nicotiana benthamiana ).

[0089] In some embodiments, the glabridin is biosynthesized from a substrate selected from the group consisting of daidzein, formononetin, vestitone, and medicarpin.

[0090] In some embodiments, the biological host is Nicotiana benthamiana, and the engineered biological host heterologously expresses: prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 10, pterocarpan reductase having an amino acid sequence as shown in SEQ ID NO: 11, oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13, and demethylase having an amino acid sequence as shown in SEQ ID NO: 15, and the substrate for the biosynthesis of the glabridin is medicarpin.

[0091] In some embodiments, the biological host is yeast, and the engineered biological host heterologously expresses: 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO: 1, isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO: 3, isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 5, vestitone reductase having an amino acid sequence as shown in SEQ ID NO: 7, prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 16, pterocarpan reductase having an amino acid sequence as shown in SEQ ID NO: 11, oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13, and demethylase having an amino acid sequence as shown in SEQ ID NO: 14, and the substrate for the biosynthesis of the glabridin is daidzein.

[0092] 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, vestitone reductase with an amino acid sequence as shown in SEQ ID NO:8, prenyltransferase with an amino acid sequence as shown in SEQ ID NO:10, pterocarpan 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 biosynthesis of the glabridin is daidzein.

[0093] An embodiment of the 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:

[0094] 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:2;

[0095] Isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:4;

[0096] Isoflavone reductase with an amino acid sequence as shown in SEQ ID NO:6;

[0097] Vestitone reductase with an amino acid sequence as shown in SEQ ID NO:8;

[0098] Prenyltransferase with an amino acid sequence as shown in SEQ ID NO:10;

[0099] Pterocarpan reductase with an amino acid sequence as shown in SEQ ID NO:11 or 12;

[0100] Oxidative cyclase with an amino acid sequence as shown in SEQ ID NO:13;

[0101] Demethylase with an amino acid sequence as shown in SEQ ID NO:15.

[0102] An embodiment of the fourth aspect of the present invention provides the use of 4'-O-methyltransferase with an amino acid sequence as shown in SEQ ID NO:1 in the synthesis of formononetin or glabridin.

[0103] An embodiment of the fifth aspect of the present invention provides the use of isoflavone 2'-hydrogenase with an amino acid sequence as shown in SEQ ID NO:3 in the synthesis of 2'-hydroxyformononetin or glabridin.

[0104] The embodiment of the sixth aspect of the present invention provides the use of isoflavone reductase with the amino acid sequence shown in SEQ ID NO:5 in the synthesis of vestitone or glabridin.

[0105] The embodiment of the seventh aspect of the present invention provides the use of vestitone reductase with the amino acid sequence shown in SEQ ID NO:7 in the synthesis of medicarpin or glabridin.

[0106] The embodiment of the eighth aspect of the present invention provides the use of prenyltransferase with the amino acid sequence shown in SEQ ID NO:9 or 16 in the synthesis of glabridin.

[0107] The embodiment of the ninth aspect of the present invention provides the use of demethylase with the amino acid sequence shown in SEQ ID NO:14 in the synthesis of glabridin.

[0108] It can be understood that the uses in the embodiments of the fourth to ninth aspects of the present invention include but are not limited to intracellular catalysis, extracellular catalysis, enzymatic catalysis, or cell-free catalysis.

[0109] The biological hosts applicable to the uses in the embodiments of the fourth to ninth aspects of the present invention are the same as those in the embodiment of the second aspect, and will not be elaborated here.

[0110] Term

[0111] As used herein, the "engineered bacterium" or "engineered plant" or "modified biological host" refers to a fungal cell line or plant in which an exogenous gene is highly expressed by genetic engineering methods.

[0112] As used herein, the "starting strain" and "chassis strain" or "biological host" refer to the original strain or plant used for modification.

[0113] As used herein, the "gene circuit" refers to a gene expression system containing all necessary elements required for expressing a target polypeptide, and usually includes the following elements: promoter, encoded reporter gene, terminator.

[0114] The "comprising" or "including" described in the present invention is an open-ended writing. When used to describe the sequence of a protein or nucleic acid, 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 has the same or similar activity as the original sequence.

[0115] The "comprising" or "including" described in the present invention is an open-ended description, including the specified components or steps described, as well as other specified components or steps that will not be substantially affected.

[0116] As used in the present invention, "and / or" includes all combinations of the items connected by this term, and each combination should be regarded as having been separately listed herein. For example, "A and / or B" includes "A", "A and B", and "B". 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".

[0117] The corresponding relationships between the abbreviations used in this application and their full names are shown in Table 1 below.

[0118] Table 1

[0119]

[0120] Sources of experimental materials

[0121] Nicotiana benthamiana Nicotiana benthamiana ) The wild type (WT) is described in the article "Yuan J, Yu Z, Li Y, Shah SHA, Xiao D, Hou X, Li Y. Ectopic expression of BrIQD35 promotes drought stress tolerance in Nicotiana benthamiana. Plant Biol (Stuttg). 2022 Aug;24(5):887-896.", and the public can obtain it from the applicant and it can only be used for repeating the experiments of the present invention.

[0122] Streptomyces coelicolor S. coelicolor A3(2), is described in the article "Hopwood DA (1999) Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145(9):2183–2202.", and the public can obtain it from the applicant and it can only be used for repeating the experiments of the present invention.

[0123] Yarrowia lipolytica ATCC 201249, is described in the article "Du H-X, Xiao W-H, Wang Y, Zhou X, Zhang Y, Liu D, et al. (2016) Engineering Yarrowia lipolytica for Campesterol Overproduction. PLoS ONE 11(1): e0146773. ", and the public can obtain it from the applicant and it can only be used for repeating the experiments of the present invention.

[0124] The following specific examples of the heterologous biosynthesis of liquiritigenin are used to further illustrate the present invention. The purpose of the specific examples is only for illustration and not for limiting the scope of the present invention. Without departing from the scope of the claims, those skilled in the art can modify various aspects of the present invention, but these modifications also fall within the protection scope of the present invention. For example, replacing the cell expression system and vector plasmid used in this example with other commonly used expression systems and vectors in the art can be understood and implemented by other technicians in the art.

[0125] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0126] Example 1: Expression and functional verification of 4'-O-methyltransferase

[0127] Select the 4'-O-methyltransferase (GgOMT, amino acid sequence SEQ ID NO:2) from Glycyrrhiza glabra. Gene synthesis is carried out according to the amino acid sequence, and the nucleotide sequence is shown in SEQ ID NO:17. After PCR amplification of the gene, it is ligated to the Agrobacterium expression vector pBI121 using Gibson assembly. The recombinant plasmid is introduced into the Agrobacterium competent cell EHA105 by electroporation. Pick the Agrobacterium clone transformed with the expression plasmid into 1 ml of LB medium containing the corresponding antibiotic and culture it at 28 °C on a shaker at 250 rpm for 24 hours. Add 100 μl of 0.5 M MES and 2 μl of 100 mM AS to 5 ml of LB medium containing the corresponding antibiotic, then inoculate 50 μl of the Agrobacterium liquid and culture it at 28 °C on a shaker at 250 rpm until OD600 = 1.0. Centrifuge the cells at 4000 rpm for 10 minutes to collect the cells, resuspend them in 10 mM MgCl2 to OD600 = 1.0, and add 100 mM AS at a ratio of 2 μl per milliliter of the cell suspension, and let it stand for more than 3 hours.

[0128] Take Nicotiana benthamiana Nicotiana benthamiana () that is in the vigorous growth period. Draw the cell suspension into a syringe, remove the needle, press the front side of the leaf with your finger, and infiltrate the cell suspension into the leaf from the back side, and at the same time inject 1 mM daidzein solution, which is the reaction substrate. After 24 - 48 hours, sample and detect the product.

[0129] Cut the leaves into pieces and break them by bead beating at -20 °C. Extract the broken precipitate and the separated supernatant with an equal volume of ethyl acetate respectively. Evaporate the obtained organic phase to dryness and redissolve it in methanol. Detect the production of formononetin by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0130] The fermentation products of the engineered strain were quantitatively determined using an instrument, UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution process was as follows: 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. Qualitative and quantitative determinations were performed using the external standard method. The detection results showed the production of formononetin.

[0131]

[0132] Example 2: Expression and functional verification of isoflavone 2'-hydrogenase

[0133] 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. After PCR amplification of the gene, it was ligated to the Escherichia coli expression vector pET28a using Gibson assembly. The recombinant plasmid was introduced into Escherichia coli competent cells BL21 by electrotransformation. Clones transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotic at 37 °C on a shaker at 220 rpm for 12 hours. 0.1 ml of the bacterial solution was inoculated into 5 ml of LB medium containing the corresponding antibiotic. After culturing for 3 h, the OD value was measured using an ultraviolet spectrophotometer. When OD600 = 0.6 - 0.8, the shaker temperature was reduced to 16 °C, and 1 mM IPTG was added to the bacterial solution for induction. After 10 h, 1 mM formononetin, the reaction substrate, was added, the shaker temperature was adjusted to 30 °C, and samples were taken for product detection after reacting for 24 - 48 h.

[0134] The reaction broth was centrifuged, the supernatant was taken, extracted with an equal volume of ethyl acetate, the obtained organic phase was evaporated to dryness and redissolved in methanol. The content of 2'-hydroxyformononetin in the product was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0135] The fermentation products of the engineered strain were quantitatively determined using an instrument UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution procedure was as follows: 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. Qualitative and quantitative determinations were performed using the external standard method. The detection results showed that 0.96 mM of 2'-hydroxyformononetin could be obtained.

[0136]

[0137] Example 3: Expression and functional verification of isoflavone reductase

[0138] The isoflavone reductase from Glycyrrhiza glabra (GgIFR, amino acid sequence SEQ ID NO:6) was selected, and gene synthesis was carried out according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:19. After PCR amplification of the gene, it was ligated to the Saccharomyces cerevisiae expression vector pYES2 using Gibson assembly. The recombinant plasmid was introduced into Escherichia coli competent cells DH5α by electrotransformation. The clones transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotic at 37 °C in a shaker at 220 rpm for 12 hours. After centrifugation to obtain the bacterial cells, 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 °C in a shaker at 250 rpm for 16 hours. Then 20 g / L of galactose was added for induction, and at the same time, the reaction substrate 1 mM of 2'-hydroxyformononetin could be added. After reacting at 30 °C in a shaker at 220 rpm for 24 - 48 h, samples were taken for product detection.

[0139] The reaction broth was centrifuged, the supernatant was taken, extracted with an equal volume of ethyl acetate, the obtained organic phase was evaporated to dryness and redissolved in methanol. The content of the product vestitone was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0140] The fermentation products of the engineered strain were quantitatively determined using an instrument UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution procedure was as follows: 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. Qualitative and quantitative determinations were performed using the external standard method. The test results showed that 0.88 mM of vistolone could be obtained.

[0141]

[0142] Example 4: Expression and functional verification of vistolone reductase

[0143] Vistolone reductase (GgVR, amino acid sequence SEQ ID NO:8) derived from Glycyrrhiza glabra was selected, and gene synthesis was carried out according to the amino acid sequence. The nucleotide sequence is shown in SEQ ID NO:20. After PCR amplification of the gene, it was ligated to the Saccharomyces cerevisiae expression vector pAK203 using Gibson assembly. The recombinant plasmid was introduced into Escherichia coli competent cells DH5α by electrotransformation. Clones transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotic at 37 °C in a shaker at 220 rpm for 12 hours. After centrifugation to obtain the cells, the plasmid was extracted. The plasmid was transformed into Streptomyces coelicolor cells by protoplast transformation. The positive clones obtained after transformation were inoculated into the medium and cultured at 30 °C in a shaker at 250 rpm for 24 hours. Then thiostrepton was added for induction, and at the same time, 1 mM of vistolone as the reaction substrate was added. After reacting at 30 °C in a shaker at 220 rpm for 24 - 48 h, samples were taken for product detection.

[0144] The reaction broth was centrifuged, the supernatant was taken, extracted with an equal volume of ethyl acetate, the obtained organic phase was evaporated to dryness and redissolved in methanol. The content of the product vistolone was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0145] The fermentation products of the engineered strain were quantitatively determined using an instrument, UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution procedure was as follows: 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. Qualitative and quantitative determinations were performed using the external standard method. The test results showed that 0.9 mM of medicarpin could be obtained.

[0146]

[0147] Example 5: Expression and functional verification of isoprenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase

[0148] Isoprenyltransferase (GgPT, amino acid sequence SEQ ID NO:10) from Glycyrrhiza glabra, pterocarpan reductase (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 (GgDMT, amino acid sequence SEQ ID NO:15) from Glycyrrhiza glabra were selected. Gene synthesis was performed according to the amino acid sequences, and the corresponding nucleotide sequences were SEQ ID NO:21, 22, 23, and 24 in sequence. After PCR amplification of the genes, they were ligated to the Agrobacterium expression vector pBI121 using Gibson assembly. The recombinant plasmid was introduced into Agrobacterium competent cells EHA105 by electroporation. Agrobacterium clones transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotics at 250 rpm on a shaker at 28 °C for 24 hours. 100 μl of 0.5 M MES and 2 μl of 100 mM AS were added to 5 ml of LB medium containing the corresponding antibiotics, and then 50 μl of the Agrobacterium bacterial solution was inoculated and cultured at 250 rpm on a shaker at 28 °C until OD600 = 1.0. The cells were collected by centrifugation at 4000 rpm for 10 minutes and resuspended in 10 mM MgCl2 to OD600 = 1.0, and 100 mM AS was added at a ratio of 2 μl per milliliter of the bacterial solution, and left standing for more than 3 hours.

[0149] Take Nicotiana benthamiana Nicotiana benthamiana that is in the vigorous growth period. Use a syringe to suck in the bacterial liquid, remove the needle, press the front side of the leaf with your finger, and let the bacterial liquid penetrate from the back side of the leaf. At the same time, inject 1 mM medicarpin solution, which is the substrate of the reaction. After placing for 24 - 48 hours, take samples for detecting the products.

[0150] Cut the leaves into pieces and perform bead beating at -20 °C. Extract the broken precipitate and the obtained supernatant with an equal volume of ethyl acetate respectively. Evaporate the obtained organic phase to dryness and redissolve it with methanol. Detect the production of glabridin by ultra - performance liquid chromatography (UPLC) or liquid chromatography - mass spectrometry (LC - MS).

[0151] Use the instrument UPLC (Agilent 1260) to quantitatively determine the fermentation products of the engineered strain. Select a Poroshell 120 EC - C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm. The detection wavelength is 254 nm, the column oven temperature is 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution process is as follows: 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 is 0.2 mL / min, and the injection volume is 1 μL. Use the external standard method for qualitative and quantitative determination. The detection results show the production of glabridin.

[0152]

[0153] Example 6: Construction and verification of the pathway for synthesizing glabridin using daidzein as a substrate

[0154] Select 4'-O-methyltransferase from Pueraria lobata (PlOMT9, amino acid sequence SEQ ID NO:1), isoflavone 2'-hydrogenase from Glycyrrhiza pallidiflora Maxim (GeCYP81E1, amino acid sequence SEQ ID NO:3), isoflavone reductase from Medicago sativa L. (MsIFR, amino acid sequence SEQ ID NO:5), vestitone reductase from Medicago sativa L. (MsVR, amino acid sequence SEQ ID NO:7), isoprenyltransferase from Psoralea corylifolia L. (tPcM4DT, amino acid sequence SEQ ID NO:16), pterocarpan reductase from Glycyrrhiza glabra L. (GgPTR, amino acid sequence SEQ ID NO:11), oxidative cyclase from Glycyrrhiza glabra L. (GgOC, amino acid sequence SEQ ID NO:13), and demethylase from Fusarium solani f. sp. cucurbitae (NhDMT, amino acid sequence SEQ ID NO:14), and perform gene synthesis according to the amino acid sequences. The corresponding nucleotide sequences are SEQ ID NO:25, 26, 27, 28, 29, 30, 31, 32 in sequence. After PCR amplification of the genes, use OE-PCR and Gibson assembly to ligate them into an expression cassette, and construct it onto the common 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 competent Escherichia coli cells DH5α by transformation. The clones transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotic at 37 °C with shaking at 220 rpm for 12 hours. After centrifugation to obtain the cells, the plasmid was extracted. The plasmid was then transformed into Yarrowia lipolytica ATCC201249, and the positive clones obtained after transformation were inoculated into SD-Ura medium and cultured at 30 °C with shaking at 250 rpm for 16 hours. The reaction substrate daidzein at 5 mM was added, and samples were taken for product detection after reacting at 30 °C with shaking at 220 rpm for 96 h.

[0155] The reacted culture was disrupted by bead beating, extracted with an equal volume of ethyl acetate, and the obtained organic phase was evaporated to dryness and redissolved in methanol. The content of glabridin in the product was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0156] The fermentation products of the engineered strain were quantitatively determined using the instrument UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and a size of 3.0×100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution procedure was as follows: 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. Qualitative and quantitative determination was performed using the external standard method. The detection results showed that 1.8 mM of glabridin could be obtained.

[0157] Example 7: Construction and verification of the pathway for synthesizing glabridin using daidzein as a substrate

[0158] Select 4'-O-methyltransferase (GgOMT, amino acid sequence SEQ ID NO: 2), isoflavone 2'-hydrogenase (GgI2'H, amino acid sequence SEQ ID NO: 4), isoflavone reductase (GgIFR, amino acid sequence SEQ ID NO: 6), vestitone reductase (GgVR, amino acid sequence SEQ ID NO: 8), prenyltransferase (GgPT, amino acid sequence SEQ ID NO: 10), pterocarpan reductase (GgPTR, amino acid sequence SEQ ID NO: 12), oxidative cyclase (GgOC, amino acid sequence SEQ ID NO: 13), and demethylase (GgDMT, amino acid sequence SEQ ID NO: 15) from Glycyrrhiza glabra. Gene synthesis was carried out according to the amino acid sequences, and the corresponding nucleotide sequences were SEQ ID NO: 33, 34, 19, 35, 36, 37, 38, 39 in sequence. After PCR amplification of the genes, they were ligated into the expression cassette HOL-P using OE-PCR and Gibson assembly TDH3 -GgOMT-T VMA1 -P FBA1 -GgI2’H-T 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 ligated it to the vector pUC19. The recombinant plasmid was introduced into Escherichia coli competent cells DH5α by transformation. Colonies transformed with the expression plasmid were picked and cultured in 1 ml of LB medium containing the corresponding antibiotics at 220 rpm on a shaker at 37 °C for 12 hours. After centrifugation to obtain the bacterial cells, the plasmid was extracted. The DNA fragment ① HOL-P 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 respectively, and then co-transformed into Saccharomyces cerevisiae BY4741. The positive clones obtained after transformation were inoculated into SD-Ura medium and cultured at 30 °C on a shaker at 250 rpm for 16 hours. The reaction substrate daidzein at 1 mM was added, and samples were taken for product detection after reacting at 30 °C on a shaker at 220 rpm for 96 h.

[0159] The cultured product after the reaction was broken by beads, extracted with an equal volume of ethyl acetate, the obtained organic phase was evaporated to dryness and redissolved in methanol. The content of glabridin in the product was detected by ultra-high performance liquid chromatography (UPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0160] The fermentation products of the engineered strains were quantitatively determined using the instrument UPLC (Agilent 1260). A Poroshell 120 EC-C18 chromatographic column with a particle size of 2.7 μm and 3.0 × 100 mm was selected. The detection wavelength was 254 nm, the column oven temperature was 35 °C, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile, and the gradient elution procedure was as follows: 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. Qualitative and quantitative determination was carried out using the external standard method. The detection results showed that 0.6 mM of glabridin could be obtained.

[0161] The enzymes, biological hosts, substrates and products used in the above examples are shown in Table 3 below. As can be seen from Table 3, a series of enzymes derived from Glycyrrhiza glabra L. var. glabra Regel for synthesizing Glycyrrhiza glabra L. var. glabra Regel disclosed for the first time in the present invention have corresponding activities in a variety of biological hosts, and the heterologous biosynthesis of glabridin is realized for the first time, and the yield of glabridin can reach more than 60%.

[0162] Table 3

[0163]

[0164] Sequence information:

[0165] >SEQ ID NO: 1 (PlOMT9)

[0166] MGSNNGRKASEIFQGQALLYRHMFAFVDSMCLKSIVELGIPNIIHKHGQPITLSELMSILQVPPAKVGHVQSLMRYLAHHGFFERLRIHEKDAYALTAASELLVKGTEPYLAPMVECMLDPTLSASFHQMKKWVYEEDLSVFDISLGSNLWDFLTKNPAYNEIFNEAMASDSQMSNLALRDCKLVFEGLESIVDVGGGTGTTAKIICEAFPNLKCIVFDRPQVVENLLENNNLTYVGGDMFKSIPKANAVLLKWILHDWTDKDCRKILENCKEAISNNSKRGKIIIIDIVINEKQDEHKVTELKLLFDVAMACVLNGKERNEEEWKKLFMEVGLQDYKISPLTGYLSLIEIYP*

[0167] >SEQ ID NO: 2 (GgOMT)

[0168] MDFSTNGSEESELYHAQIHLYKHVYNFVSSMALKSAMELGIADVIHNHGKPITLPQLASALKLHPSKVGVLYRFLRLLTHNGFFAKTTVPSQNGKEGEEEETAYALTPPSKLLVKGKPTCLASIVRGALHPSSLDMWRSSEKWFNEDKELTLFESATGESFWDFLNKDSESGTLSMFQEAMAADSQMFKLALKECRHVFEGLESLVDVGGGTGGVTKLIHEEFPHLKCTVFDQPQVVGNLSGNENLKFVGGDMFKSIPPADAVLLKWVLHDWNDELSLKILKNSKEAISGKGKEGKVIIIDISIDEASGDRELTELQLDYDLVMLTMFNGKEREKKEWEKLISDAGFSSYKITPICGFKSLIEVFP*

[0169] >SEQ ID NO: 3 (GeCYP81E1)

[0170] MEILSLLSYSVFYLALFFIFNIVIRARKFKNLPPGPPSLPIIGNLHHLKRPLHRTFKGLSEKYGHVFSLWFGSRLVVVVSSASEFQQCFTKNDVVLANRPRFLSGKYIFYNYTTLGSTSYGEHWRNLRRITALDVLSNHRINSFSGIRRDETQRLITRLADDSSTNFAEMELSSRLYDMTFNNIMRMISGKRYYGEDCDTSDLQEASQFRDMVSELLQLSGANNKTDFMPLLRFLDFENLEKRLKDISGKTDAFLRGLIEEHRTKKERANTMIDHLLNLQDSQPEYYTDQIIKGLALAMLLAGTDSSAVTLEWSMSNLLNHPEVLKKVKDELDTHVGQDRLVDESDLPKLTYLKNVINETLRLYTPAPLLLPHSTSDECNIGGYKVPQDTIVLINAWAIHRDPELWTEATTFKPERFEKKGELEKLIAFGMGRRACPGEGLAIRAISMTLALLIQCFDWKLINGDKIDLAERDGFTLTKLVPLKAMCKSRPVINKVFKQ*

[0171] >SEQ ID NO: 4 (GgI2’H)

[0172] MEILSLLSYSVFYVALFFICNLLFQARKFKNLPPGPPSLPIIGNLHHLKRPLHRTFKGLSEKYGHVISLWFGSRLVVVVSSLSVFQECFTKNDIVLADRPRFLSGKYIFYNYTTVGSSSYGEHWRNLRRITSLDVLSTHRINSFSGVRRDETQRLIQKLAEESSTDFAEIELTSKFYDMTFNNIMRMISGKRYYGDDCDMMDMEEAKQFRAMVSELLQLSGANNKTDFMPVLRLVDFENLEKRLKKISSKTDTFLRGLIQEHRNKKQHTNTMVDHLLSLQESQPEYYTDQIIKGLALGMLLAGTDSSAVTLEWALSCLLNHPEALKKARDELETHVGQDRLLEESDLTKLPYLKNIIYETLRLYTPAPLLLPHSSSDECIIGGFKVPRETIILINAWSIHRDPQIWSEATSFKPERFEKEGELDKLIAFGLGRRACPGEALALRGISLTLGLLIQCFEWKLVGDKEIDMREESGFTLSRLIPLKAMCKARPVANKLVNTQAVSLGD*

[0173] >SEQ ID NO:5(MsIFR)

[0174] MATENKILILGPTGAIGRHIVWASIKAGNPTYALVRKTPGNVNKPKLITAANPETKEELIDNYQSLGVILLEGDINDHETLVKAIKQVDIVICAAGRLLIEDQVKIIKAIKEAGNVKKFFPSEFGLDVDRHDAVEPVRQVFEEKASIRRVIEAEGVPYTYLCCHAFTGYFLRNLAQLDATDPPRDKVVILGDGNVKGAYVTEADVGTFTIRAANDPNTLNKAVHIRLPKNYLTQNEVIALWEKKIGKTLEKTYVSEEQVLKDIQESSFPHNYLLALYHSQQIKGDAVYEIDPAKDIEASEAYPDVTYTTADEYLNQFV*

[0175] >SEQ ID NO:6(GgIFR)

[0176] MNMIEVGGYPGNPINTKQSNAQQHKPTASLTTYQNKKKKNTHPSFFNSPYFYFNFNIPMAAENKILILGPTGAIGRHIVWASVKAGNPTFALVRKTNGPVNKPKLITAANPESKEQLLESYQNAGVTLLEGDINDHESLVKAIKQVDVVICATGRLLIDDQVKIIAAIKEAGNVKRFFPSEFGLDVDRHDSVEPVREVFEDKARIRRVIEAEGVPYTYLCCHAFTGYFLRNLAQLDATVPPRDKVVILGDGNVKGAYVTEADVGTYTIRAANDPRTLNKAVHIRLPANYLTANEVVSLWEKKIGKTLEKTYVPEEKVLKDIQESSFPHNYLLALYHSQQIKGDAVYEIDPAKDVEAYDLYSDVKYTTADEYLNQFV*

[0177] >SEQ ID NO:7(MsVR)

[0178] MAEGKGRVCVTGGTGFLGSWIIKSLLENGYSVNTTIRADPERKRDVSFLTNLPGASEKLHFFNADLSNPDSFAAAIEGCVGIFHTASPIDFAVSEPEEIVTKRTVDGALGILKACVNSKTVKRFIYTSSGSAVSFNGKDKDVLDESDWSDVDLLRSVKPFGWNYAVSKTLAEKAVLEFGEQNGIDVVTLILPFIVGRFVCPKLPDSIEKALVLVLGKKEQIGVTRFHMVHVDDVARAHIYLLENSVPGGRYNCSPFIVPIEEMSQLLSAKYPEYQILTVDELKEIKGARLPDLNTKKLVDAGFDFKYTIEDMFDDAIQCCKEKGYL*

[0179] >SEQ ID NO:8(GgVR)

[0180] MAEGKGRVCVTGGTGFLGSWLIKRLLEDGYGVNTTIRSDPERKRDISFLTNLPGASERLKIFNADLSDPESFGPAIEGCVGVFHTASPIDFAVSEPEEIVTKRTVDGTLGILKACKNSKTVKRVVYTSSGAAVSWGGTEKDVLDESDWSDVDMLRSVKPYSWSYAVSKTMTEKAVLEFGEQHGLDVVTLILPFIVGRFICPKIPDSVEKALVLVLGKREQIGVTRFHMVHVEDVARAYIFLLEHPNPKGRYNCSPFIVPIEEVSELLSAKYPEYQIPTVEELKEIKGAKLPDLKSKKLVDAGFEFKYTIEDMFDDAIQCCKEKGYL*

[0181] >SEQ ID NO:9(PcM4DT)

[0182] MDWGLAISSSSKAYSVTTGANLWRSKHVTNNIHYPSSCITKASQHKKKTQIEYNVLRFQRPSLGHGYNFSGGGGSTYQECNRKYAVKAVYDQPKDFELEASNPKNILDSAKKFLAAFYYFSYPYTMIGITLCAFCSSALAVEKLSDISLPFFIGVLQAVIPQLFIEIYLSGVNQLYDLEIDKINKPHLPMASGQFSFKTGVIMSAAFLALSFGFTWLTGSWPLIWNLVVIASSWTAYSIDVPFMRWKRYPLVAAMCMIATWGLALPISFFHHMQTFVLQRPIGFPRSLGFLVAFMTFYSMGIALSKDIPDVEGDKEHGINSFSVLLGQKRIFWICVSLFEMAFGVGLLGGVTSPHFWTKIITGLGNVILGSILWYQAKSFDLTDKASTGSFYMYIWKLLYAALLLMAFVR*

[0183] >SEQ ID NO:10(GgPT)

[0184] MSSLFGSNVFESISTYRSYAPKASQHKRKIQKEYNFLRSRQPSLKHIYKGIEGGSTQQECNRKYVVKTVPKPSFESDPRSIDQKDILECIKNFLDALYMFTTPYSFFSSALYIISVSFIVVEKLSDISPLFFTGVLQAVVPNLFVHIYMTGLNQLCDVEIDKINKPYLPLVSGKISFATGVIVVASCLFLSLWLGWIVGSWPSTCVVISIAMIWTAYSINVPLLRWKKYPVLAAMCIFLSLAVVNPIGYFLHMQTFVFKRPASFSRPLIFVIAFKSFFSLAVALFKDVPDIEGDQTFGVQSFVARFGKKRVFWICISLIEMAYGFALLMGATSAFLWSKIVTVLGHAVLASFVLYRAKSIDLRSKTSITSFYMLIWKLMYMEYFLMPLVR*

[0185] >SEQ ID NO:11(GgPTR)

[0186] MAETTTSKSKILFIGGTGYIGKFIVEASVNAGHPTFVLIRDSTLSNPAKSPIIDKFKSLAVNLVFGDLYDHQSLVKAIKQVDVVISTVGHLQLGDQDKIISAIKESGNVKRFFPSEFGNDVDRTHAVEPAKSAFATKAKIRRTIEAEGIPYTYVSSNFFAGYFLPTLSQPGATAAPRDKVIILGDGNPKAVANKEEDIATYTIKAVDDPRTLNKILYIRPPANTLSFNELVSLWEKKIGKTLGRVYVPEEQLLKQIQESSPPINVILSIGHSVYVKGDHTNFEIEPAFGVEASALYPDVKYTTVDEYLNQFV*

[0187] >SEQ ID NO:12(GgPTR)

[0188] MDSTAASKILVIGGTGYMGRFLVEASAKAGHPTFALVRHSTVTNPDKSSIIHSFNTLGVNLLLGDINDHQSLVKAIKQADVVISTVNHQYISDQYKIISAIKEAGNIKRFFPSEFGNDVVRTHGVDGAKALFDIKAKFRRTIEAEGIPHTYVVANFLTQHFLPTRSRLLAIAAPLDKVVILGDGNTKATFNTEEGVATFTIRAVDDPRTLNKILYIRPPANTLSYNDLVSLWEKKTGNTLERVYVPEEQVLRLIQESSYPLNMALSICHAAYVKGDHTNYEIEPSFGVEALELYPDVKFTTVDDYLEQNRDCTPFYLNQLIPITNGTKF*

[0189] >SEQ ID NO: 13 (GgOC)

[0190] MAFTYSTKKLALLSIITIFISSFPEPSTSVDLESGFLQCFSSGLGNSNSTAELVLTKNSSSYASLLQSSIRNLRFMGTSVPKPTLIVTPRDLFHIQTSIKCSVKQGLQIRVRSGGHDYEGLSYVSNDDVPFLIIDLTNLRSITIDIKDETAWVQSGATLGELYYAIAKKSNVHGFPAGSCPTVGVGGHFSGGGFGTIFRKYGLAADNVIDAQMVDVNGKILKNRTLMGEDLFWAIRGGGGSSFGVVTAWKVKLVHVPPKVTVFNIPKTLDQNASTLFHKWQIVADKLPGELFLHSVMGVSNANSISSSSGGGRTVLVSFTGLYLGTVENLLPLMQNNFAELGLQHNNCTEMSWIQSVLFFAGYDPINDSLEVLLQRNQTFGSFKAKSDYVMEPIPTSGLEGLWNMLLEENSSPTLILTPYGGRMSEISESETPFPHRNGSIYGIQYLVYWDSSEETPKHIAWMRRVYSYMVLYVSKFPRAAYLNYRDLDIGVNRGNTSYDYEEAKSWGLKYFKCNFERLAKVKAEVDPSNFFRHEQSIPLLF*

[0191] >SEQ ID NO: 14 (NhDMT)

[0192] MLVDTGLGLISELQAKLGWAVLLQIVPITIVAYNLLWFIYASFFSSLRKIPGPFLARISRVWEMKKTATGNIHEIMMDLHRRHGAIVRIGPRRYDFDTMEALKIIYRIGNALPKADYYKPFGLPSFPNLFDEQNPARHSAIKKQVASLYTMTALLSYEEGVDGQTAILKEQLQRFCDQKQVIDLPRFLQYYAFDVIGVITVGKSMGMMESNSDTNGACSALDGMWHYASMMAYIPNMHAWWLRLSSLLPIEVPIKGLTEYVERRIIQYRLKAAEFGDDAALKGENNFLAKLLLMEKKGTVTPVETQQAVGLNIGAGSDTTANALSTILYYLYTNPRTLHTLREELERYVKDGPISFQQSQSMPYLQAVIKEALRLHPGVGTQLTRVVPKGGLVIEGQFFPEGTEVGVNGWALYHNKAIFGNDASIFRPERWLEANENINIGGSFAFGAGSRSCIGKNISILEMSKAIPQIVRNFDIEINHGDMTWKNECWWFVKPEYKAMIKPRRCCLSRDESLV*

[0193] >SEQ ID NO:15 (GgDMT)

[0194] MEHFYMSLLLLFVTLVSLSLFFLIFYHNKHNMNNNNNLPPGKMGYPVIGESLEFLSMGWKGHPEKFIFDRMVRYSSELIKTSILGVPTVIFCGPACNKFLFSNENKLVTAWWPDSVNKIFPTTSNSKEESKKMRKLLPQFLKPEALQRYVGIMDTLAQRHFASLWEEKTHVTVYPLAKRYTFMLACRLFMSVEDENHVAKFREPFHLLASGIISVPIDLPWTPFNRGIKASNFIRKELLKIIRQRKVDLAQGVASPTQDILSHMLLTCDDENGEFMTELNIADKILGLLIGGHDTASAACTFIVKYLAELPHIYDRVYQEQMEIANSKSPGELLNWDDINKMRYSWNVASEVMRVAPPLQGGFREAINDFVFNGFSIPKGWKLYWSANSTHKNPEYFPAPEKFDPTRFEGNGPAAYTFVPFGGGPRMCPGKEYARLEILVFMHNLVKRFKWEMLIPEEKIVVDPLPMPANDLPIRLYPHNT*

[0195] >SEQ ID NO:16(tPcM4DT)

[0196] ASQHKKKTQIEYNVLRFQRPSLGHGYNFSGGGGSTYQECNRKYAVKAVYDQPKDFELEASNPKNILDSAKKFLAAFYYFSYPYTMIGITLCAFCSSALAVEKLSDISLPFFIGVLQAVIPQLFIEIYLSGVNQLYDLEIDKINKPHLPMASGQFSFKTGVIMSAAFLALSFGFTWLTGSWPLIWNLVVIASSWTAYSIDVPFMRWKRYPLVAAMCMIATWGLALPISFFHHMQTFVLQRPIGFPRSLGFLVAFMTFYSMGIALSKDIPDVEGDKEHGINSFSVLLGQKRIFWICVSLFEMAFGVGLLGGVTSPHFWTKIITGLGNVILGSILWYQAKSFDLTDKASTGSFYMYIWKLLYAALLLMAFVR*

[0197] >SEQ ID NO:17(GgOMT)

[0198]

[0199] >SEQ ID NO: 18 (GgI2’H)

[0200]

[0201] >SEQ ID NO: 19 (GgIFR)

[0202]

[0203] >SEQ ID NO: 20 (GgVR)

[0204] ATGGCCGAGGGCAAGGGCCGGGTATGTGTCACGGGCGGCACCGGCTTCCTGGGATCGTGGCTGATCAAGCGTTTGCTCGAGGACGGCTACGGCGTCAACACCACGATCCGGAGTGACCCGGAGCGCAAGCGGGACATCAGCTTCCTCACCAACCTGCCCGGGGCGAGCGAGCGGCTGAAGATCTTCAACGCCGACCTGTCCGACCCCGAATCCTTCGGCCCCGCGATTGAAGGCTGCGTGGGTGTCTTCCACACCGCCTCCCCGATCGACTTCGCCGTCTCGGAGCCCGAGGAGATCGTGACCAAGCGCACCGTCGACGGGACGCTGGGCATCCTCAAGGCCTGCAAGAACTCCAAGACCGTCAAGCGGGTCGTGTACACGTCCAGCGGCGCCGCGGTCAGCTGGGGCGGGACCGAGAAGGACGTGCTCGACGAGAGCGACTGGTCGGATGTGGACATGCTGCGCTCGGTGAAGCCGTACTCCTGGTCCTACGCCGTGAGCAAGACGATGACCGAAAAGGCGGTGCTGGAGTTCGGCGAGCAGCACGGCCTGGACGTCGTCACCCTGATCCTGCCGTTCATCGTCGGCCGCTTCATCTGCCCCAAGATCCCCGACTCCGTCGAGAAGGCGCTGGTCCTGGTGCTCGGCAAGCGCGAGCAGATCGGCGTGACCCGGTTCCACATGGTCCACGTCGAGGACGTGGCCAGGGCCTACATCTTCCTGCTGGAGCACCCGAACCCGAAGGGGCGCTACAACTGCTCCCCCTTCATCGTGCCGATCGAGGAGGTCTCGGAGCTCCTCTCCGCGAAGTACCCAGAGTACCAGATCCCGACGGTGGAGGAACTCAAGGAGATCAAGGGTGCCAAGCTCCCGGACCTCAAATCGAAGAAGCTGGTCGACGCGGGGTTCGAGTTCAAGTACACCATCGAGGACATGTTCGACGACGCCATCCAGTGCTGCAAGGAGAAGGGATACCTGTAA

[0205] >SEQ ID NO: 21 (GgPT)

[0206]

[0207] >SEQ ID NO: 22 (GgPTR)

[0208] ATGGCTGAAACAACTACCTCAAAATCAAAGATTCTTTTCATTGGAGGAACTGGCTACATTGGGAAATTTATTGTTGAAGCTTCTGTTAATGCTGGTCATCCAACTTTTGTGTTAATAAGAGATTCAACTCTGAGCAATCCAGCGAAGTCTCCCATAATTGATAAATTCAAGAGTCTCGCAGTGAACCTCGTTTTTGGGGACTTGTATGATCACCAGAGTCTTGTGAAAGCAATCAAACAAGTTGATGTTGTGATCTCCACCGTTGGACATCTTCAGCTTGGAGATCAAGATAAGATAATATCAGCCATTAAAGAGTCTGGTAATGTTAAGAGGTTTTTCCCTTCTGAATTTGGGAATGATGTCGACCGTACACATGCTGTAGAGCCCGCTAAGTCTGCCTTTGCAACCAAGGCAAAAATCCGCAGGACAATAGAGGCCGAGGGAATTCCATACACTTACGTATCGTCCAACTTCTTTGCTGGATATTTCTTGCCTACTCTATCTCAACCTGGTGCAACTGCCGCTCCAAGAGACAAGGTTATAATCTTAGGGGATGGAAACCCTAAAGCTGTCGCTAACAAGGAGGAAGATATTGCTACATATACAATCAAGGCGGTGGATGATCCAAGAACATTGAACAAAATACTTTATATTCGGCCGCCGGCAAATACTTTATCTTTCAATGAATTGGTGAGTCTGTGGGAAAAGAAAATTGGTAAAACTCTTGGCCGAGTTTATGTGCCAGAAGAGCAGCTCTTGAAGCAGATTCAAGAATCGAGTCCTCCAATTAATGTCATACTAAGCATCGGCCACTCAGTATATGTTAAAGGTGATCATACAAATTTTGAGATTGAGCCTGCATTTGGTGTCGAAGCATCTGCACTGTATCCTGACGTAAAGTACACGACGGTTGACGAATATTTGAATCAATTTGTTTGA

[0209] >SEQ ID NO:23 (GgOC)

[0210]

[0211] >SEQ ID NO: 24 (GgDMT)

[0212]

[0213] >SEQ ID NO: 25 (PlOMT9)

[0214]

[0215] >SEQ ID NO: 26 (GmCYP81E1)

[0216]

[0217] >SEQ ID NO: 27 (MsIFR)

[0218] ATGGCTACCGAAAACAAGATCTTGATTTTGGGTCCAACTGGTGCCATCGGTAGACACATTGTCTGGGCTTCCATCAAGGCTGGTAACCCTACCTACGCCTTAGTCAGAAAGACCCCAGGTAATGTTAACAAGCCAAAGTTGATCACTGCTGCTAACCCAGAAACCAAAGAAGAATTGATTGACAACTACCAATCTTTGGGTGTCATCTTGTTGGAAGGTGACATCAACGACCACGAAACATTGGTCAAGGCTATTAAGCAAGTCGATATTGTTATCTGTGCTGCCGGCCGTTTATTGATCGAAGACCAAGTCAAGATAATCAAAGCTATCAAGGAAGCAGGTAACGTCAAGAAGTTCTTCCCATCTGAATTCGGTTTAGACGTTGACAGACATGATGCTGTTGAACCAGTTAGACAAGTTTTTGAAGAAAAAGCTTCCATTAGAAGAGTTATTGAAGCTGAAGGTGTTCCATACACTTATTTGTGTTGTCACGCTTTCACCGGTTACTTCTTGAGAAACTTGGCTCAATTGGACGCCACCGACCCTCCAAGAGACAAGGTTGTTATTTTAGGTGATGGTAACGTTAAGGGTGCTTACGTTACTGAAGCTGATGTTGGTACTTTTACTATCCGTGCTGCTAATGACCCAAACACTTTAAACAAAGCCGTCCATATCAGATTGCCAAAGAACTACTTGACTCAAAACGAAGTCATTGCTTTGTGGGAAAAGAAGATTGGTAAGACCTTGGAAAAGACTTACGTCTCAGAAGAACAAGTTTTGAAGGATATCCAAGAATCTTCTTTCCCACACAACTATCTACTGGCTTTGTACCACTCTCAACAAATCAAGGGTGATGCTGTATACGAAATTGATCCAGCTAAGGACATTGAAGCCTCCGAGGCTTACCCAGACGTCACCTACACCACTGCCGATGAATACTTGAACCAATTCGTCTGA

[0219] >SEQ ID NO: 28 (MsVR)

[0220] ATGGCCGAAGGTAAAGGTAGAGTCTGTGTCACCGGTGGTACTGGCTTCTTGGGTTCCTGGATTATAAAGTCTTTATTAGAAAACGGTTACTCCGTCAACACCACTATCAGAGCAGATCCAGAGAGAAAGAGAGATGTCAGTTTCTTGACTAATTTGCCAGGTGCCTCTGAAAAATTGCACTTCTTCAACGCCGACTTGTCTAACCCAGATTCTTTTGCTGCTGCTATTGAAGGTTGTGTTGGTATCTTCCACACTGCTAGCCCAATCGACTTTGCTGTTTCCGAACCAGAAGAAATCGTTACCAAGAGAACCGTTGACGGTGCTCTTGGAATCTTGAAAGCTTGTGTTAACTCTAAGACTGTCAAGCGTTTCATTTACACCTCTTCTGGTTCTGCTGTTTCTTTCAATGGTAAGGACAAGGATGTTTTGGACGAATCCGACTGGTCTGACGTCGACTTGTTGAGATCTGTCAAGCCATTCGGTTGGAACTACGCTGTCTCCAAGACCCTAGCTGAAAAGGCTGTATTGGAATTCGGTGAACAAAACGGTATTGACGTTGTTACTTTGATTTTGCCTTTCATTGTCGGTCGTTTCGTCTGCCCTAAGTTGCCAGACTCCATTGAAAAGGCTTTGGTCCTAGTTTTGGGTAAGAAAGAACAAATTGGTGTTACCAGATTTCACATGGTTCACGTTGATGACGTCGCCAGAGCCCATATCTATTTATTGGAAAACTCCGTTCCAGGTGGTAGATACAACTGTTCTCCATTCATCGTTCCAATTGAAGAAATGTCACAATTGTTGTCCGCTAAGTACCCAGAATACCAAATCTTGACTGTGGATGAATTGAAGGAAATCAAGGGTGCTAGATTACCAGATTTGAACACTAAGAAGTTAGTCGATGCTGGTTTCGACTTCAAGTACACAATCGAAGACATGTTCGATGATGCTATCCAATGTTGTAAGGAAAAGGGTTACTTGTGA

[0221] >SEQ ID NO: 29 (tPcM4DT)

[0222]

[0223] >SEQ ID NO: 30 (GgPTR)

[0224] ATGGCCGAAACCACCACTTCTAAGTCTAAAATATTGTTTATTGGTGGTACCGGTTACATTGGCAAGTTCATTGTTGAAGCCAGTGTCAATGCCGGTCACCCAACTTTCGTCTTGATTAGAGATAGCACTTTGTCTAACCCAGCGAAGTCCCCAATCATTGATAAATTCAAGTCTTTGGCTGTCAACTTGGTTTTCGGTGACTTGTACGACCACCAATCTTTGGTCAAGGCCATCAAACAAGTTGATGTAGTGATCTCCACAGTCGGTCATTTGCAATTGGGTGACCAAGACAAGATTATTTCCGCTATCAAGGAATCCGGTAATGTCAAGAGATTCTTCCCATCAGAATTCGGTAACGACGTTGACAGAACCCACGCTGTCGAACCAGCTAAATCCGCTTTCGCTACCAAGGCTAAGATCAGACGTACTATCGAAGCTGAAGGTATCCCATACACTTACGTTTCTTCCAACTTTTTTGCTGGTTACTTCTTGCCAACCTTATCCCAACCAGGTGCTACTGCTGCTCCAAGAGACAAGGTTATCATCTTGGGTGACGGTAACCCAAAGGCTGTTGCTAACAAGGAAGAAGATATTGCTACTTACACTATCAAGGCAGTCGATGACCCACGTACCTTAAACAAGATTTTGTACATCAGACCACCAGCCAACACTCTATCTTTCAACGAATTGGTTTCCTTATGGGAAAAGAAGATCGGTAAGACCTTGGGTAGAGTCTATGTCCCAGAAGAACAACTGTTGAAGCAAATTCAAGAGTCTTCTCCACCTATCAACGTTATCTTATCTATTGGTCACTCTGTTTACGTCAAGGGTGACCACACCAACTTCGAAATTGAACCTGCTTTCGGTGTTGAAGCCTCTGCTTTGTATCCAGATGTTAAGTACACCACTGTTGATGAATACTTGAACCAATTCGTTTGA

[0225] >SEQ ID NO:31(GgOC)

[0226]

[0227] >SEQ ID NO: 32 (NhDMT)

[0228]

[0229] >SEQ ID NO: 33 (GgOMT)

[0230]

[0231] >SEQ ID NO: 34 (GgI2’H)

[0232]

[0233] >SEQ ID NO: 35 (GgVR)

[0234] ATGGCTGAAGGTAAGGGTAGAGTCTGTGTCACCGGTGGTACTGGTTTCCTAGGTTCATGGTTAATCAAGCGTTTATTGGAAGACGGTTACGGCGTCAACACTACCATCAGATCCGACCCAGAAAGAAAGAGAGACATTTCTTTCTTGACTAACTTGCCAGGTGCCTCCGAAAGATTAAAAATCTTCAATGCTGACTTGTCCGACCCAGAATCCTTCGGACCAGCTATTGAAGGTTGTGTTGGTGTCTTCCACACTGCTTCCCCAATTGACTTCGCCGTCAGTGAACCAGAAGAAATTGTCACCAAGCGTACTGTTGATGGTACTTTGGGTATCTTGAAGGCTTGCAAGAACTCCAAGACTGTTAAGAGAGTTGTCTACACCTCTTCTGGTGCTGCTGTCTCTTGGGGTGGTACCGAAAAGGATGTCTTGGACGAATCTGACTGGTCCGATGTGGACATGTTGAGATCTGTCAAGCCATACTCTTGGTCTTACGCTGTTTCCAAGACCATGACTGAAAAGGCCGTCTTGGAATTCGGTGAACAACACGGTTTGGATGTTGTTACATTGATTTTGCCATTCATTGTCGGTAGATTTATCTGTCCAAAGATCCCTGACTCTGTTGAGAAAGCTTTGGTCCTCGTTTTGGGTAAGAGAGAACAAATCGGTGTTACCAGATTCCACATGGTTCATGTTGAAGACGTTGCTAGAGCTTACATCTTTTTGCTTGAACACCCAAACCCAAAGGGGAGATACAACTGTTCTCCATTTATTGTTCCAATCGAAGAAGTTTCTGAATTGTTGTCTGCCAAATACCCAGAATACCAAATTCCAACCGTTGAAGAATTAAAAGAAATCAAGGGTGCTAAGTTGCCTGATTTGAAGTCTAAAAAGTTAGTTGATGCTGGTTTCGAATTCAAGTACACTATTGAAGATATGTTCGACGATGCCATCCAATGTTGTAAGGAAAAGGGTTATTTGTGA

[0235] >SEQ ID NO: 36 (GgPT)

[0236]

[0237] >SEQ ID NO: 37 (GgPTR)

[0238] ATGGACTCTACTGCTGCCTCCAAGATCTTGGTTATTGGTGGTACTGGTTACATGGGTCGTTTTCTGGTCGAAGCTTCTGCCAAGGCCGGTCATCCTACCTTTGCTTTAGTACGTCACAGTACTGTTACCAACCCAGACAAGTCCTCTATTATTCACTCTTTCAACACCCTAGGCGTCAATCTATTGTTGGGTGACATTAATGATCATCAATCTTTGGTCAAGGCCATCAAGCAAGCTGATGTTGTCATCTCTACCGTTAACCACCAATATATTTCTGACCAATACAAGATTATTAGCGCTATCAAGGAAGCAGGTAACATCAAACGTTTCTTCCCATCTGAATTCGGTAACGACGTTGTCAGAACTCACGGTGTCGACGGTGCTAAGGCTTTGTTCGATATCAAGGCTAAATTCAGAAGAACCATCGAAGCTGAAGGTATCCCTCACACTTACGTCGTTGCCAACTTCTTGACTCAACACTTCTTGCCAACTAGATCCAGATTATTGGCTATTGCTGCTCCATTAGATAAGGTTGTCATATTGGGTGATGGTAACACCAAGGCCACATTCAACACTGAAGAAGGTGTTGCTACCTTCACTATCAGAGCTGTCGACGACCCAAGAACTTTGAACAAGATCTTATACATTAGACCACCAGCTAACACTTTGTCCTACAACGATTTGGTTTCCTTGTGGGAAAAGAAGACCGGTAACACCTTGGAAAGAGTTTATGTTCCAGAAGAACAAGTTTTGAGATTGATCCAAGAATCTTCTTACCCATTGAACATGGCTTTGTCCATCTGTCACGCTGCTTACGTTAAGGGTGACCACACCAACTACGAAATTGAACCATCCTTCGGTGTTGAAGCCTTGGAATTGTACCCAGATGTCAAGTTCACCACTGTGGATGACTACTTGGAACAAAACAGAGACTGTACTCCATTCTACTTAAATCAATTGATTCCAATCACTAACGGTACCAAATTTTGA

[0239] >SEQ ID NO: 38 (GgOC)

[0240]

[0241] >SEQ ID NO: 39 (GgDMT)

[0242]

[0243] The nucleotide sequences provided above are only for illustrative purposes of the examples, and any codon-optimized nucleic acid sequences obtained based on the amino acid sequences are regarded as within the protection scope of the present invention.

[0244] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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.

[0245] In the description of this specification, the descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection 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 can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0246] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, replacements, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for the biosynthesis of glabridin, characterized in that, Comprising: Providing a biological host; Causing the biological host to express an enzyme selected from the group consisting of: 4'-O-methyltransferase, isoflavone 2'-hydrogenase, isoflavone reductase, vestitone reductase, prenyltransferase, pterocarpan reductase, oxidative cyclase, and demethylase to obtain an engineered biological host; and Culturing the engineered biological host to obtain the glabridin, wherein the substrate for the biosynthesis of the glabridin is daidzein, wherein the engineered biological host heterologously expresses: 4'-O-methyltransferase having an amino acid sequence as shown in SEQ ID NO: 1, isoflavone 2'-hydrogenase having an amino acid sequence as shown in SEQ ID NO: 3, isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 5, vestitone reductase having an amino acid sequence as shown in SEQ ID NO: 7, prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 16, pterocarpan reductase having an amino acid sequence as shown in SEQ ID NO: 11, oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13, and demethylase having an amino acid sequence as shown in SEQ ID NO: 14; or the engineered biological host heterologously expresses: 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, isoflavone reductase having an amino acid sequence as shown in SEQ ID NO: 6, vestitone reductase having an amino acid sequence as shown in SEQ ID NO: 8, prenyltransferase having an amino acid sequence as shown in SEQ ID NO: 10, pterocarpan reductase having an amino acid sequence as shown in SEQ ID NO: 12, oxidative cyclase having an amino acid sequence as shown in SEQ ID NO: 13, and demethylase having an amino acid sequence as shown in SEQ ID NO: 15, the biological host is yeast.

2. The biosynthetic method of glabridin according to claim 1, wherein The yeast includes Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), and Pichia pastoris ( Pichia pastoris ).

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