(-)-alpha-bisabolol synthase mutant and application thereof

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a (-)-alpha-bisabolol synthase mutant and application thereof. On the basis of wild type (-)-alpha-bisabolol synthase, three types of (-)-alpha-bisabolol synthase mutants are constructed through substitution, deletion and addition mutation modes, the (-)-alpha-bisabolol synthase activity of homologous mutants with different consistency with the mutants obtained through the different mutation modes is investigated, and the (-)-alpha-bisabolol synthase activity of the homologous mutants with different consistency with the mutants obtained through the different mutation modes is obtained. The invention also provides a yield ratio value of (-)-alpha-bisabolol in fermentation production of (-)-alpha-bisabolol relative to a wild type. It is proved that the mutant obtains the (-)-alpha-bisabolol yield which is equal to or higher than that of the wild type (-)-alpha-bisabolol synthase. It is proved that the mutation mode and the obtained mutant can be used for batch production and popularization of (-)-alpha-bisabolol, and therefore development of multiple downstream industries such as beauty makeup and medicine is assisted.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a (-)-α-bisabolol synthase mutant and application thereof. Background Art

[0002] (-)-α-Bisabolol is a natural sesquiterpene compound, mainly used as a skin care product and cosmetic ingredient, and has potential medicinal value. For example, it has a faint sweet floral scent and is often used as a fragrance. In addition, it has soothing, antibacterial, antioxidant, anti-aging and skin whitening properties. The current production method of (-)-α-bisabolol from natural plants such as chamomile is not only costly but also unsustainable, and chemical synthesis can only produce a racemic bisabolol mixture, which is difficult to bring out the medicinal value of (-)-α-bisabolol. Heterologous expression of (-)-α-bisabolol synthase in chassis microorganisms through synthetic biology can open up a path for the sustainable production of (-)-α-bisabolol, but natural terpene synthases are often less active and difficult to meet the needs of industrial production. Therefore, using enzyme engineering to improve the activity of (-)-α-bisabolol synthase can break through the bottleneck of (-)-α-bisabolol synthesis, which is of great significance for the industrial production of (-)-α-bisabolol synthase. Summary of the invention

[0003] The purpose of the present disclosure is to provide a (-)-α-bisabolol synthase mutant, and to use the mutant enzyme to increase the expression amount of (-)-α-bisabolol with chirality selection in an expression system, thereby achieving the purpose of efficient and continuous production of (-)-α-bisabolol.

[0004] In order to achieve the above objectives, the present disclosure provides the following technical solutions:

[0005] In a first aspect, the present disclosure provides a (-)-α-bisabolol synthase mutant selected from (a) or (b):

[0006] (a) a first protein having (-)-α-bisabolol synthase activity derived from a (-)-α-bisabolol synthase with an amino acid sequence as shown in SEQ ID No: 1, by substitution, deletion or addition of at least one amino acid;

[0007] (b) a second protein having more than 83% identity with (a) and having (-)-α-bisabolol synthase activity, wherein the amino acid sequence of the second protein is not identical to SEQ ID No: 1, and the identity includes but is not limited to 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0008] In an optional embodiment, the substitution described in (a) includes a third protein having (-)-α-bisabolol synthase activity derived from amino acid residues 320 to 550 of SEQ ID No: 1 by substitution of at least one amino acid residue; for example, the number of amino acid residue substitutions can be 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0009] Furthermore, the substituted site includes at least one of position 321, 323, 399, 425, 465, 469, 473, 541 or 544 of SEQ ID No:1.

[0010] Furthermore, the substitution includes at least one conservative substitution and at least one non-conservative substitution in position 321, 323, 399, 425, 465, 469, 473, 541 or 544 of SEQ ID No: 1. The amino acid residue substitution includes at least one of I321V, L323I, L399Y, C425N, L465C, D469N, T473S, S541A or L544I. For example, it can be any one of the following combinations: I321V and C425N; I321V and S541A; L399Y and T473S; C425N and S541A; L465C and L544I; T473S and S541A; I321V, C425N and S541A; L323I, L465C, S541A and L544I; I321V, L465C, D469N, T473S, S541A and L544I.

[0011] In another optional embodiment, the (-)-α-bisabolol synthase mutant is a (-)-α-bisabolol synthase having an amino acid sequence as shown in SEQ ID No: 1, wherein 1 to 29 amino acid residues are deleted at the N-terminus, or 1 to 19 amino acid residues are deleted at the C-terminus.

[0012] In a second aspect, the present disclosure provides a fusion protein, the fusion protein comprising the (-)-α-bisabolol synthase mutant described in the first aspect and a functional protein connected to its N-terminus or C-terminus. Optionally, the functional protein comprises a fusion tag.

[0013] In a third aspect, the present disclosure provides a biomaterial, comprising any one of (a) to (c):

[0014] (a) a nucleic acid molecule encoding the (-)-α-bisabolol synthase mutant described in the first aspect, or encoding the fusion protein described in the second aspect;

[0015] (b) a recombinant vector containing the nucleic acid molecule described in (a);

[0016] (c) A recombinant cell comprising the nucleic acid molecule described in (a) and / or the recombinant vector described in (b), wherein the original cell of the recombinant cell comprises bacteria or fungi.

[0017] In an alternative embodiment, the original cell of the (c) recombinant cell contains a mevalonate pathway gene, and / or the gene ERG20 encoding farnesyl pyrophosphate synthase.

[0018] Preferably, the mevalonate pathway genes include: gene ERG10 encoding acetoacetyl-CoA thiolase, gene ERG13 encoding HMG-CoA synthase, gene tHMG1 encoding HMG-CoA reductase, gene ERG12 encoding mevalonate kinase, gene ERG8 encoding mevalonate-5-phosphate kinase, gene MVD1 encoding mevalonate pyrophosphate decarboxylase, and gene IDI1 encoding isoprene pyrophosphate isomerase.

[0019] In a fourth aspect, the present disclosure provides use of the (-)-α-bisabolol synthase mutant, fusion protein or biomaterial described in the aforementioned three aspects in the preparation of (-)-α-bisabolol.

[0020] In a fifth aspect, the present disclosure provides a method for preparing (-)-α-bisabolol, the method comprising culturing the (c) recombinant cells in the biological material described in the third aspect, and isolating and obtaining (-)-α-bisabolol.

[0021] The present invention discloses three types of (-)-α-bisabolol synthase mutants constructed on the basis of wild-type (-)-α-bisabolol synthase (SEQ ID No: 1) through substitution, deletion and addition mutations, (1) by amino acid substitution at different sites or different site combinations within its specific sequence fragment, (2) by deleting part of its N-terminal or C-terminal amino acid fragment, (3) by trying to add one or more amino acid residues at its N-terminal or C-terminal, or connecting other functional proteins to obtain a new fusion protein, and investigates the (-)-α-bisabolol synthase activity of homologous mutants with different consistency from the mutants obtained by the above different mutation methods, and the yield ratio relative to the wild type when used for fermentation production of (-)-α-bisabolol. It has been confirmed that the above mutants have achieved a (-)-α-bisabolol yield equivalent to or higher than that of the wild-type (-)-α-bisabolol synthase. It has been proved that the above mutation method and the obtained mutants, as well as the recombinant cells that biosynthesize the above mutants, can be used for the mass production and promotion of (-)-α-bisabolol, thereby promoting the development of many downstream industries such as beauty, cosmetics, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 It is the synthesis pathway of (-)-α-bisabolol in yeast cells;

[0024] Figure 2 Schematic diagram of the element map of the plasmid vector pZY900 used in Example 1. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments.

[0026] (I) Definitions or terms

[0027] The following abbreviations are used for the relevant definitions or terms involved in this disclosure. Unless otherwise defined, all scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art. The following terms are provided below.

[0028] As used herein, the terms "a" and "an" and "the" and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0029] As used herein, the conjunction term "and / or" between a variety of described elements is understood to include both single options and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within the meaning, thus satisfying the requirements of the term "and / or" used herein. The concurrent applicability of multiple options is also understood to be the meaning of the term, thus satisfying the requirements of the term "and / or".

[0030] As used herein, the term "(-)-α-bisabolol" is an organic compound derived from Matricariachamomilla var.Recutita, with a molecular formula of C 15 H 26O, a non-toxic sesquiterpene alcohol found in natural essential oils, has anticancer activity. (-)-α-Bisabolol exerts anticancer effects on A549 NSCLC cells by inducing cell cycle arrest, mitochondrial death, and inhibiting the PI3K / Akt signaling pathway. (-)-α-Bisabolol is mainly used in skin protection and skin care cosmetics. (-)-α-Bisabolol is used as an active ingredient to protect and care for allergic skin. (-)-α-Bisabolol is suitable for use in sunscreen products, after-sunbathing baths, baby products, and after-shave care products. In addition, (-)-α-Bisabolol can also be used in oral hygiene products, such as toothpaste and mouthwash.

[0031] As used herein, the terms "first", "second", etc., may be used herein to describe different proteins, only as a distinction between different proteins, and do not indicate an order or connection relationship, etc. For example, without departing from the scope of the present disclosure, the first protein may be referred to as the fourth protein or the fifth protein, and similarly, the second protein may be referred to as the sixth protein or the seventh protein, which means that both the first protein and the second protein are proteins, but they are not the same protein.

[0032] As used herein, the term "nucleic acid molecule", which may also be referred to as "polynucleotide", "nucleic acid", refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are usually linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA. As used herein, an isolated nucleic acid molecule is a nucleic acid molecule separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. "Isolated" nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include nucleic acid molecules isolated or contained in a recombinant vector or recombinant cell that encode the provided (-)-α-bisabolol synthase mutant.

[0033] As used herein, the term "conservative substitution" or "conservative sequence modification" of a sequence refers to nucleotide and amino acid sequence modifications that do not eliminate the (-)-α-bisabolol synthetic activity of the amino acid sequence encoded by the nucleotide sequence or containing the amino acid sequence. These conservative sequence modifications include conservative nucleotide and amino acid substitutions as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., gene synthesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residue in (-)-α-bisabolol synthase is preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and amino acid conservative substitutions having (-)-α-bisabolol synthesis activity are well known in the art. As described herein, the representation of substitution is "single letter abbreviation of amino acid before substitution-substitution position-single letter abbreviation of amino acid after substitution", for example, "S541A" means that serine at position 541 is replaced by alanine. Amino acids that can be conservatively substituted are shown in Table 1 below:

[0034] Table 1 Examples of conservative amino acid substitutions

[0035]

[0036]

[0037] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0038] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate and amplify a vector. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, yeast cells.

[0039] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors disclosed herein contain polynucleotide sequences and additional sequence elements, such as for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express antibodies and antibody fragments of the present disclosure include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES) and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. The expression vectors disclosed herein may also contain the following polynucleotides, which encode markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0040] As used herein, "expression vector" includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0041] As used herein, the term "homology" has a meaning recognized in the art and is a central concept in comparative biology. The basic meaning of homology is that the two samples being compared (such as an amino acid sequence or a nucleotide sequence) have a common ancestor. In general, if two traits (states) in two species meet any of the following two conditions, the two traits can be called a pair of homologous traits: 1. They are the same as a trait found in the ancestral group of these species; 2. They are different traits with an ancestor-descendant relationship. Usually, identity and similarity are used as indicators to measure the degree of homology between two sequences. Among them, identity refers to whether the residues at the same position of the two sequences are the same, that is, the percentage of the number of identical residues at the corresponding positions in the length of the same comparison to the total length. Similarity refers to the percentage of identical and similar residues at the corresponding positions to the total number. This indicator includes identical and similar residues, so it may be more tolerant than the consistency indicator. In practical applications, those skilled in the art can choose which indicator to use according to different needs. Generally speaking, if you need to measure the similarity between two sequences more strictly, you can choose to use a consistency index; if you need to measure the similarity between two sequences more loosely, you can choose to use a similarity index.

[0042] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. For example, a protein domain can be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an "amino terminal fusion protein" or a "carboxyl terminal fusion protein", respectively. In an optional embodiment, the fusion protein is a single-chain polypeptide that can be completely encoded by a nucleic acid sequence and includes at least two protein domains covalently linked by a peptide connection or optionally covalently linked by a peptide linker.

[0043] As used herein, the term "functional protein" refers to a naturally occurring protein, a functional variant thereof, or an engineered derivative thereof.

[0044] (II) Detailed technical solution

[0045] In a first aspect, the present disclosure provides a (-)-α-bisabolol synthase mutant selected from (a) or (b):

[0046] (a) a first protein having (-)-α-bisabolol synthase activity derived from a (-)-α-bisabolol synthase with an amino acid sequence as shown in SEQ ID No: 1, by substitution, deletion or addition of at least one amino acid;

[0047] (b) a second protein having more than 83% identity with (a) and having (-)-α-bisabolol synthase activity, wherein the amino acid sequence of the second protein is not identical to SEQ ID No: 1, and the identity includes but is not limited to 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0048] In an optional embodiment, the substitution described in (a) includes a third protein having (-)-α-bisabolol synthase activity derived from amino acid residues 320 to 550 of SEQ ID No: 1 by substitution of at least one amino acid residue; for example, the number of amino acid residue substitutions can be 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0049] Furthermore, the substituted site includes at least one of position 321, 323, 399, 425, 465, 469, 473, 541 or 544 of SEQ ID No:1.

[0050] Furthermore, the substitution includes at least one conservative substitution and at least one non-conservative substitution in position 321, 323, 399, 425, 465, 469, 473, 541 or 544 of SEQ ID No: 1. The amino acid residue substitution includes at least one of I321V, L323I, L399Y, C425N, L465C, D469N, T473S, S541A or L544I. For example, it can be any one of the following combinations: I321V and C425N; I321V and S541A; L399Y and T473S; C425N and S541A; L465C and L544I; T473S and S541A; I321V, C425N and S541A; L323I, L465C, S541A and L544I; I321V, L465C, D469N, T473S, S541A and L544I, as shown in Table 2:

[0051] Table 2 Location and type of mutants

[0052] name Mutation location and type Mutant 1 (SEQ ID NO: 3) I321V Mutant 2 (SEQ ID NO: 5) L323I Mutant 3 (SEQ ID NO: 7) L399Y Mutant 4 (SEQ ID NO: 9) L465C Mutant 5 (SEQ ID NO: 11) C425N Mutant 6 (SEQ ID NO: 13) D469N Mutant 7 (SEQ ID NO: 15) T473S Mutant 8 (SEQ ID NO: 17) S541A Mutant 9 (SEQ ID NO: 19) L544I Mutant 10 (SEQ ID NO: 21) I321V / C425N Mutant 11 (SEQ ID NO: 23) I321V / S541A Mutant 12 (SEQ ID NO: 25) L399Y / T473S Mutant 13 (SEQ ID NO: 27) C425N / S541A Mutant 14 (SEQ ID NO: 29) L465C / L544I Mutant 15 (SEQ ID NO: 31) T473S / S541A Mutant 16 (SEQ ID NO: 33) I321V / C425N / S541A Mutant 17 (SEQ ID NO: 35) L323I / L465C / S541A / L544I Mutant 18 (SEQ ID NO: 37) I321V / L465C / D469N / T473S / S541A / L544I

[0053] In an optional embodiment, the present disclosure provides new mutants having different identities from the amino acid sequences of the above mutants, wherein the identity adjustment is achieved by amino acid residue substitution, and the mutation sites listed in Table 2 are not involved in the identity adjustment process. As an example, the amino acid sequences of the above mutants 1, 3, 5, 8, 11, 13, 16 and 18 after the identity ratios are adjusted are shown in SEQ ID NOs: 39 to 53 (odd numbers), respectively, and the nucleotide sequences are shown in SEQ ID NOs: 40 to 54 (even numbers), respectively.

[0054] In another optional embodiment, the (-)-α-bisabolol synthase mutant is a (-)-α-bisabolol synthase having an amino acid sequence as shown in SEQ ID No: 1, wherein 1 to 29 amino acid residues are deleted at the N-terminus, or 1 to 19 amino acid residues are deleted at the C-terminus.

[0055] In a second aspect, the present disclosure provides a fusion protein, the fusion protein comprising the (-)-α-bisabolol synthase mutant described in the first aspect and a functional protein connected to its N-terminus or C-terminus. Optionally, the functional protein comprises a fusion tag.

[0056] Wherein, the fusion tag protein includes a purification tag protein and / or a reporter tag protein, wherein the purification tag protein includes a His tag (histidine tag), a GST tag (glutathione sulfhydryl transferase tag), an MBP tag (maltose binding protein tag), a FLAG tag, an Avi tag (a short peptide with a single biotinylated lysine site), a SUMO tag (a small molecule ubiquitin-like modified protein), a Halo tag (a genetically modified derivative of a dehalogenase) or a SNAP tag (derived from an O6-methylguanine-DNA methyl transfer reaction). The reporter tag protein includes a c-Myc tag, an HA tag or a luciferase or fluorescent tag protein.

[0057] In a third aspect, the present disclosure provides a biomaterial, comprising any one of (a) to (c):

[0058] (a) a nucleic acid molecule encoding the (-)-α-bisabolol synthase mutant described in the first aspect, or encoding the fusion protein described in the second aspect;

[0059] (b) a recombinant vector containing the nucleic acid molecule described in (a);

[0060] (c) A recombinant cell comprising the nucleic acid molecule described in (a) and / or the recombinant vector described in (b), wherein the original cell of the recombinant cell comprises bacteria or fungi.

[0061] In an alternative embodiment, the original cell of the (c) recombinant cell contains a mevalonate pathway gene, and / or the gene ERG20 encoding farnesyl pyrophosphate synthase.

[0062] Preferably, the mevalonate pathway genes include: gene ERG10 encoding acetoacetyl-CoA thiolase, gene ERG13 encoding HMG-CoA synthase, gene tHMG1 encoding HMG-CoA reductase, gene ERG12 encoding mevalonate kinase, gene ERG8 encoding mevalonate-5-phosphate kinase, gene MVD1 encoding mevalonate pyrophosphate decarboxylase, and gene IDI1 encoding isoprene pyrophosphate isomerase.

[0063] In a fourth aspect, the present disclosure provides use of the (-)-α-bisabolol synthase mutant, fusion protein or biomaterial described in the aforementioned three aspects in the preparation of (-)-α-bisabolol.

[0064] In a fifth aspect, the present disclosure provides a method for preparing (-)-α-bisabolol, the method comprising culturing the (c) recombinant cells in the biological material described in the third aspect, and isolating and obtaining (-)-α-bisabolol.

[0065] Some embodiments of the present disclosure are described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. The following examples are used to further illustrate the present disclosure, but should not be construed as limiting the present disclosure. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present disclosure should be equivalent replacement methods and are included in the protection scope of the present disclosure. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0066] Example 1 Construction of an engineered yeast strain expressing (-)-α-bisabolol synthase

[0067] Reference to the synthesis pathway of (-)-α-bisabolol in yeast cells (e.g. Figure 1 As shown), plasmid pZY900 (see patent 202210473488.4) is used to construct a yeast transformation fragment, which is characterized by: △LEU2:LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20, using promoters GAL1 and GAL10 to control the expression gene ERG20 and the insertion gene respectively, the screening marker is Leu2, and the chromosome site of the insertion is Leu2, such as Figure 2 shown.

[0068] Primers pXL01-1-F / R were used to amplify the synthetic gene (SEQ ID NO: 2) encoding (-)-α-bisabolol synthase (SEQ ID NO: 1), and the target gene was inserted into pZY900 using Golden Gate assembly to obtain plasmid pXL01. After restriction digestion and gel recovery using MssI and NdeI, the fragment was transformed into the competent yeast strain JCR27 by the PEG / LiAC method (the construction of yeast strain JCR27 is shown in the literature Siemon, Thomas et al. "Semisynthesis of Plant Derived Englerin A Enabled by Microbe Engineering of Guaia-6, 10 (14) -diene as Building Block." Journal of the American Chemical Society vol. 142, 6 (2020): 2760-2765. doi: 10.1021 / jacs.9b12940), and the obtained positive strain was named JXL01. The strain JXL01 was inoculated into 5 mL of seed culture medium (peptone (20 g / L), yeast powder (10 g / L), glucose (20 g / L)) and cultured at 30 °C and 200 rpm for 20-24 h. 600 =0.1 was transferred to 45 mL fermentation medium (peptone (20 g / L), yeast powder (10 g / L), glucose (10 g / L), galactose (10 g / L)), and covered with 5 mL organic phase (isopropyl myristate) after transfer, and fermented at 30°C and 200 rpm for 72 h. The yield of (-)-α-bisabolol in the fermentation broth was determined by gas phase mass spectrometry GC-MS, and the equipment used for the GC-MS detection was a TRACE GC ULTRA gas chromatograph equipped with an AS 3000 automatic injection and a split / splitless injector, and a TSQQUANTUM XLS mass spectrometer equipped with a triple quadrupole detector from Thermo Fisher Scientific. The GC-MS detection parameters are as follows: the chromatographic column is a TR-5MS column (30m×0.25mm×0.25μm), the carrier gas is high-purity helium, the flow rate is 1mL / min, acetone is used as the needle wash liquid, the injection volume is 1μL, and the split ratio is 50. The injection port temperature is 250°C, and the ion transfer tube temperature is 270°C. Detection procedure: the initial column temperature is 70°C, and the temperature is increased to 180°C at 10°C / min; the temperature is increased to 300°C at 20°C / min and maintained for 2min.

[0069] Example 2 Construction and fermentation of substitution mutants

[0070] The operation steps were as described in Example 1, except that the synthetic gene (SEQ ID NO: 2) encoding (-)-α-bisabolol synthase (SEQ ID NO: 1) was replaced with synthetic genes (SEQ ID NO: 4-38 (even numbers)) encoding (-)-α-bisabolol synthase mutants (SEQ ID NO: 3-37 (odd numbers)). The obtained positive strains were named JXL02-JXL19. The strains JXL01-19 were inoculated into 5 mL of seed culture medium (peptone (20 g / L), yeast powder (10 g / L), glucose (20 g / L)) and cultured at 30°C, 200 rpm for 20-24 h. Subsequently, they were transferred to 45 mL of fermentation medium (peptone (20 g / L), yeast powder (10 g / L), glucose (10 g / L), galactose (10 g / L)) at a final OD600 of 0.1. After the transfer, they were covered with 5 mL of organic phase (isopropyl myristate) and fermented at 30°C, 200 rpm for 72 h. The same GC-MS determination method as in Example 1 was used to determine the yield of (-)-α-bisabolol in the fermentation broth, and the increase in the yield of (-)-α-bisabolol of yeast engineering expressing different mutants compared to the yeast engineering bacteria expressing the wild enzyme (increase = (experimental group - control) / control) and the yield ratio (experimental group / control group) were calculated. The results are shown in the following table.

[0071] Table 3 Activity parameters of (-)-α-bisabolol synthase mutants and related strains

[0072]

[0073]

[0074] As can be seen from Table 3, the (-)-α-bisabolol yields obtained by the mutants provided in this example were significantly improved compared to the wild-type (-)-α-bisabolol synthase.

[0075] Example 3 Comparison of yields of different homologous nucleotide sequences

[0076] Based on the amino acid sequence design of different (-)-α-bisabolol synthase mutants, different homologous amino acid sequences were designed with consistency as an indicator (Table 4). According to the operating steps described in Example 1, only the synthetic gene (SEQ ID NO: 2) encoding (-)-α-bisabolol synthase (SEQ ID NO: 1) was replaced with a gene encoding a homologous amino acid sequence with different consistency, and primers (pXL01-1-F, pXL01-1-R) were used for amplification. After obtaining the positive strain, fermentation was performed, and the difference in the yield of (-)-α-bisabolol before and after the design was compared.

[0077] Table 4 Comparison of activities of homologous amino acid sequences with different identities

[0078] consistency Designed amino acid sequence Optimized nucleotide sequence Corresponding mutants before design The yield ratio of the mutant before design 95.10％ SEQ ID NO:39 SEQ ID NO:40 Mutant 1 (SEQ ID NO: 3) 1.2 93.36％ SEQ ID NO:41 SEQ ID NO:42 Mutant 3 (SEQ ID NO: 7) 1.3 91.08％ SEQ ID NO:43 SEQ ID NO:44 Mutant 5 (SEQ ID NO: 11) 1.0 89.16％ SEQ ID NO:45 SEQ ID NO:46 Mutant 8 (SEQ ID NO: 17) 1.2 87.06％ SEQ ID NO:47 SEQ ID NO:48 Mutant 11 (SEQ ID NO: 23) 1.0 85.14％ SEQ ID NO:49 SEQ ID NO:50 Mutant 13 (SEQ ID NO: 27) 1.0 83.04％ SEQ ID NO:51 SEQ ID NO:52 Mutant 16 (SEQ ID NO: 33) 1.0 81.12％ SEQ ID NO:53 SEQ ID NO:54 Mutant 18 (SEQ ID NO: 37) 0.1

[0079] As can be seen from Table 4, based on the substitution mutants provided in the present disclosure, when the consistency adjustment is performed and the consistency ratio reaches more than 83%, the (-)-α-bisabolol yield obtained is significantly improved compared with the wild-type (-)-α-bisabolol synthase.

[0080] Example 4 Construction and fermentation of deletion mutants

[0081] The operation steps are as described in Example 1, except that the synthetic gene (SEQ ID NO: 2) encoding (-)-α-bisabolol synthase (SEQ ID NO: 1) is replaced with the gene of the mutant corresponding to (-)-α-bisabolol synthase and the corresponding primers (Table 5) are used to obtain positive strains expressing mutants with different amino acid truncation lengths and ferment them, and the difference in the yield of (-)-α-bisabolol before and after truncation is compared. When the N-terminal is truncated within 30 amino acids, there is no significant change in the yield before and after truncation, and when the N-terminal is truncated by 30 amino acids, the yield drops sharply, as shown in Table 6. When the C-terminal is truncated within 20 amino acids, there is no significant change in the yield before and after truncation, and when the C-terminal is truncated by 20 amino acids, the yield drops sharply, as shown in Table 7.

[0082] Table 5 Primer list

[0083]

[0084]

[0085] Table 6 Comparison of the activities of mutants with different N-terminal truncation lengths

[0086] Truncated mutant N-terminal truncation length Forward Primer Post Primer Yield ratio after truncation / before truncation Mutant 1 (SEQ ID NO: 3) 5 1delN5-F pXL01-1-R 1.0 Mutant 2 (SEQ ID NO: 5) 10 2delN10-F pXL01-1-R 1.0 Mutant 3 (SEQ ID NO: 7) 15 3delN15-F pXL01-1-R 1.0 Mutant 4 (SEQ ID NO: 9) 20 4delN20-F pXL01-1-R 1.0 Mutant 6 (SEQ ID NO: 13) 25 5delN25-F pXL01-1-R 1.0 Mutant 7 (SEQ ID NO: 15) 26 6delN26-F pXL01-1-R 1.0 Mutant 8 (SEQ ID NO: 17) 27 7delN27-F pXL01-1-R 1.0 Mutant 11 (SEQ ID NO: 23) 28 9delN28-F pXL01-1-R 0.9 Mutant 17 (SEQ ID NO: 35) 29 13delN29-F pXL01-1-R 0.9 Mutant 18 (SEQ ID NO: 37) 30 13delN30-F pXL01-1-R 0.2

[0087] Table 7 Comparison of activities of mutants with different C-terminal truncation lengths

[0088] Mutants C-terminal truncation length Forward Primer Post Primer Yield ratio after truncation / before truncation Mutant 1 (SEQ ID NO: 3) 5 pXL01-1-F 1delC5-R 1.0 Mutant 6 (SEQ ID NO: 13) 10 pXL01-1-F 5delC10-R 1.0 Mutant 11 (SEQ ID NO: 23) 15 pXL01-1-F 9delC10-R 1.0 Mutant 12 (SEQ ID NO: 25) 16 pXL01-1-F 10delC16-R 1.0 Mutant 14 (SEQ ID NO: 29) 17 pXL01-1-F 11delC17-R 1.0 Mutant 15 (SEQ ID NO: 31) 18 pXL01-1-F 12delC18-R 1.0 Mutant 17 (SEQ ID NO: 35) 19 pXL01-1-F 13delC19-R 1.0 Mutant 18 (SEQ ID NO: 37) 20 pXL01-1-F 14delC20-R 0.1

[0089] As can be seen from Tables 6 and 7, in the truncation mutation modification, there are significant differences in the lengths that can be truncated between the N-terminus and the C-terminus of the wild-type (-)-α-bisabolol synthase. When the N-terminus is truncated by 29 amino acid residues, the activity of the (-)-α-bisabolol synthase and the resulting (-)-α-bisabolol yield are not significantly affected, while when the N-terminus is truncated to 30 amino acid residues, the yield of (-)-α-bisabolol is significantly reduced. When the C-terminus is truncated to 20 amino acid residues, the yield of (-)-α-bisabolol is significantly reduced.

[0090] Example 5 Construction and fermentation of fusion protein (added mutant)

[0091] The operation steps were as described in Example 1, except that the synthetic gene (SEQ ID NO: 2) encoding the (-)-α-bisabolol synthase (SEQ ID NO: 1) was replaced with a gene (SEQ ID NO: 56 or 60) encoding a mutant fusion protein (SEQ ID NO: 55 or 59) with an N-terminal MBP tag protein added thereto, or a gene (SEQ ID NO: 57 or 61) encoding a mutant fusion protein (SEQ ID NO: 58 or 62) with an C-terminal SUMO tag protein added thereto, or a gene (SEQ ID NO: 64) encoding a mutant fusion protein (SEQ ID NO: 63) with an N-terminal FLAG tag protein added thereto, or a gene (SEQ ID NO: 66) encoding a mutant fusion protein (SEQ ID NO: 65) with an C-terminal FLAG tag protein added thereto, and the corresponding primers (Table 3) were used to obtain a positive strain and then fermented to compare the difference in the yield of (-)-α-bisabolol before and after the addition of the additional protein sequence. Adding additional proteins to both ends of (-)-α-bisabolol synthase not only does not affect the activity of the enzyme, but also has a certain promoting effect on the activity of the enzyme.

[0092] Table 8 Comparison of the activities of different fusion mutant fusion proteins

[0093]

[0094] Comparison sequence list

[0095] SEQ ID NO:1

[0096] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0097] SEQ ID NO:2

[0098]

[0099] SEQ ID NO:3

[0100] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0101] SEQ ID NO:4

[0102]

[0103] SEQ ID NO:5

[0104] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVIDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0105] SEQ ID NO:6

[0106]

[0107] SEQ ID NO:7

[0108] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSYLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0109] SEQ ID NO:8

[0110]

[0111] SEQ ID NO:9

[0112] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0113] SEQ ID NO:10

[0114]

[0115] SEQ ID NO:11

[0116] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0117] SEQ ID NO:12

[0118]

[0119] SEQ ID NO:13

[0120] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMNDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0121] SEQ ID NO:14

[0122]

[0123] SEQ ID NO:15

[0124] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0125] SEQ ID NO:16

[0126]

[0127] SEQ ID NO:17

[0128] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0129] SEQ ID NO:18

[0130]

[0131] SEQ ID NO:19

[0132] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0133] SEQ ID NO:20

[0134]

[0135] SEQ ID NO:21

[0136] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0137] SEQ ID NO:22

[0138]

[0139] SEQ ID NO:23

[0140] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0141] SEQ ID NO:24

[0142]

[0143] SEQ ID NO:25

[0144] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSYLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0145] SEQ ID NO:26

[0146]

[0147] SEQ ID NO:27

[0148] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0149] SEQ ID NO:28

[0150]

[0151] SEQ ID NO:29

[0152] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0153] SEQ ID NO:30

[0154]

[0155] SEQ ID NO:31

[0156] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0157] SEQ ID NO:32

[0158]

[0159] SEQ ID NO:33

[0160] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0161] SEQ ID NO:34

[0162]

[0163] SEQ ID NO:35

[0164] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVIDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0165] SEQ ID NO:36

[0166]

[0167] SEQ ID NO:37

[0168] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMNDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0169] SEQ ID NO:38

[0170]

[0171] SEQ ID NO:39

[0172] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIVGDQFLEYKEKFNVATEKQLICELKEEVRNELMIRACNEASRYIKLDQLIDVVERLGLAYHFEKEIECSLQEIYVTYGHKWTNYQNIESLSLWFRLLRQNCFNVSSDIFENHIDEKGNFQESMCNDPQGMLALYESAYMRVEGEIILDKANEFTKLHLGIISNMPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEALLKLAKLDFNVLQEMIRDELSQICKWKKDLDIHNKLPYVRDRLIEGYFWILGKYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAIQIHYIKEMAKIGTFSLLLEAKWLKEGYYPTSDEYLSNSLVTCGYALMTARSYVAPDDGIVTEDAFKWVATHPPIVKAACKLLRLQDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVIVQESLMPYDVPFPLLIPAISLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0173] SEQ ID NO:40

[0174]

[0175] SEQ ID NO:41

[0176] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNEVIFHDSIWGDQFLEYKEKFNVASEKQLIEELKEEVRNELMIRACNEASRYIKLIQLNDVVERLGLAYHFEKEVEESLQHIYVTYGHKWTNYNNIESLSGWFTLLRQWGPNVSSDITENHIDEKGNQQESLCNDPQGMLCLYEAAYMRVEGEIILDKALEFYKLHLGIISNDPSCDSSLATEQKQALKQPLRRRLPRLENVRYIAIYQQKASHSEVLLHLAKLDFNVLQEMHKIELSQICKWWWDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSCTRMFLMPTCMWLIVLDDTFDNYCTYEALEIFRQACERWSITCLDELPEYMKNIYHEQFRVHQEMEESLEKEGKAYQIHYSKEMAKIHTRSYLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKIIRLMDDIATMKEYQERGHIASSIKCYRKEKGPSEEEAKMDFLKQVEFGWKVMNQESLMPTDVPFPLLIPAINLYRVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0177] SEQ ID NO:42

[0178]

[0179] SEQ ID NO:43

[0180] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHCSIWGDQCHEYKELFNTATMKQLIEELKEEVRNELMILACAEASRYEKLIQLIDVVERWGWAKRFEKEIEESHQHIYVTYGHKWTSYNNIESLSLWFRLLRQNGFNVASDIFENHDDEKGNFQWSLCNDGPGMLALYEYYYMRVEGEEHLDKALEFVKLHLGVISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYEAIYQQKASHSEVLLKLNMLAFNVFQEMHKDELCQICKWWKDLDIWNKLPYVRDRLIEGYFWKLGIYFEFQHSRTRMFLMKTDMWLIVLLDEFDNYGTLEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIAYIKEMAKEGTRSLLLEAKWLTEGYMPTLDEYLPDSLVTNGYALMTARSYVARDDGICTNDAFKWVATHPPIVKAACKILRLMDDIATHKFEQERGHISSSKECYRKETGASEEEACMDFLKQVEDAWKVINQESLMPTDVQFHLLIPAINLARVSDTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0181] SEQ ID NO:44

[0182]

[0183] SEQ ID NO:45

[0184] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRESTIFKGSIWGDQFLDYKESFNFAFEKQLIGELKEEVREELMIRACNEASRYIKLEQLIDVVERKGLAPHFEKEIEESGQHIYVTYGHKWTHYNNIESLSLWFRLLRQNGFNVSSRIFENHIDEKGAFQEGLWNDPQAMLALYEFAYMRVEGEYILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKAIHIEVLLKLRRLFFNVLQEMHKDELSQTCKWWKPLDIRNKLPYVRDRLIEGYFWILGISFEPQESRTRMVLPKTCMWLIVLSWTFDNYGTYEELEIFSQAVERHSITCLVELPEYMKPIDHEAFRVNQEMEESLEKNGKAYAIHYIKEMAQEGTRSLLLMAKWLKEGSMKTLDLYLSNSLVTCGRALMTARSYVARDDGIATEDAFKWVATHPPIVKMGCKILKLMDDIATHKEEQERGHIASSIECYRKETGASEESACMDFHKFVEDLWKVINQESLMESDVPFPDLIPAIILARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0185] SEQ ID NO:46

[0186]

[0187] SEQ ID NO:47

[0188] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNMVILHDSIWGDQFLEYYEKQQVATEKQLIEEKKEEVRNELMIRCCNEASRYIKLIQLIDVVERLGNAYHFEKKIEESLQQIYTTYGHKWTNMNNIESLSLWFRLLRWNGFNVSSDIEENHIDEKGILQESLCNDPQGMLALYEAWYMRVEGECILDKALEFTKNHLGMISNDPSCMSSLRTEIKQALQQPLRRRLVRLEAVRWICIYQQRTHKSEVLLKLIKGDFNVLQEMHKDELSQPPKWEKDLDIRQKLPQVRNRLINGGFWILHIYNEPQHSYTRVFLMCTCMWLVRLDDTFDNYGTYEELTIFTQVVERWSITCLDELPEYMKLKYHEQFRVAQEVEPSLEWEGKATQIHYIKEMGKEGTRSLLLEAKWLSLGYMPTLFEYLSSSEVTCKYALMPARSYDARDDGIVTEDAFFWVATHPPIVAAACKILSLMDDIATHKEEQERGHIAISIEKYRKETGASEEETCDDWLKQVEYGWKVIVQESLMPTDVPFPKLIPAENLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0189] SEQ ID NO:48

[0190]

[0191] SEQ ID NO:49

[0192] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRWSVIFHDSIVNDQFLFYKEKFNVAREKQLIEEMDWEVRDHLVTRACNKASRYIKLSTLIDVWERLGLAYHFEKEIEESLYRIYVTYGHKWTNYNNIEARSLWFKLCRQDGFNVSADIFENIIDEKGNFQEFLCNHPQGTLALWETAYMRVEFEIILDRWLEITHLHIGIISNDPSCDSSLRTEIKQALKQPLRRRLLRLEAVRYIAIYPQKASHSEVLEKLAKLQFPVLFGMLKEELSMILRWWKDLDIRNSLPHVRDRLEEGYFWILGIYFEPQHSRTRMFLMKDRMWTIVLDDTQDNSGTYECQEIFTQKVGRWSIACHDELPEYMKLIYHEQFRVHQEMEMELEKEGFAGQIHYIKEMAKEGTRSLLLEKKWLKEKYMPTVDEYLSNSLVPNSYALMTAWSYVARDDGIVTEDAFKWVATNPCIVKTARHILRLMDSIATHKEEQERGHIASSFECYRKGTGASCEEACMIFLKQREDGWKVINQESLMPTDCPFPLKIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0193] SEQ ID NO:50

[0194]

[0195] SEQ ID NO:51

[0196] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSKIFHDKIWGDQFLEYKEFPNVITEKQRIEELKWEVHNELMDRACNENSRYIVLIRLIDVVERLWLDSHFEKEIEESLRHIYVTYGIKWWNYFNIESLSLWFRLLFQNGFNVSSDIFENHIDELGYFQGKLCRDGQGMLALYESAKMRVEGEICLDKYLEFTKLHLGIRVNDPSCDSSHRTEIVQVEKQPLRRRLPRLEAVRYCAIYQQKLSHQAVWEKLMKLDFNPLQEMHRDEMSMHCKWWKDLDIRNKLPYPRPRDIEGYFWILGIYFEPQHSRTGMFLMKTCMWLVVLDDTFDNYGTYEELEIQTQAVEKWSSTCSDELPEYMHLIYHEQFRVHQEMDYSEEKEGKAYQRHYIDEPAKADMRSLLWEAKWLKEMYMCTLDEYLSNSNVTNGYHLMTAHSYVAYCDGGGWEDLFKWVPTHPPIVKKACKILHLCDDIATHKEPQEHGHRASSIHCYRKETGASKVEGKMCFLKQVEDGWKVINQSMLMATCVPFLLLIPAINHARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0197] SEQ ID NO:52

[0198]

[0199] SEQ ID NO:53

[0200] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHVSRWGDKFLEYKEKFNVAEEKQLIGELKEHVRNELMIRHEGENSGYIELIQLIDQCEQLGLACHKEKEKEESLQHIYVTYGHKWTNYNNIEMLSHWEHLLRQNYFNVSSDEFENHIDEKGNFQESLCNDPQGVLALYEAGYDRGEMEIILDKALEFTKLYLGIIENGPSCDSSLREEQKQALKQPLRRRFKRLEAVQFIGIYQQKASQSEVLLKLAKLDMNVDAEMHKDELCQICKWIKDWDIRDGLPYVTDRLIEGKFWILQIYFEPQHSRRRMQLSMTCMWLVVLDLTFDIYAMYEELEIFTRAVERWTITCLDELPEYMKLPYHEQFYVMQNMQFSLEKHGKAPQIHYIIEEQTEGARTLLLEAKWTAKGYMWWLGEYNSNSQVTCGYALMTAFSYVAIDDGIVGEDAFKLNATHPPIEKAADKICRLMNDIASHKYEQERSHIASSREKYRAEIYFSEEEAATDFLKQVEDGWKVINVESLMPTIVGFTLSIPALFLAPVADTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0201] SEQ ID NO:54

[0202]

[0203] SEQ ID NO:55

[0204] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMNDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0205] SEQ ID NO:56

[0206]

[0207] SEQ ID NO:57

[0208] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKICRLMNDIASHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTIYKDNDGYNHADKEVIGYIKSLFVHPMIVSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG

[0209] SEQ ID NO:58

[0210]

[0211] SEQ ID NO:59

[0212] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTMSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTIYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0213] SEQ ID NO:60

[0214]

[0215] SEQ ID NO:61

[0216] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVSDTIYKDNDGYNHADKEVIGYIKSLFVHPMIVSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG

[0217] SEQ ID NO:62

[0218]

[0219] SEQ ID NO:63

[0220] MDYKDHDGDYKDHDIDYKDDDDKMSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIGYIKSLFVHPMIV

[0221] SEQ ID NO:64

[0222] ATGGACTACAAAGATCACGACGGAGATTATAAAGACCACGATATTGATTACAAGGACGATGATGA

[0223] CAAGATGTCTACTCTGTCCGTCTCTACTCCATCTTTTTCATCTTCTCCATTGAGCTCTGTCAACAAGAA

[0224] CAGTACCAAGCAACACGTTACCCGGAATTCTGTTATTTTCCACGACTCTATCTGGGGTGACCAATTTTT

[0225] AGAATATAAAGAAAAGTTCAACGTTGCCACCGAAAAGCAATTGATTGAAGAATTGAAGGAAGAAGTT

[0226] AGAAACGAATTGATGATCAGGGCTTGTAACGAAGCGTCTAGATACATCAAGTTGATCCAATTAATTGA

[0227] TGTTGTTGAAAGACTGGGCTTGGCTTACCACTTCGAAAAGGAAATTGAAGAATCGCTACAACACATC

[0228] TACGTCACCTACGGTCATAAATGGACCAACTACAACAACATCGAATCCTTGTCTTTGTGGTTCCGTTTG

[0229] TTGAGACAACGGTTTCAACGTCTCCTCCGATATCTTTGAAAACCACATTGACGAAAAGGGTAACTT

[0230] CCAAGAATCTTTATGCAACGATCCTCAAGGTATGTTGGCTTTGTACGAAGCTGCCTACATGCGTGTTGA

[0231] AGGTGAAATCATTTTGGACAAGGCTTTAGAATTCACCAAGTTGCACTTGGGTATCATCTCCAATGACC

[0232] CATCTTGTGATTCTAGCTTGAGAACTGAAATTAAGCAAGCTTTGAAGCAACCATTGAGAAGACGTCTT

[0233] CCAAGATTAGAAGCTGTCAGATATATTGCTATCTACCAACAAGGCCTCCCACTCTGAAGTCTTATTG

[0234] AAACTTGCTAAATTGGACTTTAATGTTTTGCAAGAAATGCACAAGGACGAGCTCTCTCAAATTTGTAA

[0235] GTGGTGGAAGGACTTGGACATAAGAAACAAGTTGCCATACGTGAGAGACAGATTGATTGAAGGGTAT

[0236] TTCTGGATCTTAGGTATCTACTTCGAACCACAACACTCCAGAACCAGAATGTTCTTGATGAAGACCTG

[0237] TATGTGGTTGGTGGTCTTGGATGATACTTTCGACAACTACGGTACTTACGAAGAACTGGAGATCTTCA

[0238] CTCAAGCCGTCGAAAGATGGTCTATCACTTGTTTGGATGAATTGCCAGAATACATGAAGTTGATTTATC

[0239] ACGAACAATTCAGAGTCCACCAAGAGATGGAAGAATCCTTGGAAAAGGAAGGTAAGGCTTACCAAA

[0240] TTCATTACATTAAGGAAATGGCCAAGGAAGGTACTAGATCTCTATTGTTAGAAGCTAAGTGGTTGAAG

[0241] GAAGGTTACATGCCTACCTTGGATGAATACTTGTCCAACTCCTTGGTTACAAATGGTTACCGCTTTGATG

[0242] ACTGCTCGTTCTTACGTTGCTAGAGATGACGGTATCGTCACTGAAGACGCTTTCAAGTGGGTCGCTAC

[0243] TCATCCACCAATCGTCAAGGCTGCTTGCAAGATCTTGAGATTGATGGACGACATTGCTACCCACAAGG

[0244] AAGAACAAGAAAGAGGTCACATTGCTTCCTCTATTGAATGTTACAGAAAGGAAACCGGTGCTTCAGA

[0245] AGAAGAAGCCTGTATGGATTTCCTTAAGCAGGTTGAAGATGGTTGGAAGGTTATCAACCAAGAATCTT

[0246] TGATGCCAACTGACGTTCCATTCCCATTGTTGATTCCAGCTATTAACTTGGCCAGAGTTGCGGATACTT

[0247] TGTACAAGGATAACGACGGTTACAACCATGCCGACAAAGAAGTTATCGGTTACATCAAATCCTTATTC|GTTCACCCAATGATTGTTTGA

[0248] SEQ ID NO:65

[0249] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVR

[0250] NELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQN

[0251] GFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLR

[0252] TEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRN

[0253] KLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSITCL

[0254] DELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSN

[0255] SLVTNGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYR

[0256] KETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARVADTLYKDNDGYNHADKEVIG

[0257] YIKSLFVHPMIVDYKDHDGDYKDHDIDYKDDDDK

[0258] SEQ ID NO:66

[0259] ATGTCTACTCTGTCCGTCTCTACTCCATCTTTTTCATCTTCTCCATTGAGCTCTGTCAACAAGAAC

[0260] AGTACCAAGCAACACGTTACCCGGAATTCTGTTATTTTTCCACGACTCTATCTGGGGTGACCAATTTTTA

[0261] GAATATAAAGAAGAATTGAAGGAAGAAGTTA

[0262] GAAACGAATTGATGATCAGGGCTTGTAACGAAGCGTCTAGATACATCAAGTTGATCCAATTAATTGAT

[0263] GTTGTTGAAAGACTGGGCTTGGCTTACCACTTCGAAAAGGAAATTGAAGAATCGCTACAACACATCT

[0264] ACGTCACCTACGGTCATAAATGGACCAACTACAACAACATCGAATCCTTGTCTTTGTGGTTCCGTTTG

[0265] TTGAGACAACGGTTTCAACGTCTCCTCCGATATCTTTGAAAACCACATTGACGAAAAGGGTAACTT

[0266] CCAAGAATCTTTATGCAACGATCCTCAAGGTATGTTGGCTTTGTACGAAGCTGCCTACATGCGTGTTGA

[0267] AGGTGAAATCATTTTGGACAAGGCTTTAGAATTCACCAAGTTGCACTTGGGTATCATCTCCAATGACC

[0268] CATCTTGTGATTCTAGCTTGAGAACTGAAATTAAGCAAGCTTTGAAGCAACCATTGAGAAGACGTCTT

[0269] CCAAGATTAGAAGCTGTCAGATATATTGCTATCTACCAACAAGGCCTCCCACTCTGAAGTCTTATTG

[0270] AAACTTGCTAAATTGGACTTTAATGTTTTGCAAGAAATGCACAAGGACGAGCTCTCTCAAATTTGTAA

[0271] GTGGTGGAAGGACTTGGACATAAGAAACAAGTTGCCATACGTGAGAGACAGATTGATTGAAGGGTAT

[0272] TTCTGGATCTTAGGTATCTACTTCGAACCACAACACTCCAGAACCAGAATGTTCTTGATGAAGACCTG

[0273] TATGTGGTTGGTGGTCTTGGATGATACTTTCGACAACTACGGTACTTACGAAGAACTGGAGATCTTCA

[0274] CTCAAGCCGTCGAAAGATGGTCTATCACTTGTTTGGATGAATTGCCAGAATACATGAAGTTGATTTATC

[0275] ACGAACAATTCAGAGTCCACCAAGAGATGGAAGAATCCTTGGAAAAGGAAGGTAAGGCTTACCAAA

[0276] TTCATTACATTAAGGAAATGGCCAAGGAAGGTACTAGATCTCTATTGTTAGAAGCTAAGTGGTTGAAG

[0277] GAAGGTTACATGCCTACCTTGGATGAATACTTGTCCAACTCCTTGGTTACAAATGGTTACGCTTTGATG

[0278] ACTGCTCGTTCTTACGTTGCTAGAGATGACGGTATCGTCACTGAAGACGCTTTCAAGTGGGTCGCTAC

[0279] TCATCCACCAATCGTCAAGGCTGCTTGCAAGATCTTGAGATTGATGGACGACATTGCTACCCACAAGG

[0280] AAGAACAAGAAAGAGGTCACATTGCTTCCTCTATTGAATGTTACAGAAAGGAAACCGGTGCTTCAGA

[0281] AGAAGAAGCCTGTATGGATTTCCTTAAGCAGGTTGAAGATGGTTGGAAGGTTATCAACCAAGAATCTT

[0282] TGATGCCAACTGACGTTCCATTCCCATTGTTGATTCCAGCTATTAACTTGGCCAGAGTTGCGGATACTT

[0283] TGTACAAGGATAACGACGGTTACAACCATGCCGACAAAGAAGTTATCGGTTACATCAAATCCTTATTC

[0284] GTTCACCCAATGATTGTTGACTACAAAGATCACGACGGAGATTATAAAGACCACGATATTGATTACAA

[0285] GGACGATGATGACAAGTGA

Claims

1. A (-)-α-bisabolol synthase mutant selected from (a) or (b): (a) a first protein having (-)-α-bisabolol synthase activity derived from a (-)-α-bisabolol synthase with an amino acid sequence as shown in SEQ ID No: 1, by substitution, deletion or addition of at least one amino acid; (b) a second protein having more than 83% identity with (a) and having (-)-α-bisabolol synthase activity, wherein the amino acid sequence of the second protein is not the same as SEQ ID No:

1.

2. The (-)-α-bisabolol synthase mutant according to claim 1, wherein The (-)-α-bisabolol synthase mutant is a third protein having (-)-α-bisabolol synthase activity derived from the amino acid residues 320 to 550 of the (-)-α-bisabolol synthase shown in SEQ ID No: 1 by substitution of at least one amino acid residue; Preferably, the number of amino acid residue substitutions is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

3. The (-)-α-bisabolol synthase mutant according to claim 2, wherein The amino acid residue substitution sites include at least one of positions 321, 323, 399, 425, 465, 469, 473, 541 or 544 of SEQ ID No:

1.

4. The (-)-α-bisabolol synthase mutant according to claim 2 or 3, wherein The (-)-α-bisabolol synthase mutant has at least one conservative substitution and at least one non-conservative substitution at position 321, 323, 399, 425, 465, 469, 473, 541 or 544 of the (-)-α-bisabolol synthase with an amino acid sequence as shown in SEQ ID No:

1.

5. The (-)-α-bisabolol synthase mutant according to any one of claims 2 to 4, wherein The amino acid residue substitution includes at least one of I321V, L323I, L399Y, C425N, L465C, D469N, T473S, S541A or L544I; Optionally, the amino acid residue substitutions include any one of the following combinations: I321V and C425N; I321V and S541A; L399Y and T473S; C425N and S541A; L465C and L544I; T473S and S541A; I321V, C425N and S541A; L323I, L465C, S541A and L544I; I321V, L465C, D469N, T473S, S541A and L544I.

6. The (-)-α-bisabolol synthase mutant according to claim 1, wherein The (-)-α-bisabolol synthase mutant is a (-)-α-bisabolol synthase with an amino acid sequence as shown in SEQ ID No: 1, wherein 1 to 29 amino acid residues are deleted at the N-terminus, or 1 to 19 amino acid residues are deleted at the C-terminus.

7. A fusion protein comprising the (-)-α-bisabolol synthase mutant according to any one of claims 1 to 6 and a functional protein connected to its N-terminus or C-terminus; Optionally, the functional protein includes a fusion tag.

8. Biological material, comprising any one of (a) to (c): (a) a nucleic acid molecule encoding the (-)-α-bisabolol synthase mutant according to any one of claims 1 to 6, or encoding the fusion protein according to claim 7; (b) a recombinant vector containing the nucleic acid molecule described in (a); (c) A recombinant cell comprising the nucleic acid molecule described in (a) and / or the recombinant vector described in (b), wherein the original cell of the recombinant cell comprises bacteria or fungi.

9. The biomaterial according to claim 8, wherein The original cell of the recombinant cell (c) contains a mevalonate pathway gene, and / or a gene ERG20 encoding farnesyl pyrophosphate synthase; Preferably, the mevalonate pathway genes include: gene ERG10 encoding acetoacetyl-CoA thiolase, gene ERG13 encoding HMG-CoA synthase, gene tHMG1 encoding HMG-CoA reductase, gene ERG12 encoding mevalonate kinase, gene ERG8 encoding mevalonate-5-phosphate kinase, gene MVD1 encoding mevalonate pyrophosphate decarboxylase, and gene IDI1 encoding isoprene pyrophosphate isomerase.

10. Use of the (-)-α-bisabolol synthase mutant according to any one of claims 1 to 6, the fusion protein according to claim 7, or the biomaterial according to claim 8 or 9 in the preparation of (-)-α-bisabolol.

11. A method for preparing (-)-α-bisabolol, comprising culturing the (c) recombinant cells in the biological material according to claim 8, and isolating and obtaining (-)-α-bisabolol.

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