(-)-a-bisabolol synthase mutants and uses thereof

Abstract

The application relates to the technical field of enzyme engineering, in particular to a (-)-alpha-bisabolol synthase mutant and application thereof. On the basis of a wild-type (-)-alpha-bisabolol synthase (SEQ ID No: 1), three types of (-)-alpha-bisabolol synthase mutants are constructed through substitution, deletion and addition mutation modes, and the (-)-alpha-bisabolol synthase activity of homologous mutants with different consistency obtained through the above different mutation modes is investigated, and the yield ratio relative to the wild type when used for fermentative production of (-)-alpha-bisabolol is investigated. It is proved that the above mutants all achieve the same or higher (-)-alpha-bisabolol yield as the wild-type (-)-alpha-bisabolol synthase. It is proved that the above mutation modes and the obtained mutants and the recombinant cells for biosynthesizing the mutants can be used for batch production and popularization of (-)-alpha-bisabolol, thereby helping the development of many downstream industries such as beauty and medicine.

Description

(-)-α-bisabolol synthase mutant and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023114918130 filed on November 10, 2023 and PCT application No. PCT / CN2023 / 130915 filed on November 10, 2023, and this application cites the full text of the above-mentioned Chinese patent application and PCT application. Technical Field

[0003] The present disclosure relates to the field of enzyme engineering technology, and in particular to a (-)-α-bisabolol synthase mutant and applications thereof. Background Art

[0004] (-)-α-Bisabolol is a natural sesquiterpene compound primarily used as an ingredient in skincare and cosmetic products and also possesses potential pharmaceutical value. For example, its faint sweet floral fragrance makes it a popular fragrance. It also possesses soothing, antibacterial, antioxidant, anti-aging, and skin-whitening properties. Current production methods for (-)-α-bisabolol from natural plants such as chamomile are both costly and unsustainable. Chemical synthesis currently yields only racemic bisabolol mixtures, making it difficult to fully realize the pharmaceutical potential of (-)-α-bisabolol. Heterologous expression of (-)-α-bisabolol synthase in microbial substrates through synthetic biology approaches could pave the way for the sustainable production of (-)-α-bisabolol. However, the low activity of natural terpene synthases often hinders 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.

[0005] Summary of the Invention

[0006] The present disclosure aims to provide a (-)-α-bisabolol synthase mutant, and to utilize the mutant enzyme to increase the expression level of (-)-α-bisabolol with chirality selection in an expression system, thereby achieving the purpose of efficient and sustainable production of (-)-α-bisabolol.

[0007] In order to achieve the above objectives, the present disclosure provides the following technical solutions:

[0008] In a first aspect, the present disclosure provides a (-)-α-bisabolol synthase mutant selected from (a) or (b):

[0009] (a) a first protein having (-)-α-bisabolol synthase activity derived from the (-)-α-bisabolol synthase with the amino acid sequence shown in SEQ ID No: 1 by substitution, deletion or addition of at least one amino acid;

[0010] (b) a second protein having greater 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%.

[0011] In an optional embodiment, the substitution in (a) includes a third protein having (-)-α-bisabolol synthase activity derived from amino acid residues 90 to 550 of SEQ ID No: 1 by substitution of at least one amino acid residue.

[0012] Preferably, the (-)-α-bisabolol synthase mutant is a fourth protein having (-)-α-bisabolol synthase activity derived by replacing at least one amino acid residue at amino acid residues 90 to 270 and / or 320 to 550 of the (-)-α-bisabolol synthase shown in SEQ ID No: 1.

[0013] Preferably, the (-)-α-bisabolol synthase mutant is a fifth protein having (-)-α-bisabolol synthase activity derived by replacing at least one amino acid residue at positions 93 to 265, 321 to 366, 399 to 437, 465 to 473 and / or 522 to 544 of the (-)-α-bisabolol synthase whose amino acid sequence is shown in SEQ ID No: 1.

[0014] Optionally, the number of amino acid residue substitutions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0015] Furthermore, the substituted sites include at least one of positions 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 541 or 544 of SEQ ID No: 1.

[0016] Furthermore, the substitution includes at least one conservative substitution or at least one non-conservative substitution at position 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 541 or 544 of SEQ ID No: 1. The amino acid residue substitution includes at least one of E93Q, Q159K, D265K, I321V, L323I, F366L, L399Y, C425N, A437G, L465C, D469N, T473S, M522R, S541A or L544I. For example, any one of the following combinations may be used: 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. 4I; D265K and I321V; D265K and S541A; D265K, I321V, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, M522R, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, L465C, D469N, T473S, M522R, S541A, and L544I.

[0017] In some preferred embodiments, the (-)-α-bisabolol synthase mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-53, 91-115 having an identity of more than 83% with the odd-numbered sequences.

[0018] 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 with 1 to 29 amino acid residues deleted at the N-terminus, or 1 to 19 amino acid residues deleted at the C-terminus.

[0019] In a second aspect, the present disclosure provides a fusion protein comprising the (-)-α-bisabolol synthase mutant described in the first aspect and a functional protein linked to its N-terminus or C-terminus. Optionally, the functional protein comprises a fusion tag.

[0020] In some preferred embodiments, the fusion protein comprises an amino acid sequence selected from among SEQ ID NOs: 55-65 that has more than 83% identity with the odd-numbered sequences.

[0021] In a third aspect, the present disclosure provides a biomaterial comprising any one of (a) to (c):

[0022] (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;

[0023] (b) a recombinant vector containing the nucleic acid molecule described in (a);

[0024] (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.

[0025] In an alternative embodiment, the original cell of the recombinant cell (c) contains a mevalonate pathway gene, and / or the gene ERG20 encoding farnesyl pyrophosphate synthase.

[0026] 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.

[0027] 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.

[0028] In a fifth aspect, the present disclosure provides a method for preparing (-)-α-bisabolol, comprising culturing the (c) recombinant cells in the biomaterial described in the third aspect, and isolating and obtaining (-)-α-bisabolol.

[0029] The present invention discloses three types of (-)-α-bisabolol synthase mutants constructed based on the wild-type (-)-α-bisabolol synthase (SEQ ID No: 1) through substitution, deletion and addition mutations. (1) amino acid substitutions are made at different sites or different combinations of sites within a specific sequence fragment; (2) partial amino acid fragments are deleted at the N-terminus or C-terminus; (3) one or more amino acid residues are added at the N-terminus or C-terminus, or new fusion proteins are obtained by connecting to other functional proteins. The (-)-α-bisabolol synthase activity of homologous mutants with different identities from the mutants obtained by the above-mentioned different mutation methods is investigated, as well as the yield ratio relative to the wild-type when used for fermentation production of (-)-α-bisabolol. It is confirmed that the above-mentioned mutants all achieve (-)-α-bisabolol yields equivalent to or higher than that of the wild-type (-)-α-bisabolol synthase. It has been demonstrated that the above-mentioned mutation method and the resulting mutants, as well as the recombinant cells that biosynthesize the above-mentioned mutants, can be used for the mass production and promotion of (-)-α-bisabolol, thereby contributing to the development of many downstream industries such as beauty, cosmetics, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. 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.

[0031] Figure 1 shows the synthesis pathway of (-)-α-bisabolol in yeast cells;

[0032] FIG2 is a schematic diagram of the element map of the plasmid vector pZY900 used in Example 1. DETAILED DESCRIPTION

[0033] 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 combination with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments.

[0034] (I) Definitions or terms

[0035] The following abbreviations shall apply to the relevant definitions or terms involved in this disclosure. Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. The following terms are provided below.

[0036] As used herein, the terms "a," "an," "the," and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0037] As used herein, the conjunction term "and / or" between multiple described elements is understood to include both individual options and combined options. For example, when two elements are linked together 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 and therefore meets the requirements of the term "and / or" as used herein. The concurrent applicability of multiple options is also understood to be within the meaning of the term and therefore meets the requirements of the term "and / or."

[0038] As used herein, the term "(-)-α-bisabolol" is an organic compound derived from Matricaria chamomilla var. Recutita, with the molecular formula C 15 H 26 O, a non-toxic sesquiterpene alcohol found in natural essential oils, exhibits 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 primarily used in skin protection and skin care cosmetics. As an active ingredient, (-)-α-Bisabolol protects and cares for sensitive skin. It is suitable for use in sunscreens, after-sunbath washes, baby products, and after-shave care products. Furthermore, (-)-α-Bisabolol can be used in oral hygiene products such as toothpaste and mouthwash.

[0039] As used herein, the terms "first," "second," and the like may be used herein to describe different proteins only to distinguish between the 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 the first protein and the second protein are both proteins, but are not the same protein.

[0040] As used herein, the term "nucleic acid molecule," also 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), typically 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 material 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 encoding the provided (-)-α-bisabolol synthase mutants.

[0041] 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 nucleotide sequence or the amino acid sequence contained therein. 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 an amino acid residue is replaced with an amino acid residue having a similar side chain. 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, a predicted nonessential amino acid residue in (-)-α-bisabolol synthase is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that possess (-)-α-bisabolol synthesis activity are well known in the art. As described herein, substitutions are represented by the single-letter abbreviation of the amino acid before substitution - the position of substitution - the single-letter abbreviation of the amino acid after substitution. For example, "S541A" indicates that serine at position 541 is replaced by alanine. Amino acids that can be conservatively substituted are shown in Table 1 below:

[0042] Table 1. Examples of conservative amino acid substitutions

[0043] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0044] 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.

[0045] 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, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention 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 the 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 the genes carried on the expression vector. The expression vectors disclosed herein may also contain the following polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0046] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region 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 origins of replication, 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, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the 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, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0047] As used herein, the term "homology" has a generally accepted meaning in the art and is a central concept in comparative biology. The fundamental meaning of homology is that the two samples being compared (e.g., an amino acid sequence or a nucleotide sequence) share a common ancestor. Generally speaking, if two traits (states) in two species meet either of the following two conditions, these two traits are considered a pair of homologous traits: 1. They are identical to a trait found in the ancestral group of these species; 2. They are different traits with an ancestor-descendant relationship. Typically, identity and similarity are used as metrics to measure the degree of homology between two sequences. Identity refers to whether the residues at the same position in the two sequences are identical, that is, the percentage of the total length of the aligned sequence with identical residues at corresponding positions. Similarity refers to the percentage of identical and similar residues at corresponding positions. This metric includes both identical and similar residues and, therefore, may be more tolerant than identity. In practical applications, those skilled in the art can choose which metric to use based on their specific needs. Generally speaking, if you need to measure the similarity between two sequences more strictly, you can choose to use the consistency index; if you need to measure the similarity between two sequences more loosely, you can choose to use the similarity index.

[0048] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. For example, one protein domain can be located at the amino-terminal (N-terminal) portion or the carboxyl-terminal (C-terminal) portion of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxyl-terminal fusion protein," respectively. In alternative embodiments, a fusion protein is a single-chain polypeptide that can be completely encoded by a nucleic acid sequence and comprises at least two protein domains covalently linked by a peptide linkage or optionally covalently linked by a peptide linker.

[0049] As used herein, the term "functional protein" refers to a naturally occurring protein, a functional variant thereof, or an engineered derivative thereof.

[0050] (II) Detailed technical plan

[0051] In a first aspect, the present disclosure provides a (-)-α-bisabolol synthase mutant selected from (a) or (b):

[0052] (a) a first protein having (-)-α-bisabolol synthase activity derived from the (-)-α-bisabolol synthase with the amino acid sequence shown in SEQ ID No: 1 by substitution, deletion or addition of at least one amino acid;

[0053] (b) a second protein having greater 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%.

[0054] In an optional embodiment, the substitution in (a) includes a third protein having (-)-α-bisabolol synthase activity derived from amino acid residues 90 to 550 of SEQ ID No: 1 by substitution of at least one amino acid residue.

[0055] Preferably, the (-)-α-bisabolol synthase mutant is a fourth protein having (-)-α-bisabolol synthase activity derived by replacing at least one amino acid residue at amino acid residues 90 to 270 and / or 320 to 550 of the (-)-α-bisabolol synthase shown in SEQ ID No: 1.

[0056] Preferably, the (-)-α-bisabolol synthase mutant is a fifth protein having (-)-α-bisabolol synthase activity derived by replacing at least one amino acid residue at positions 93 to 265, 321 to 366, 399 to 437, 465 to 473 and / or 522 to 544 of the (-)-α-bisabolol synthase whose amino acid sequence is shown in SEQ ID No: 1.

[0057] For example, the number of amino acid residue substitutions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0058] Furthermore, the substituted sites include at least one of positions 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 541 or 544 of SEQ ID No: 1.

[0059] Furthermore, the substitution includes at least one conservative substitution or at least one non-conservative substitution at position 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 541 or 544 of SEQ ID No: 1. The amino acid residue substitution includes at least one of E93Q, Q159K, D265K, I321V, L323I, F366L, L399Y, C425N, A437G, L465C, D469N, T473S, M522R, S541A or L544I. For example, it may 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; D265K and I321V; D265K and S541A; D265K, I321V, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, M522R, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, L465C, D469N, T473S, M522R, S541A, and L544I, as shown in Table 2:

[0060] Table 2. Location and type of mutants

[0061] In an optional embodiment, the present disclosure provides new mutants having different identities with the amino acid sequences of the above mutants, wherein the adjustment of the identity is achieved by substitution of amino acid residues. As an example, the amino acid sequences of the above mutants 1, 3, 5, 8, 11, 13, 16 and 18 after adjustment of the identity ratio are shown in SEQ ID NOs: 39 to 53 (odd numbers), and the nucleotide sequences are shown in SEQ ID NOs: 40 to 54 (even numbers).

[0062] 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 with 1 to 29 amino acid residues deleted at the N-terminus, or 1 to 19 amino acid residues deleted at the C-terminus.

[0063] In a second aspect, the present disclosure provides a fusion protein comprising the (-)-α-bisabolol synthase mutant described in the first aspect and a functional protein linked to its N-terminus or C-terminus. Optionally, the functional protein comprises a fusion tag.

[0064] 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 modifier protein), a Halo tag (a genetically modified derivative of a dehalogenase), or a SNAP tag (derived from an O6-methylguanine-DNA methyltransferase reaction). The reporter tag protein includes a c-Myc tag, an HA tag, or a luciferase or fluorescent tag protein.

[0065] In some preferred embodiments, the fusion protein comprises an amino acid sequence selected from among SEQ ID NOs: 55-65 that has more than 83% identity with the odd-numbered sequences.

[0066] In a third aspect, the present disclosure provides a biomaterial comprising any one of (a) to (c):

[0067] (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;

[0068] (b) a recombinant vector containing the nucleic acid molecule described in (a);

[0069] (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.

[0070] In an alternative embodiment, the original cell of the recombinant cell (c) contains a mevalonate pathway gene, and / or the gene ERG20 encoding farnesyl pyrophosphate synthase.

[0071] 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.

[0072] 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.

[0073] In a fifth aspect, the present disclosure provides a method for preparing (-)-α-bisabolol, comprising culturing the (c) recombinant cells in the biomaterial described in the third aspect, and isolating and obtaining (-)-α-bisabolol.

[0074] 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 principles of the present disclosure should be equivalent replacement methods and are included in the scope of protection of the present disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0075] Example 1. Construction of an engineered yeast strain expressing (-)-α-bisabolol synthase

[0076] With reference to the synthesis pathway of (-)-α-bisabolol in yeast cells (as shown in FIG1 ), plasmid pZY900 (see patent 202210473488.4) was used to construct a yeast transformation fragment, which is characterized by: △LEU2:LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20, with promoters GAL1 and GAL10 controlling the expression of gene ERG20 and the insertion gene, respectively, the screening marker is Leu2, and the inserted chromosomal site is Leu2, as shown in FIG2 .

[0077] 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 enzyme 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 BuildingBlock." Journal of the American Chemical Society vol. 142, 6(2020): 2760-2765. doi: 10.1021 / jacs.9b12940). 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 of fermentation medium (peptone (20 g / L), yeast extract (10 g / L), glucose (10 g / L), and galactose (10 g / L)). After transfer, the mixture was overlaid with 5 mL of organic phase (isopropyl myristate) and fermented at 30°C and 200 rpm for 72 h. The yield of (-)-α-bisabolol in the fermentation broth was determined using gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis was performed using a Thermo Fisher Scientific TRACE GC ULTRA gas chromatograph equipped with an AS 3000 autosampler and a split / splitless injector, and a TSQ QUANTUM XLS mass spectrometer equipped with a triple quadrupole detector. The GC-MS detection parameters were as follows: a TR-5MS column (30 m × 0.25 mm × 0.25 μm), high-purity helium carrier gas at a flow rate of 1 mL / min, acetone as a needle wash, an injection volume of 1 μL, and a split ratio of 50. The injection port temperature was 250°C, and the ion transfer tube temperature was 270°C. The detection procedure was as follows: the initial column temperature was 70°C, which was increased at 10°C / min to 180°C; the temperature was then increased at 20°C / min to 300°C and held for 2 minutes.

[0078] Example 2. Construction and fermentation of substitution mutants

[0079] 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, 92-112 (even numbers)) encoding (-)-α-bisabolol synthase mutants (SEQ ID NO: 3-37, 91-111 (odd numbers)). The obtained positive strains were named JXL02-JXL30. Strain JXL01-30 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. It was then 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, it was covered with 5 mL of organic phase (isopropyl myristate) and fermented at 30°C and 200 rpm for 72 h. The same GC-MS assay method as in Example 1 was used to determine the yield of (-)-α-bisabolol in the fermentation broth, and the increase in (-)-α-bisabolol yield (increase = (experimental group - control) / control) and the yield ratio (experimental group / control group) of yeast strains expressing different mutants compared to yeast strains expressing the wild-type enzyme were calculated. The results are shown in Table 3.

[0080] Table 3. Activity parameters of (-)-α-bisabolol synthase mutants and related strains

[0081] 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.

[0082] Example 3. Comparison of yields of different homologous nucleotide sequences

[0083] Based on the amino acid sequence design of different (-)-α-bisabolol synthase mutants, different homologous amino acid sequences were designed using consistency as an indicator (Table 4). According to the operating procedures described in Example 1, only the synthetic gene (SEQ ID NO: 2) encoding (-)-α-bisabolol synthase (SEQ ID NO: 1) was replaced with genes (SEQ ID NO: 40-54, 114-116 (even numbers)) encoding homologous amino acid sequences with different consistency (Table 4, SEQ ID NO: 39-53, 113-115 (odd numbers)). Primers (pXL01-1-F, pXL01-1-R) were used for amplification. After obtaining positive strains, fermentation was carried out, and the difference in (-)-α-bisabolol production before and after the design was compared. For the amino acid sequence design of the wild-type synthase, different homologous amino acid sequences (SEQ ID NO: 113, SEQ ID NO: 115) were designed based on consistency. Amplification was performed using primers (pXL113-1-F, pXL113-1-R) and (pXL115-1-F, pXL115-1-R), respectively. After obtaining positive strains, fermentation was performed, and the difference in (-)-α-bisabolol production before and after the design was compared.

[0084] Table 4. Comparison of activities of homologous amino acid sequences with different identities

[0085] As can be seen from Table 4, based on the substitution mutants provided by the present disclosure, when the consistency adjustment is performed and the consistency ratio reaches 83% or more, the (-)-α-bisabolol yield obtained is significantly improved compared with the wild-type (-)-α-bisabolol synthase.

[0086] Example 4. Construction and fermentation of deletion mutants

[0087] The procedure was 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 the gene for the corresponding deletion mutant of (-)-α-bisabolol synthase (Tables 6 and 7), and the corresponding primers (Table 5) were used. Positive strains expressing mutants with different amino acid truncation lengths were generated and fermented. The difference in (-)-α-bisabolol yield before and after truncation was compared. When the N-terminal truncation was within 30 amino acids, there was no significant change in yield before and after truncation, but there was a significant decrease in yield after truncation, as shown in Table 6. When the C-terminal truncation was within 20 amino acids, there was no significant change in yield before and after truncation, but there was a significant decrease in yield after truncation, as shown in Table 7.

[0088] Table 5. Primer list

[0089] Table 6. Comparison of the activities of mutants with different N-terminal truncation lengths

[0090] Table 7. Comparison of activities of mutants with different C-terminal truncation lengths

[0091] As can be seen from Tables 6 and 7, there are significant differences in the lengths that can be truncated between the N-terminus and C-terminus of the wild-type (-)-α-bisabolol synthase during truncation mutation modification. When the N-terminus is truncated to 29 amino acid residues, the activity of the (-)-α-bisabolol synthase and the resulting (-)-α-bisabolol yield are not significantly affected. However, when the N-terminus is truncated to 30 amino acid residues, the (-)-α-bisabolol yield is significantly reduced. Furthermore, when the C-terminus is truncated to 20 amino acid residues, the (-)-α-bisabolol yield is significantly reduced.

[0092] Example 5. Construction and fermentation of fusion protein (added mutant)

[0093] 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 a gene encoding a mutant fusion protein (SEQ ID NO: 55 or 59) with an N-terminal MBP tag (SEQ ID NO: 56 or 60), a mutant fusion protein (SEQ ID NO: 57 or 61) with a C-terminal SUMO tag (SEQ ID NO: 58 or 62), a mutant fusion protein (SEQ ID NO: 63) with an N-terminal FLAG tag (SEQ ID NO: 64), or a mutant fusion protein (SEQ ID NO: 65) with a C-terminal FLAG tag (SEQ ID NO: 66). Corresponding primers (Table 8) were used to obtain positive strains and fermentation was performed. The difference in (-)-α-bisabolol production before and after the addition of the additional protein sequences was compared. 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.

[0094] Table 8. Comparison of the activities of different fusion mutant fusion proteins

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 90 to 550 of the (-)-α-bisabolol synthase shown in the amino acid sequence of SEQ ID No: 1 by substitution of at least one amino acid residue; Preferably, the (-)-α-bisabolol synthase mutant is a fourth protein having (-)-α-bisabolol synthase activity derived by substitution of at least one amino acid residue at positions 90 to 270 and / or 320 to 550 of the (-)-α-bisabolol synthase as shown in SEQ ID No: 1; Preferably, the (-)-α-bisabolol synthase mutant is a fifth protein having (-)-α-bisabolol synthase activity derived from the amino acid sequence of SEQ ID No: 1, wherein at least one amino acid residue is substituted at positions 93 to 265, 321 to 366, 399 to 437, 465 to 473 and / or 522 to 544 of the (-)-α-bisabolol synthase; Preferably, the number of amino acid residue substitutions is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

3. The (-)-α-bisabolol synthase mutant according to claim 2, wherein The amino acid residue substitution sites include at least one of positions 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 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 or at least one non-conservative substitution at position 93, 159, 265, 321, 323, 366, 399, 425, 437, 465, 469, 473, 522, 541 or 544 of the (-)-α-bisabolol synthase as shown in the amino acid sequence of 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 comprises at least one of E93Q, Q159K, D265K, I321V, L323I, F366L, L399Y, C425N, A437G, L465C, D469N, T473S, M522R, 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, S541 A and L544I; D265K and I321V; D265K and S541A; D265K, I321V, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, M522R, and S541A; E93Q, Q159K, D265K, I321V, F366L, A437G, L465C, D469N, T473S, M522R, S541A, and L544I; Preferably, the (-)-α-bisabolol synthase mutant comprises an amino acid sequence selected from the group consisting of the odd-numbered sequences in SEQ ID NOs: 3-53, 91-115 having a greater than 83% identity.

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; Optionally, the fusion tag includes a purification tag protein and / or a reporter tag protein, wherein the purification tag protein is selected from a His tag, a GST tag, an MBP tag, a FLAG tag, an Avi tag, a SUMO tag, a Halo tag and / or a SNAP tag; the reporter tag protein includes a c-Myc tag, an HA tag, a luciferase and / or a fluorescent tag protein; Preferably, the fusion protein comprises an amino acid sequence selected from the group consisting of amino acids having more than 83% identity with the odd-numbered sequences in SEQ ID NOs: 55-65.

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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