Valencene oxidase mutants and their use in preparing nootkatone

Abstract

This invention discloses a Valenciane oxidase mutant and its application in the preparation of naringin. The invention constructs a gene library of the HPO mutant using site-directed mutagenesis. In vitro catalytic experiments using resting cells show that, under the same conditions, the optimal HPO mutant (HPO_G302G-V480A-V482A-A484A, whose amino acid sequence is shown in SEQ ID No. 4 and nucleotide sequence in SEQ ID No. 5) exhibits a catalytic efficiency 2.54 times that of the wild type, thereby increasing substrate utilization efficiency and improving the yield of the target compound, naringin. Furthermore, the bio-preparation method for naringin described in this invention is simple to operate, environmentally friendly, and uses mild reaction conditions, showing great application potential. In addition, this invention is the first report of successfully constructing a beneficial HPO mutant for high-yield naringin production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the application of a Valencia ene oxidase (HPO) mutant with an amino acid sequence as shown in SEQ ID No. 3 in the sequence listing and its recombinant expression cells in the biological preparation of terpineone. It also relates to the nucleic acid sequence encoding the enzyme, the recombinant expression vector containing the encoded nucleic acid sequence, and the method for preparing terpineone. Background Technology

[0002] Nootkatone, also known as naringin, was first isolated from the heartwood of Alaskan yellow cypress (Callitropsis nootkatensis) and is also commonly found in fruits such as grapefruit and sweet orange. Due to its strong and persistent citrus aroma with a woody note, it has become a high-value and high-demand aromatic compound in the food and flavor industries. In addition, naringin is an effective and environmentally friendly insect repellent, effective against mosquitoes, ticks, and other insects. Recent studies have discovered that naringin and its derivatives also possess physiological activities such as inhibiting cancer cell proliferation, anti-platelet aggregation, and activating energy metabolism, thus making them potential drug progenitor molecules.

[0003] However, the cost and yield of extracting this compound from natural sources are high. Therefore, in industrial production, the oxidation of inexpensive and readily available valencene is commonly used to prepare naringone. However, chemical oxidation methods inevitably rely on unsafe reagents, such as tert-butyl peracetate, chromium trioxide, tert-butyl chromate, or other heavy metal salts, and cause serious environmental damage (Hunter GLK, et al. Conversion of valencene tonootkatone[J]. Journal of Food Science, 1965, 30(5): 876-878.). In recent years, thanks to the rapid development of synthetic biology, the biocatalysis of valencene to naringone has become a new direction of exploration and research trend.

[0004] In 2007, Shunji Takahashi et al. isolated an enzyme (NCBI accession number EF569601.1) from cell suspension cultures of Hyoscyamus muticus that can catalyze the hydroxylation of the allyl C-2 position of Valenciaene. The full English name of this enzyme is Hyoscyamus muticus premnaspirodiene oxygenase, abbreviated as HPO. This was the first time that HPO was discovered and its function was characterized in the literature. Subsequent studies have confirmed that HPO does indeed catalyze the hydroxylation of naringin to produce naringin. In 2020, Xiangfeng Meng et al. found that in the Saccharomyces cerevisiae strain W303-1A, naringin synthase (CnVS, NCBI accession number JX040471), HPO, AtCPR (the redox chaperone of HPO, NCBI accession number NM_118585), and alcohol dehydrogenase ZSD1 from Zingiber zerumbet (NCBI accession number AB480831) were co-expressed, which could achieve de novo synthesis of naringin, but the yield was only 59.78 mg / L. At the same time, a large amount of naringin remained in the fermentation broth (Meng X, et al. Metabolic engineering Saccharomyces cerevisiae for de novo production of the sesquiterpenoid(+)-nootkatone[J]. Microbial Cell Factories, 2020, 19.). It is evident that due to insufficient expression levels and catalytic activity of HPO, the key rate-limiting enzyme in this process, especially its catalytic activity being inhibited at higher valenciaene concentrations, naringin still faces a severe production bottleneck.

[0005] Protein engineering, as a known powerful means to improve enzyme catalytic performance, has been successfully applied to the study of improving the catalytic performance of lipases, cellulosic enzymes, etc., and has also been used by researchers to explore the design and modification of HPO. In 2019, Ouyang Xiaodan et al. constructed a mutant of HPO (V482I-A484I) using overlap extension PCR technology, and co-expressed the mutant gene and AtCPR in Saccharomyces cerevisiae CEN.PK2-1Ca strain. Through resting cell experiments, it was found that the HPO mutant (V482I-A484I) did not increase the yield of terpineol, that is, the ability of the HPO mutant to catalyze the hydroxylation of Valenciaene was not improved (Ouyang Xiaodan. Construction and optimization of the synthetic pathway of Valenciaene and its derivatives in Saccharomyces cerevisiae [D]. South China University of Technology, 2019.). In the same year, Liu Hui et al. continued to explore the possibility of improving the catalytic ability of HPO through site-directed mutagenesis. The researchers designed 12 HPO mutants and transferred them into a yeast strain that had previously been constructed in the laboratory to produce high-yield Valenciaene. Gas chromatography detection showed that no naringin or naringone was produced, meaning that none of the 12 HPO mutants had catalytic activity (Liu Hui. Design and construction of metabolic pathway for the synthesis of plant sesquiterpenoid naringone by Saccharomyces cerevisiae [D]. Shandong University, 2019.).

[0006] In summary, although experimental studies and literature reports indicate that the catalytic performance of HPO is a crucial factor limiting the increase of naringin production, HPO itself, as a plant-derived P450 enzyme, has a complex catalytic mechanism, is difficult to isolate and purify (plant membrane protein), lacks protein crystal structure information, and lacks high-throughput screening methods adapted to directed evolution of HPO. Despite numerous researchers' efforts to improve its catalytic performance through bioengineering since its discovery in 2007, no literature reports to date have successfully obtained HPO mutants capable of high naringin production. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a Valencia ene oxidase mutant.

[0008] Another object of the present invention is to provide the application of the above-mentioned Valencia ene oxidase mutant in the preparation of naringin.

[0009] Valencia ene oxidase HPO was first discovered in 2007 from henbane (Hyoscyamus muticus) and its ability to biosynthesize naringin was confirmed. However, the catalytic performance of the wild-type enzyme (its amino acid sequence is shown in SEQ ID No. 1, and its nucleotide sequence is shown in SEQ ID No. 2) is insufficient when heterologously expressed in Saccharomyces cerevisiae, thus limiting the increase in naringin production.

[0010] The technical problem this invention aims to solve is that while Valenciaene oxidase (HPO) is a key rate-limiting enzyme in the biosynthesis of naringin, its catalytic performance cannot meet the demands of industrial production. This invention provides a series of highly efficient HPO mutants. These mutants exhibit advantages such as strong specific conversion ability and high conversion rate in the biological preparation of naringin using Saccharomyces cerevisiae as a cell factory.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] In this invention, the Valenciane oxidase HPO is derived from Hyoscyamus muticus, whose full English name is Hyoscyamus muticus premnaspirodiene oxygenase. The NCBI accession number of the protein is ABS00393.1, and its amino acid sequence is shown in SEQ ID No.1, containing a total of 502 amino acids. The NCBI accession number of the HPO gene is EF569601.1, and its length is 1509 bp.

[0013] In this invention, the nucleotide sequence of the Valencia ene oxidase HPO (wild type) is shown in SEQ ID No. 2, containing a total of 1509 nucleotides. It was obtained by optimizing the yeast codons based on the sequence shown in NCBI accession number EF569601.1. This is a necessary step for heterologous expression of the HPO gene in Saccharomyces cerevisiae.

[0014] In this invention, an HPO mutant sequence is further provided that has higher transformation efficiency and substrate tolerance compared to the Valencia ene oxidase HPO (wild type) shown in SEQ ID No. 2 in the sequence listing.

[0015] Preferably, the Valencia ene oxidase HPO mutant includes single-site mutants and multi-site mutants, and its amino acid sequence is shown in SEQ ID No. 3. In SEQ ID No. 3, at least one site marked with "X" has a mutated amino acid residue compared to the corresponding site in SEQ ID No. 1. The amino acids at positions 302 and 484 of SEQ ID No. 3 are synonymous mutations, i.e., the amino acids are different and the codons are changed. Specifically, the codon at position 302 of SEQ ID No. 3 is mutated from GGC(G) in SEQ ID No. 2 to GGG(G), and the codon at position 484 of SEQ ID No. 3 is mutated from GCT(A) in SEQ ID No. 2 to GCG(A).

[0016] Preferably, the amino acid sequence of the Valencia ene oxidase HPO mutant (including single-site mutants and multi-site mutants) has at least 55%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1.

[0017] In this invention, a recombinant expression vector containing the above-mentioned Valencia oxidase HPO wild-type or HPO mutant encoding gene is provided.

[0018] This invention provides a recombinant expression cell containing the recombinant expression vector of the Valencia ene oxidase HPO gene described in this invention, and provides a method for preparing terpineone using the recombinant expression cell.

[0019] The conversion efficiency described in this invention is expressed as the proportion of the precursor material Valenciaene converted into the intermediate product terpineol within a certain time period.

[0020] According to the present invention, experiments have confirmed that the Valenciaene oxidase HPO mutant, as shown in SEQ ID No. 3, exhibits superior efficiency in catalyzing the conversion of Valenciaene to naringol compared to the wild-type HPO, which has been reported and widely used in the biosynthesis of naringol. In particular, this difference is further demonstrated in in vitro experiments using recombinant expression cells catalyzing the conversion of Valenciaene to naringol under near-neutral pH conditions. Experimental results show that, under the same conditions, the substrate target conversion rate obtained using the HPO mutant described in this invention is higher, and its conversion efficiency for Valenciaene can be at least 1.1 times, preferably 2.0 to 2.5 times, that of the wild-type HPO shown in SEQ ID No. 2.

[0021] As used herein, the term "mutation" means that at least one nucleotide or amino acid in the HPO mutant gene or amino acid sequence differs from the compared HPO wild-type sequence, and that the mutation of the enzyme can be achieved by site-directed mutagenesis using methods conventional in the art.

[0022] Compared with the wild-type Valenciaene oxidase HPO shown in SEQ ID No. 2, the modified HPO mutant provided by the present invention has better tolerance to the substrate Valenciaene, that is, the enzyme can still maintain good activity in the presence of high concentrations of substrate.

[0023] It has been specifically found that mutations at the following positions in SEQ ID No. 1 are beneficial for obtaining HPO mutants with high transformation efficiency: 302, 365, 480, 482, and 484. In a preferred embodiment, compared with SEQ ID No. 1, good results have been achieved with HPO mutants selected from one or more of the following sites combined with mutations: 302G (synonymous mutation), 365A, 480A, 480T, 480G, 482A, 482C, and 484A (synonymous mutation). It should be noted that, in principle, there is no limitation on the number of mutant amino acid residues in HPO, provided that the enzyme retains sufficient catalytic performance as a Valenciaene oxidase.

[0024] Preferably, the Valencia ene oxidase HPO mutant is HPO_G302G-V480A-V482A-A484A, whose amino acid sequence is shown in SEQ ID No. 4 and whose nucleotide sequence is shown in SEQ ID No. 5.

[0025] As used herein, the term "nucleic acid molecule" has the meaning commonly understood by those skilled in the art. A nucleic acid molecule may include polynucleotides such as those shown in NCBI accession number EF569601.1 or SEQ ID No. 2, or may include polynucleotides that also include additional coding and / or non-coding sequences. In specific embodiments of the present invention, the nucleic acid molecule may be a DNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the DNA encoding the amino acid sequence shown in SEQ ID No. 1.

[0026] As used herein, the terms "vector" and "expression vector" have the meanings commonly understood by those skilled in the art. A "vector" refers to a nucleic acid medium into which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, it is called an expression vector. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector preferably contains one or more selectivity markers that allow for convenient selection of cells such as transformed, transfected, or transduced cells. In expressing a foreign gene using *Saccharomyces cerevisiae*, the foreign gene to be expressed is first ligated to a vector. The vector can be any conventional vector in the art, such as commercially available plasmids, bacteriophages, or viral vectors. This invention preferably uses plasmids YEp352 and p414 to ligate the *Valencia ene oxidase* HPO gene with a suitable regulatory sequence to achieve constitutive expression of HPO.

[0027] This invention also provides recombinant expression cells containing the vector of this invention. These recombinant expression cells can be prepared by transforming the recombinant expression vector of this invention into host cells. The host cells can be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated and passaged, and that the gene carried by it can be effectively expressed. The host cells can be from any organism, specifically selected from prokaryotic cells, eukaryotic cells, archaea, protists, plant cells (including algae), and cells derived from animals; further, they can be *Saccharomyces cerevisiae*, *Pichia pastoris*, *Yarrowia solanacearum*, or *Escherichia coli*, etc. This invention preferably uses *Saccharomyces cerevisiae*, more preferably *Saccharomyces cerevisiae* strain CEN.PK2-1Ca.

[0028] This invention provides a method for determining the catalytic performance of HPO mutants. Specifically, it employs an in vitro catalytic experiment using resting cells, by adding the precursor valenciaene exogenously and transforming it in vitro using the recombinant expression cells described in this invention to obtain garnetol. The valenciaene can be obtained through chemical synthesis, plant extraction, or biosynthesis. Specific reaction conditions, such as substrate concentration, pH, buffer composition, and amount of recombinant expression transformant, can be selected according to conventional conditions for such reactions in the art. The biotransformation reaction can be carried out under shaking or stirring conditions. The biotransformation reaction time is preferably 24 hours. After the reaction, the content of garnetol in the reaction mixture can be determined using conventional extraction separation and gas chromatography detection methods in the art, thereby allowing for a comparison of the catalytic performance of HPO mutants and wild-type HPO. In a specific embodiment, it can be observed that the recombinant expression cells exhibit superior transformation efficiency for exogenously added valenciaene.

[0029] This invention also provides a method for the in situ biosynthesis of naringin using a *Saccharomyces cerevisiae* strain containing the HPO mutant gene expression vector described in this invention. Specifically, naringin is prepared by culturing recombinant *Saccharomyces cerevisiae* cells expressing the relevant enzymes in a reactor containing culture medium. The method utilizes the *Saccharomyces cerevisiae*'s own mevalonate pathway (MVA pathway), which first generates mevalonate (MVA) from acetyl-CoA. Then, in the next two steps, MVA is phosphorylated by mevalonate kinase (MK) and mevalonate 5-phosphate kinase (MPK) to form 5-mevalonate pyrophosphate (MVAPP). Finally, MVAPP is decarboxylated and dehydrated by ATP-dependent 5-mevalonate pyrophosphate decarboxylase (MVD) to form isopentenyl pyrophosphate (IPP). IPP is isomerized by IPP isomerase (IDI) to generate dimethylallyl pyrophosphate (DMAPP). Saccharomyces cerevisiae further utilizes endogenous isoprene synthase (IspA) to continuously add IPP / DMAPP to the active isoprene diphosphate (IPP) unit to form farnesyl pyrophosphate (FPP), and heterologously expresses the Valenciaene synthase CnVS from Alaskan cypress (Callitropsis nootkatensis) described in international patent application number PCT / NL2010 / 050848 to convert FPP into Valenciaene. Subsequently, using the HPO mutant provided by this invention and the AtCPR derived from Arabidopsis thaliana described in Urban, Pd et al.'s 1997 article (Urban, P., et al. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450CYP73A5. J. Biol. Chem. 1997, 272, 19176–19186.), Valenciaene was further catalytically converted into the intermediate product naringol. Naringol can be further dehydrogenated to the target product naringone under the catalysis of the highly efficient alcohol dehydrogenase ADH. In a preferred embodiment, this in-situ synthesis method of naringone was used for fed-batch fermentation in shake flasks, resulting in a high yield of naringone and its proportion in the total terpenes of the final fermentation product.

[0030] The bioreactor used is, in principle, selected from equipment capable of ensuring cell proliferation and biochemical reactions by living cells and enzymes, such as small shake flasks and fermenters. The culture medium used is primarily suitable for yeast cell growth, commonly used in the field; YPD medium and SD auxotrophic medium are preferred in this invention. The fermentation temperature is preferably selected to maintain good catalytic performance of HPO. In one specific embodiment, a fermentation temperature of 25°C is preferred to reduce the volatilization of Valenciaene and maintain the catalytic stability of HPO.

[0031] If necessary, the naringin or its precursor valenciaene, intermediate naringol, etc., produced in the method of this invention can be extracted and separated by extraction. Specifically, the liquid organic solvent n-dodecane is suitable as the extractant. In a preferred embodiment, n-dodecane is used to coat the fermentation medium. While the recombinant Saccharomyces cerevisiae cells generate the product naringin, n-dodecane can serve as an extraction phase with a higher affinity for the product, thereby extracting naringin into n-dodecane during fermentation. Furthermore, thanks to the extractive effect of n-dodecane, the potential toxic effects of naringin, its substrate valenciaene, and intermediate naringol on Saccharomyces cerevisiae cells can be effectively reduced. Simultaneously, since naringin is a hydrophobic sesquiterpene compound, coating it with n-dodecane can effectively reduce its volatilization loss. Experiments have shown that this method facilitates in-situ product recovery without the need for cell lysis to recover naringin, greatly simplifying the product separation and purification process.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] This invention constructed a gene library of the HPO mutant using site-directed mutagenesis and modified the Valencia ene oxidase HPO using protein engineering principles to optimize its catalytic efficiency. In vitro catalytic experiments using resting cells showed that, under the same conditions, the optimal HPO mutant (HPO_G302G-V480A-V482A-A484A, with its amino acid sequence shown in SEQ ID No. 4 and its nucleotide sequence shown in SEQ ID No. 5) exhibited a catalytic efficiency 2.54 times that of the wild type, thereby increasing substrate utilization efficiency and improving the yield of the target compound, naringin. By heterologously expressing the HPO mutant and related enzymes in the high-FPP-producing *Saccharomyces cerevisiae* strain ScPK2-04, the optimal HPO mutant was verified to also exhibit excellent performance in the in situ synthesis of naringin. After fermentation at 25°C and 200 rpm in a fed-batch fermentation system for 130 h, the total terpene yield was 399.40 mg / L, of which naringin yield was 322.62 mg / L, representing the highest shake-flask yield of naringin synthesized in situ using yeast as a host reported in the literature. Furthermore, the bio-preparation method for naringin described in this invention has advantages such as simple operation, environmental friendliness, and mild reaction conditions, showing great application potential. In addition, this invention is the first report of successfully constructing a beneficial HPO mutant for high-yield naringin production. Attached Figure Description

[0034] Figure 1 This is a map of the recombinant plasmid containing the HPO gene (wild type) from Example 1.

[0035] Figure 2 This is a schematic diagram of the in vitro catalysis of the formation of garnetol from garnet by garnet oxidase HPO.

[0036] Figure 3 This is a schematic diagram of the in-situ synthesis of naringin in brewer's yeast.

[0037] Figure 4 This is a schematic diagram of the shake-flask fermentation results in Example 5.

[0038] Figure 5 This is a schematic diagram of the shake-flask fermentation results in Example 6. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used in the following examples are commercially available. All experiments in the following examples include controls and are repeated three times.

[0041] The S. cerevisiae CEN.PK2-1Ca and ScPK2-M used in the examples are both disclosed in "CN201910271558-Saccharomyces cerevisiae engineered strain for producing Valenciaene and its construction method and application".

[0042] Example 1: Construction of a recombinant expression vector for Valencia ene oxidase HPO (wild type)

[0043] Based on the Valencia ene oxidase HPO gene (NCBI accession number EF569601.1), it was synthesized by Shanghai Sangon Biotech Co., Ltd. after codon optimization. The codon-optimized nucleotide sequence is shown in SEQ ID No. 2, and it is defined as Valencia ene oxidase wild-type (HPO wild-type, HPO(wt)), constructed in YEp352-CDC19. p -TPI1 t The vector, YEp352-CDC19, was obtained. p -HPO(wt)-TPI1 t Plasmid; among which, expression cassette CDC19 p -HPO(wt)-TPI1 t Inserted between the SmaI and XbaI restriction sites in the YEp352 plasmid.

[0044] The upstream primer promoter-F (5'-TTTACAGTCGACAATGCTAGTATTTTGGAGAT-3') and the downstream primer terminator-R (5'-TTTACA) were used. CTGCAG CTATATAACAGTTGAAATT-3'), with the aforementioned YEp352-CDC19 p -HPO(wt)-TPI1 t After amplifying the target gene fragment using the plasmid as a template, the fragment was double-digested with SalI and PstI (purchased from Thermo Fisher Scientific), and then purified using a purification kit (purchased from Nanjing Novizan Biotech).

[0045] The CDS sequence of the AtCPR gene (NCBI accession number NM_118585) was synthesized by Shanghai Sangon Biotech Co., Ltd. after codon optimization. The codon-optimized nucleotide sequence is shown in SEQ ID No. 6 and was constructed in YEp352-CYC1. p -CYC1 t The vector, YEp352-CYC1, was obtained. p -AtCPR-CYC1 t Plasmid; among which, expression cassette CYC1 p -AtCPR-CYC1t The gene fragment was inserted between the PstI and HindIII restriction sites of the YEp352 plasmid. The plasmid was double-digested with SalI and PstI, and then ligated with T4 ligase (purchased from New England BioLabs) to ligate the double-digested vector and the previously double-digested and purified gene fragment. 10 μL of the ligation product was added to 20 μL of E. coli DH5α competent cells, incubated on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, followed by immediate incubation on ice for 2 minutes. 1 mL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm for 1 hour. 300 μL of the bacterial culture was plated on LB agar plates containing 100 μg / mL ampicillin and incubated at 37°C for 12 to 16 hours to obtain recombinant E. coli DH5α (YEp352-CDC19). p -HPO(wt)-TPI1 t -CYC1 p -AtCPR-CYC1 t For the plasmid construction process and diagrams, see [link to diagram]. Figure 1 A single clone of the recombinant bacteria was picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The culture was carried out at 37℃ and 220 rpm for 12 hours. Then, the plasmid was extracted using a rapid plasmid mini-extraction kit (purchased from Tiangen (Beijing) Co., Ltd.) to obtain the recombinant plasmid YEp352-CDC19 containing the described Valencia ene oxidase HPO (wild-type) gene. p -HPO(wt)-TPI1 t -CYC1 p -AtCPR-CYC1 t .

[0046] Example 2: Construction of a recombinant expression vector for Valencia ene oxidase HPO (mutant)

[0047] This example uses the recombinant plasmid YEp352-CDC19 from Example 1. p -HPO(wt)-TPI1 t -CYC1 p -AtCPR-CYC1 t Using this as a template, we constructed a recombinant expression vector for Valencia ene oxidase HPO (single point mutant and multi-site mutant).

[0048] Construction of HPO unit point mutant: using recombinant plasmid YEp352-P CDC19 -HPO(V482A)-T TPI1 -P CYC1 -AtCPR-T CYC1This will be illustrated using an example. The recombinant plasmid YEp352-CDC19 will be used as an example. p -HPO(wt)-TPI1 t -CYC1 p -AtCPR-CYC1 t Using a template, a single point mutation (GTT→GCT) of V482A was introduced into the primers, and the HPO single point mutant was constructed using homologous recombination, a method commonly used in molecular cloning (using the ClonExpress II Recombinant Cloning Kit, purchased from Nanjing Novizan Biotechnology Co., Ltd.). Subsequent transformation into E. coli DH5α competent cells and plasmid extraction were performed in the same manner as in Example 1.

[0049] Construction of HPO multi-site mutants: The construction principle is the same as that of the aforementioned HPO single-site mutants, that is, introducing mutations into the primers. When there are many mutation sites that are far apart, multiple rounds of molecular cloning can also be used for construction.

[0050] Example 3: Construction of recombinant expression cells

[0051] Using the Sc EasyComp Transformation Kit (purchased from Invitrogen, USA), the recombinant expression vectors obtained in Examples 1 and 2 were transformed into Saccharomyces cerevisiae CEN.PK2-1Ca competent cells, respectively. 200 μL of the transformation solution was plated on SD plates lacking uracil and cultured at 30°C for 2 days to obtain Saccharomyces cerevisiae ScCEN.PK2-1Ca recombinant expression cells (YEp352-CDC19). p -HPO (wt or mutant)-TPI1 t -CYC1 p -AtCPR-CYC1 t ).

[0052] Example 4: In vitro catalytic experiment of resting cells to determine the catalytic performance of HPO mutant

[0053] Recombinant Saccharomyces cerevisiae ScCEN.PK2-1Ca expression cells (YEp3 52-CDC19) obtained in Example 3 were selected. p -HPO (wt or mutant)-TPI1 t -CYC1 p -AtCPR-CYC1 t Add to SD liquid medium lacking uracil and incubate at 30°C and 220 rpm for 24 hours. Then, according to the initial OD... 600The inoculum of 0.05 was transferred to a 250 mL baffled shaker flask containing 50 mL of mono-uracil SD liquid medium. After incubation at 30 °C and 200 rpm for 24 hours, the bacterial culture was collected, centrifuged at 1060 × g for 10 minutes, and the supernatant was discarded. The recombinant expression cells were then resuspended in potassium phosphate buffer (50 mM, pH 7.4) and brought to a final volume of 1 mL. 20 μL of 100 mM Valenciaene solution was added to bring the final substrate concentration to 2 mM. Catalysis was carried out at 25 °C and 220 rpm for 24 hours. A schematic diagram of the catalytic reaction is shown below. Figure 2 As shown.

[0054] The method for detecting the product naringin is as follows:

[0055] After the reaction was complete, the entire reaction solution was transferred to a 2 mL centrifuge tube, and 1 mL of ethyl acetate was added. The mixture was shaken and extracted for 10 minutes, followed by centrifugation at 12000 rpm for 5 minutes. 500 μL of the upper ethyl acetate layer was transferred to a 1.5 mL centrifuge tube, and another 500 μL of ethyl acetate was added. Finally, the mixture was filtered through a 0.22 μm organic filter into a chromatographic vial for gas chromatography analysis. The gas chromatograph used was a Shimadzu GC-2014C gas chromatograph (Japan), with a 5% Ph-Me siloxane column (30 m × 0.10 mm × 0.10 μm), a flame ionization detector (FID), and N2 as the carrier gas.

[0056] The detection method is as follows: 1 μL sample is injected by split injection at a split ratio of 15:1, the injection port temperature is 250℃, the detector temperature is 350℃, and the total time is 20 min.

[0057] Table 1 lists the in vitro catalytic performance results of the Valencia ene oxidase HPO unit point mutant.

[0058] Table 1: Summary of catalytic activity of HPO with a single point mutation compared to SEQ ID NO.2

[0059]

[0060] a The substrate conversion efficiency obtained by using HPO with a single point mutation (=sample) / the substrate conversion efficiency obtained by using wild-type HPO (=control) is calculated as (saturated terpineol concentration [sample] / substrate concentration [sample]) / (saturated terpineol concentration [control] / substrate concentration [control]) × 100%. In this embodiment, the substrate concentration [sample] = substrate concentration [control] = 2mM.

[0061] b Wild-type HPO (nucleotide sequence as shown in SEQ ID NO.2).

[0062] cThe parentheses :() indicate the codon for the amino acid at that site in wild-type HPO.

[0063] d The parentheses :() indicate the codon used when the site is mutated to the specified amino acid.

[0064] The results in Table 1 show that the HPO single-point mutants exhibit superior catalytic conversion of the substrate valenciaene. Compared to the wild-type HPO used in this experimental system, these single-point mutants showed conversion efficiencies of 112% (e.g., HPO_V480G) to 203% (e.g., HPO_V482A), indicating that mutant proteins with specific mutations in the core amino acids associated with enzyme catalytic activity (i.e., the five amino acid sites listed in Table 1) have the potential to enhance enzyme activity.

[0065] Table 2 lists the in vitro catalytic performance results of the Valencia enyl oxidase HPO multisite mutant.

[0066] Table 2: Summary of catalytic activity of HPO with multiple site mutations compared to SEQ ID NO.2

[0067] Amino acids after multi-site mutation Substrate concentration (mM) <![CDATA[Conversion efficiency (% relative to wild type) a > WT (SEQ ID NO.2) 2 100 302G-484A 2 192 302G-365A-484A 2 134 302G-480A-484A 2 173 302G-482A-484A 2 203 302G-482C-484A 2 132 302G-365A-482A-484A 2 145 302G-480A-482A-484A 2 254 302G-365A-480A-482A-484A 2 130

[0068] a The substrate conversion efficiency obtained by using HPO with a single point mutation (=sample) / the substrate conversion efficiency obtained by using wild-type HPO (=control) is calculated as (saturated terpineol concentration [sample] / substrate concentration [sample]) / (saturated terpineol concentration [control] / substrate concentration [control]) × 100%. In this embodiment, the substrate concentration [sample] = substrate concentration [control] = 2mM.

[0069] The results in Table 2 show that the Valenciaene oxidase HPO multi-site mutant described in this invention also exhibited significantly improved enzyme activity. The optimal HPO mutant was HPO_G302G-V480A-V482A-A484A, with its amino acid sequence shown in SEQ ID No. 4 and its nucleotide sequence shown in SEQ ID No. 5. This four-site mutant showed an in vitro Valenciaene conversion efficiency 2.54 times that of the wild type. It should be noted that, combining the results in Tables 1 and 2, it can be observed that the results of combined mutations are not always "better + better = better." That is, combining beneficial single-site mutations to form a multi-site mutant does not necessarily lead to better improvement in enzyme catalytic performance. This is a complex process involving the interaction of structure and function between the amino acids in the enzyme.

[0070] Example 5: In-situ synthesis of terpineone and experimental determination of the catalytic performance of the optimal HPO mutant

[0071] In Example 4, the catalytic performance of the optimal HPO mutant, HPO_G302G-V480A-A482A-A484A, was verified through in vitro catalytic experiments using resting cells. The catalytic performance of the optimal HPO mutant in the in-situ synthesis of naringin was then determined. A schematic diagram of the in-situ synthesis of naringin by *Saccharomyces cerevisiae* is shown below. Figure 3 As shown.

[0072] Using the technology described in patent application number 201910271558.6, the host cell ScCEN.PK2-1Ca underwent genomic modification, specifically by knocking out the mevalonate pathway restriction factor rox1 and downregulating the expression intensity of erg9, an enzyme related to the downstream pathway of the sesquiterpene precursor FPP, to increase the supply of the precursor FPP, ultimately obtaining the ScPK2-M strain. Based on this ScPK2-M strain, the expression intensity of erg12 and tHMG1 was further upregulated using the method described in the literature "Chen Hefeng. Construction and Metabolic Engineering of Valenciaene Cell Factory [D]. South China University of Technology, 2019," ultimately obtaining the ScPK2-01 strain. A recombinant plasmid YEp352-PDC1 containing the valenciaene synthase CnVS gene (NCBI accession number JX040471) and the tHMG1 gene was constructed. p -VS-SAG1 t -TEF1 p -tHMG1-CYC1 t -TDH3 p -Bm6-ADH1 t (Bm6 is an alcohol dehydrogenase, the amino acid sequence of which is shown in SEQ ID No. 7 and the nucleotide sequence of which is shown in SEQ ID No. 8).

[0073] Additionally, replace TEF1p-Cas9-CYC1t in the p414-TEF1p-Cas9-CYC1t vector (a commercial plasmid from Addgene) with CDC19. p -HPO (wt or optimal type)-TPI1 t -CYC1 p -AtCPR-CYC1 t p414-CDC19 was obtained. p -HPO (wt or optimal type)-TPI1 t -CYC1 p -AtCPR-CYC1 t The recombinant plasmid, YEp352-PDC1, was used. p -VS-SAG1 t -TEF1 p -tHMG1-CYC1 t-TDH3 p -Bm6-ADH1 t and p414-CDC19 p -HPO (wt or optimal type)-TPI1 t -CYC1 p -AtCPR-CYC1 t These two recombinant plasmids were co-transformed into strain ScPK2-01 to obtain the Saccharomyces cerevisiae strains ScPK2-02 (control strain, wild-type HPO) and ScPK2-03 (experimental strain, optimal type HPO_G302G-V480A-V482A-A484A). The control strain ScPK2-02 and the experimental strain ScPK2-03 were inoculated into test tubes containing SD / ΔTrp-Ura medium (SD medium lacking tryptophan (Trp) and uracil (Ura)) and cultured at 30℃ and 220 rpm for 24 h on a shaker. Afterward, they were transferred to 50 mL Erlenmeyer flasks containing SD / ΔTrp-Ura medium and covered with 2 mL of n-dodecane organic phase. Fermentation was then carried out at 25℃ and 200 rpm on a shaker for 96 h.

[0074] After fermentation, 500 μL of the upper n-dodecane organic phase was taken and mixed with an equal volume of ethyl acetate. The contents of Valencene, Nootkatol, and Nootkatone in the fermentation broth were then determined by gas chromatography according to the following method. The instruments used were as described in Example 4. The specific detection method was as follows: 1 μL sample was injected by split injection at a split ratio of 15:1, the injection port temperature was 250°C, the detector temperature was 350°C, and the total detection time was 22.58 min.

[0075] Final fermentation as Figure 4 As shown, the experimental strain ScPK2-03 produced 66.68 mg / L of naringin, accounting for 69.70% of the total terpenoid (valenciane, naringol, and naringin combined) yield. Compared to the control strain ScPK2-02 using HPO (wt) (naringin yield was 53.07 mg / L, accounting for 59.14% of the total terpenoid yield), the naringin yield increased by 26% with a slight increase in total terpenoid yield. This indicates that the optimal HPO mutant provided by this invention, HPO_G302G-V480A-A482A-A484A, also exhibits superior catalytic performance in the in-situ synthesis of naringin, promoting the conversion of valenciane to naringol, thereby increasing the yield of naringin.

[0076] Example 6: Preparation of terpineone using a simple carbon source via fed-batch shake-flask fermentation

[0077] This embodiment lays the foundation for subsequent fermentation experiments in a small-scale fermenter (3L) by conducting fed-batch fermentation experiments in shake flasks. It also explores the catalytic performance of the optimal HPO mutant provided in this invention, HPO_G302G-V480A-A482A-A484A, in fed-batch fermentation experiments in shake flasks. The aforementioned "ScPK2-01 strain" was supplemented with defective types (uracil (Ura) and histidine (His)) to make it more suitable for high-density fermentation, resulting in the Saccharomyces cerevisiae strain ScPK2-04. Subsequently, it was transformed into two recombinant plasmids described in Example 5, YEp352-PDC1. p -VS-SAG1 t -TEF1 p -tHMG1-CYC1 t -TDH3 p -Bm6-ADH1 t and p414-CDC19 p -HPO (wt or optimal type)-TPI1 t -CYC1 p -AtCPR-CYC1 t Saccharomyces cerevisiae strains ScPK2-05 and ScPK2-06 were obtained. The only difference between the experimental strain ScPK2-06 and the control strain ScPK2-05 (containing wild-type HPO) is the introduction of the optimal HPO_G302G-V480A-V482A-A484A.

[0078] The control strain ScPK2-05 and the experimental strain ScPK2-06 were inoculated into test tubes containing SD / ΔTrp-Ura medium and cultured on a shaker at 30℃ and 220 rpm for 24 h. Then, they were transferred to 50 mL Erlenmeyer flasks containing SD / ΔTrp-Ura medium and covered with 2 mL of n-dodecane organic phase. Fermentation was carried out on a shaker at 25℃ and 200 rpm using fed-batch fermentation for a total of 130 h.

[0079] Fermentation results as follows Figure 5 As shown, in the shake-flask fed-batch fermentation experiment, the optimal HPO mutant provided by this invention, namely HPO_G302G-V480A-V482A-A484A, better demonstrates its catalytic advantages. Specific fermentation results are as follows: Figure 5As shown, the control strain ScPK2-05, which used HPO (wt), produced a total terpene yield of 325.09 mg / L, of which naringin yield was 224.17 mg / L. The experimental strain ScPK2-06, which used the optimal HPO mutant, produced a total terpene yield of 399.40 mg / L, of which naringin yield was 322.62 mg / L, representing the highest shake-flask yield of naringin synthesized in situ using yeast as a host reported in the literature. The comparison shows that the optimal HPO mutant not only increased naringin yield (by 44%) but also increased the percentage of naringin in total terpenes (from 69% to 81%). This indicates that the optimal HPO mutant not only increases naringin yield but also, due to its superior catalytic conversion performance of the substrate valenciaene, further reduces the cellular metabolic burden and potential toxicity caused by valenciaene accumulation. Therefore, it can achieve high-density fermentation while simultaneously increasing the total terpene yield of the cells. Meanwhile, since the shake-flask fed-batch fermentation experiment can partially simulate the experimental conditions of a small fermenter (3L), it can be seen that using the optimal HPO mutant can yield higher yields and higher purity of naringin. In summary, the optimal HPO mutant (HPO_G302G-V480A-V482A-A484A) provided by this invention has excellent prospects for industrial application, and this invention is also the first report of successfully constructing a beneficial HPO mutant for high-yield naringin production.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. sequence list <110> South China University of Technology <120> Valencia ene oxidase mutant and its application in the preparation of naringin <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Valencia ene oxidase HPO (wild type) <400> 1 Met Gln Phe Phe Ser Leu Val Ser Ile Phe Leu Phe Leu Ser Phe Leu 1 5 10 15 Phe Leu Leu Arg Lys Trp Lys Asn Ser Asn Ser Gln Ser Lys Lys Leu 20 25 30 Pro Pro Gly Pro Trp Lys Leu Pro Leu Leu Gly Ser Met Leu His Met 35 40 45 Val Gly Gly Leu Pro His His Val Leu Arg Asp Leu Ala Lys Lys Tyr 50 55 60 Gly Pro Leu Met His Leu Gln Leu Gly Glu Val Ser Ala Val Val Val 65 70 75 80 Thr Ser Pro Asp Met Ala Lys Glu Val Leu Lys Thr His Asp Ile Ala 85 90 95 Phe Ala Ser Arg Pro Lys Leu Leu Ala Pro Glu Ile Val Cys Tyr Asn 100 105 110 Arg Ser Asp Ile Ala Phe Cys Pro Tyr Gly Asp Tyr Trp Arg Gln Met 115 120 125 Arg Lys Ile Cys Val Leu Glu Val Leu Ser Ala Lys Asn Val Arg Ser 130 135 140 Phe Ser Ser Ile Arg Arg Asp Glu Val Leu Arg Leu Val Asn Phe Val 145 150 155 160 Arg Ser Ser Thr Ser Glu Pro Val Asn Phe Thr Glu Arg Leu Phe Leu 165 170 175 Phe Thr Ser Ser Met Thr Cys Arg Ser Ala Phe Gly Lys Val Phe Lys 180 185 190 Glu Gln Glu Thr Phe Ile Gln Leu Ile Lys Glu Val Ile Gly Leu Ala 195 200 205 Gly Gly Phe Asp Val Ala Asp Ile Phe Pro Ser Leu Lys Phe Leu His 210 215 220 Val Leu Thr Gly Met Glu Gly Lys Ile Met Lys Ala His His Lys Val 225 230 235 240 Asp Ala Ile Val Glu Asp Val Ile Asn Glu His Lys Lys Asn Leu Ala 245 250 255 Met Gly Lys Thr Asn Gly Ala Leu Gly Gly Glu Asp Leu Ile Asp Val 260 265 270 Leu Leu Arg Leu Met Asn Asp Gly Gly Leu Gln Phe Pro Ile Thr Asn 275 280 285 Asp Asn Ile Lys Ala Ile Ile Phe Asp Met Phe Ala Ala Gly Thr Glu 290 295 300 Thr Ser Ser Ser Thr Leu Val Trp Ala Met Val Gln Met Met Arg Asn 305 310 315 320 Pro Thr Ile Leu Ala Lys Ala Gln Ala Glu Val Arg Glu Ala Phe Lys 325 330 335 Gly Lys Glu Thr Phe Asp Glu Asn Asp Val Glu Glu Leu Lys Tyr Leu 340 345 350 Lys Leu Val Ile Lys Glu Thr Leu Arg Leu His Pro Pro Val Pro Leu 355 360 365 Leu Val Pro Arg Glu Cys Arg Glu Glu Thr Glu Ile Asn Gly Tyr Thr 370 375 380 Ile Pro Val Lys Thr Lys Val Met Val Asn Val Trp Ala Leu Gly Arg 385 390 395 400 Asp Pro Lys Tyr Trp Asp Asp Ala Asp Asn Phe Lys Pro Glu Arg Phe 405 410 415 Glu Gln Cys Ser Val Asp Phe Ile Gly Asn Asn Phe Glu Tyr Leu Pro 420 425 430 Phe Gly Gly Gly Arg Arg Ile Cys Pro Gly Ile Ser Phe Gly Leu Ala 435 440 445 Asn Val Tyr Leu Pro Leu Ala Gln Leu Leu Tyr His Phe Asp Trp Lys 450 455 460 Leu Pro Thr Gly Met Glu Pro Lys Asp Leu Asp Leu Thr Glu Leu Val 465 470 475 480 Gly Val Thr Ala Ala Arg Lys Ser Asp Leu Met Leu Val Ala Thr Pro 485 490 495 Tyr Gln Pro Ser Arg Glu 500 <210> 2 <211> 1509 <212> DNA <213> Artificial Sequence <220> <223> The nucleotide sequence of Valencia ene oxidase HPO (wild type) after codon optimization <400> 2 atgcagttct tctcactggt ttctatcttc ctatttctgt cctttctgtt cctgctgagg 60 aaatggaaga acagtaactc acagagtaag aagttgcctc caggtccatg gaaattacca 120 ttactaggta gtatgcttca tatggttggt ggattgcctc atcatgtact tagagatctt 180 gcaaagaaat acggaccact tatgcatctt caacttggtg aagtttctgc tgttgtggtc 240 actagccctg atatggcaaa ggaagtattg aagactcatg atatcgcatt tgcatctagg 300 cctaaactat tggcaccaga aattgtttgt tacaacagaa gcgatattgc attctgtcca 360 tacggtgact attggagaca aatgagaaag atctgtgtac tagaggtatt gagtgctaag 420 aatgtgaggt ctttcagttc gattagacgt gacgaagttc ttagactagt gaatttcgtt 480 agaagctcta cgtcagaacc tgttaacttt acggaaagac tattcttgtt tacgtcctca 540 atgacctgta gatcagcatt tggtaaagtg tttaaagagc aggagacctt catacagcta 600 atcaaggaag tgataggttt ggcaggagga tttgatgtcg cagatatatt tccatcgttg 660 aaattcttgc acgtcttgac cggaatggaa ggtaaaatta tgaaagctca ccacaaagtg 720 gacgctattg tggaagacgt tattaacgag cataagaaga acctggctat ggggaagact 780 aatggtgctt taggagggga agatctaatt gatgttctat tgagattgat gaatgacgga 840 ggtttgcaat ttcctattac aaatgacaat atcaaagcga ttatatttga catgtttgcc 900 gcgggcaccg agacttcttc atctacattg gtgtgggcta tggttcaaat gatgagaaat 960 ccaactatat tagccaaagc tcaagccgag gtcagagaag ccttcaaagg taaagaaacc 1020 ttcgatgaga atgatgtcga agaattgaaa tacctgaaat tagtcatcaa agagacattg 1080 aggttacatc caccagtccc attattagta ccacgtgagt gcagagaaga aacagaaatt 1140 aatggctata ctattccggt aaagactaag gtcatggtta atgtttgggc cttaggccgt 1200 gatccgaaat attggggatga tgccgataac tttaaaccgg aaaggtttga acaatgctcc 1260 gtagatttca ttggtaataa ctttgaatat cttccctttg gtggggggag gcgtatatgc 1320 cctggtatat cgtttggctt agctaatgtt tacttgcctt tagctcaatt attatatcac 1380 tttgattgga agttacccac aggcatggaa cccaaagact tagatttgac agaattagtt 1440 ggcgttacag ctgccaggaa atccgactta atgttagtag cgactcctta tcaaccctcc 1500 cgtgaataa 1509 <210> 3 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Valencia ene oxidase HPO mutant <220> <222> (302)..(302) <223> Any amino acid <220> <222> (365) <223> Any amino acid <220> <222> (480)..(480) <223> Any amino acid <220> <222> (482)..(482) <223> Any amino acid <220> <222> (484)..(484) <223> Any amino acid <400> 3 Met Gln Phe Phe Ser Leu Val Ser Ile Phe Leu Phe Leu Ser Phe Leu 1 5 10 15 Phe Leu Leu Arg Lys Trp Lys Asn Ser Asn Ser Gln Ser Lys Lys Leu 20 25 30 Pro Pro Gly Pro Trp Lys Leu Pro Leu Leu Gly Ser Met Leu His Met 35 40 45 Val Gly Gly Leu Pro His His Val Leu Arg Asp Leu Ala Lys Lys Tyr 50 55 60 Gly Pro Leu Met His Leu Gln Leu Gly Glu Val Ser Ala Val Val Val 65 70 75 80 Thr Ser Pro Asp Met Ala Lys Glu Val Leu Lys Thr His Asp Ile Ala 85 90 95 Phe Ala Ser Arg Pro Lys Leu Leu Ala Pro Glu Ile Val Cys Tyr Asn 100 105 110 Arg Ser Asp Ile Ala Phe Cys Pro Tyr Gly Asp Tyr Trp Arg Gln Met 115 120 125 Arg Lys Ile Cys Val Leu Glu Val Leu Ser Ala Lys Asn Val Arg Ser 130 135 140 Phe Ser Ser Ile Arg Arg Asp Glu Val Leu Arg Leu Val Asn Phe Val 145 150 155 160 Arg Ser Ser Thr Ser Glu Pro Val Asn Phe Thr Glu Arg Leu Phe Leu 165 170 175 Phe Thr Ser Ser Met Thr Cys Arg Ser Ala Phe Gly Lys Val Phe Lys 180 185 190 Glu Gln Glu Thr Phe Ile Gln Leu Ile Lys Glu Val Ile Gly Leu Ala 195 200 205 Gly Gly Phe Asp Val Ala Asp Ile Phe Pro Ser Leu Lys Phe Leu His 210 215 220 Val Leu Thr Gly Met Glu Gly Lys Ile Met Lys Ala His His Lys Val 225 230 235 240 Asp Ala Ile Val Glu Asp Val Ile Asn Glu His Lys Lys Asn Leu Ala 245 250 255 Met Gly Lys Thr Asn Gly Ala Leu Gly Gly Glu Asp Leu Ile Asp Val 260 265 270 Leu Leu Arg Leu Met Asn Asp Gly Gly Leu Gln Phe Pro Ile Thr Asn 275 280 285 Asp Asn Ile Lys Ala Ile Ile Phe Asp Met Phe Ala Ala Xaa Thr Glu 290 295 300 Thr Ser Ser Ser Thr Leu Val Trp Ala Met Val Gln Met Met Arg Asn 305 310 315 320 Pro Thr Ile Leu Ala Lys Ala Gln Ala Glu Val Arg Glu Ala Phe Lys 325 330 335 Gly Lys Glu Thr Phe Asp Glu Asn Asp Val Glu Glu Leu Lys Tyr Leu 340 345 350 Lys Leu Val Ile Lys Glu Thr Leu Arg Leu His Pro Xaa Val Pro Leu 355 360 365 Leu Val Pro Arg Glu Cys Arg Glu Glu Thr Glu Ile Asn Gly Tyr Thr 370 375 380 Ile Pro Val Lys Thr Lys Val Met Val Asn Val Trp Ala Leu Gly Arg 385 390 395 400 Asp Pro Lys Tyr Trp Asp Asp Ala Asp Asn Phe Lys Pro Glu Arg Phe 405 410 415 Glu Gln Cys Ser Val Asp Phe Ile Gly Asn Asn Phe Glu Tyr Leu Pro 420 425 430 Phe Gly Gly Gly Arg Arg Ile Cys Pro Gly Ile Ser Phe Gly Leu Ala 435 440 445 Asn Val Tyr Leu Pro Leu Ala Gln Leu Leu Tyr His Phe Asp Trp Lys 450 455 460 Leu Pro Thr Gly Met Glu Pro Lys Asp Leu Asp Leu Thr Glu Leu Xaa 465 470 475 480 Gly Xaa Thr Xaa Ala Arg Lys Ser Asp Leu Met Leu Val Ala Thr Pro 485 490 495 Tyr Gln Pro Ser Arg Glu 500 <210> 4 <211> 502 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the optimal mutant of Valencia ene oxidase HPO (HPO_G302G-V480A-V482A-A484A) <400> 4 Met Gln Phe Phe Ser Leu Val Ser Ile Phe Leu Phe Leu Ser Phe Leu 1 5 10 15 Phe Leu Leu Arg Lys Trp Lys Asn Ser Asn Ser Gln Ser Lys Lys Leu 20 25 30 Pro Pro Gly Pro Trp Lys Leu Pro Leu Leu Gly Ser Met Leu His Met 35 40 45 Val Gly Gly Leu Pro His His Val Leu Arg Asp Leu Ala Lys Lys Tyr 50 55 60 Gly Pro Leu Met His Leu Gln Leu Gly Glu Val Ser Ala Val Val Val 65 70 75 80 Thr Ser Pro Asp Met Ala Lys Glu Val Leu Lys Thr His Asp Ile Ala 85 90 95 Phe Ala Ser Arg Pro Lys Leu Leu Ala Pro Glu Ile Val Cys Tyr Asn 100 105 110 Arg Ser Asp With Ala Phe Cys Pro Tyr Gly Asp Tyr Trp Arg Gln Met 115 120 125 Arg Lys Ile Cys Will Be Glu Will Be Ala Lys Asn Will Be Arg 130 135 140 Phe Ser Ser Ile Arg Arg Asp Glu Val Leu Arg Leu Val Asn Phe Val 145 150 155 160 Arg Ser Ser Thr Ser Glu Pro Val Asn Phe Thr Glu Arg Leu Phe Leu 165 170 175 Phe Thr Ser Ser Met Thr Cys Arg Ser Ala Phe Gly Lys Val Phe Lys 180 185 190 Glu Gln Glu Thr Phe And Gln Leu And Lys Glu Val And Gly Leu Ala 195 200 205 Gly Gly Phe Asp Val Ala Asp Ile Phe Pro Ser Leu Lys Phe Leu His 210 215 220 Val Leu Thr Gly Met Glu Gly Lys Ile Met Lys Ala His His Lys Val 225 230 235 240 Asp Ala Ile Val Glu Asp Val Ile Asn Glu His Lys Lys Asn Leu Ala 245 250 255 Met Gly Lys Thr Asn Gly Ala Leu Gly Gly Glu Asp Leu Ile Asp Val 260 265 270 Leu Leu Arg Leu Met Asn Asp Gly Gly Leu Gln Phe Pro Ile Thr Asn 275 280 285 Asp Asn Ile Lys Ala Ile Ile Phe Asp Met Phe Ala Ala Gly Thr Glu 290 295 300 Thr Ser Ser Ser Thr Leu Val Trp Ala Met Val Gln Met Met Arg Asn 305 310 315 320 Pro Thr Ile Leu Ala Lys Ala Gln Ala Glu Val Arg Glu Ala Phe Lys 325 330 335 Gly Lys Glu Thr Phe Asp Glu Asn Asp Val Glu Glu Leu Lys Tyr Leu 340 345 350 Lys Leu Val Ile Lys Glu Thr Leu Arg Leu His Pro Pro Val Pro Leu 355 360 365 Leu Val Pro Arg Glu Cys Arg Glu Glu Thr Glu Ile Asn Gly Tyr Thr 370 375 380 Ile Pro Val Lys Thr Lys Val Met Val Asn Val Trp Ala Leu Gly Arg 385 390 395 400 Asp Pro Lys Tyr Trp Asp Asp Ala Asp Asn Phe Lys Pro Glu Arg Phe 405 410 415 Glu Gln Cys Ser Val Asp Phe Ile Gly Asn Asn Phe Glu Tyr Leu Pro 420 425 430 Phe Gly Gly Gly Arg Arg Ile Cys Pro Gly Ile Ser Phe Gly Leu Ala 435 440 445 Asn Val Tyr Leu Pro Leu Ala Gln Leu Leu Tyr His Phe Asp Trp Lys 450 455 460 Leu Pro Thr Gly Met Glu Pro Lys Asp Leu Asp Leu Thr Glu Leu Ala 465 470 475 480 Gly Ala Thr Ala Ala Arg Lys Ser Asp Leu Met Leu Val Ala Thr Pro 485 490 495 Tyr Gln Pro Ser Arg Glu 500 <210> 5 <211> 1509 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the optimal mutant of Valencia ene oxidase HPO (HPO_G302G-V480A-V482A-A484A) <400> 5 atgcagttct tctcactggt ttctatcttc ctatttctgt cctttctgtt cctgctgagg 60 aaatggaaga acagtaactc acagagtaag aagttgcctc caggtccatg gaaattacca 120 ttactaggta gtatgcttca tatggttggt ggattgcctc atcatgtact tagagatctt 180 gcaaagaaat acggaccact tatgcatctt caacttggtg aagttctgc tgttgtggtc 240 actagccctg atatggcaaa ggaagtattg aagactcatg atatcgcatt tgcatctagg 300 cctaaactat tggcaccaga aattgtttgt tacaacagaa gcgatattgc attctgtcca 360 tacggtgact attggagaca aatgagaaag atctgtgtac tagaggtatt gagtgctaag 420 aatgtgaggt ctttcagttc gattagacgt gacgaagttc ttagactagt gaatttcgtt 480 agaagctcta cgtcagaacc tgttaacttt acggaaagac tattcttgtt tacgtcctca 540 atgacctgta gatcagcatt tggtaaagtg tttaaagagc aggagacctt catacagcta 600 atcaaggaag tgataggttt ggcaggagga tttgatgtcg cagatatatt tccatcgttg 660 aaattcttgc acgtcttgac cggaatggaa ggtaaaatta tgaaagctca ccacaaagtg 720 gacgctattg tggaagacgt tattaacgag cataagaaga acctggctat ggggaagact 780 aatggtgctt taggaggga agatctaatt gatgttctat tgagattgat gatgacgga 840 ggtttgcaat ttcctattac aaatgacaat atcaagcga ttatatttga catgtttgcc 900 gcggggaccg agactcttc atctacattg gtgtgggcta tggttcaat gatgagaaat 960 ccaactatat tagccaaagc tcaagccgag gtcagagaag ccttcaagg taaagaaacc 1020 ttcgatgaga atgatgtcga agaattgaaa tacctgaat tagtcatcaa agagacattg 1080 aggttacatc caccagtccc attagta caccagtgagt gcagagaaga aacagaaatt 1140 aatggctata ctattccggt aaagactaag gtcatggtta atgttttgggc cttaggccgt 1200 gatccgaat attgggatga tgccgataac tttaaaccgg aaaggtttga acatgctcc 1260 gtagatttca ttggtaataa ctttgaatat cttccctttg gtggggggag gcgtatatgc 1320 cctggtatat cgtttggctt agctaatgtt tacttgcctt tagctcatt attatatcac 1380 tttgattgga agttaccac agcatgga cccaagact tegtttgac agattagct 1440 ggcgctacag cggccaggaa atccgactta atgttagccgactcctta tcaccctcc 1500 cgtgaataa 1509 <210> 6 <211> 2079 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of codon-optimized AtCPR <400> 6 atgacttctg ccttgtatgc ctctgatttg ttcaagcaat tgaagtccat tatgggtact 60 gactcattgt ccgatgatgt tgttttggtt attgctacta cctccttggc tttggttgct 120 ggttttgttg ttttattgtg gaaaaagacc accgccgata gatcaggtga attgaaacca 180 ttgatgatcc caaagtcttt gatggccaaa gatgaagatg atgatttgga tttgggttcc 240 ggtaagacta gagtttctat tttcttcggt actcaaaccg gtactgctga aggttttgct 300 aaggctttgt ctgaagaaat caaggccaga tacgaaaaag ctgccgttaa ggttatagat 360 ttggatgatt atgctgccga tgacgaccaa tacgaagaaa agttgaagaa agaaaccttg 420 gccttcttct gtgttgctac ttatggtgat ggtgaaccta ctgataatgc tgctagattt 480 tacaagtggt tcactgaaga aaacgaaaga gacatcaagt tgcaacaatt ggcttacggt 540 gttttgctt tgggtaacag acaatacgaa cacttcaaca agatcggtat cgttttggat 600 gaagaattgt gtaagaaggg tgccaagaga ttgattgaag ttggtttggg tgatgatgac 660 caatccatcg aagatgattt taacgcctgg aaagaatcct tgtggtctga attggataag 720 ttgttgaagg acgaagatga caaatctgtt gctacaccat acactgctgt tatcccagaa 780 tatagagttg ttacccatga tccaagattc accactcaaa agtctatgga atctaacgtt 840 gctaacggta acaccaccat cgatattcat catccatgta gagttgatgt cgccgtccaa 900 aaagaattgc atactcatga atctgacaga tcctgcatcc acttggaatt tgatatttcc 960 agaaccggta ttacctacga aactggtgat catgttggtg tttacgctga aaaccatgtt 1020 gaaatcgttg aagaagccgg taagttgtta ggtcattcct tagatttggt tttctccatc 1080 catgccgaca aagaagatgg ttctccattg gaatctgctg ttccaccacc atttccaggt 1140 ccatgtactt tgggtactgg tttggctaga tatgctgact tgttgaatcc accaagaaag 1200 tctgctttag ttgctttggc tgcttatgct actgaaccat ctgaagccga aaaattgaaa 1260 catttgactt ccccagatgg taaggacgaa tattctcaat ggatagttgc ctcccaaaga 1320 tccttgttgg aagttatggc tgcttttcca tctgctaaac caccattggg tgtttttttt 1380 gctgctattg ctccaagatt gcaacctaga tattactcca tttcctccag tccaagatta 1440 gctccatcaa gagttcatgt tacatccgct ttggtttatg gtccaactcc aactggtaga 1500 atccataagg gtgtttgttc tacctggatg aagaacgctg ttccagctga aaaatctcat 1560 gaatgttctg gtgccccaat tttcattaga gcttctaatt tcaagttgcc atccaaccca 1620 tctactccaa tagttatggt tggtccaggt acaggtttag ctccttttag aggtttctta 1680 caagaaagaa tggccttgaa agaagatggt gaagaattgg gttcctcctt gttgtttttt 1740 ggttgcagaa acagacaaat ggatttcatc tatgaagatg aattgaacaa cttcgttgac 1800 caaggtgtca tctccgaatt gattatggct ttttcaagag aaggtgctca aaaagaatac 1860 gtccaacaca agatgatgga aaaagccgct caagttttggg acttgatcaa agaagaaggt 1920 tacttgtacg tttgcggtga tgctaaaggt atggctagag atgttcatag aacattgcat 1980 accatcgttc aagaacaaga aggtgtctca tcttctgaag ctgaagctat cgttaagaag 2040 ttgcaaactg aaggtagata cttgagagat gtctggtga 2079 <210> 7 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Bm6 <400> 7 Met Gly Glu Leu Ser Gly Lys Thr Ala Val Val Ser Gly Gly Ser Gly 1 5 10 15 Gly Ile Gly Phe Ala Ile Cys Thr Lys Phe Ala Gln Asn Gly Ala Lys 20 25 30 Val Ile Ile Leu Asp Tyr Asn Lys Glu Thr Leu Asp Glu Met Leu Pro 35 40 45 Lys Leu Val Ala Pro Glu Gly Gln Lys His Glu Gly His Phe Tyr Asp 50 55 60 Val Thr Lys Asn Asp Arg Pro Pro Val Asp Phe Ser Lys Val Asp Ile 65 70 75 80 Leu Leu Asn Gly Ala Gly Ile Cys Asn Gly Gly Phe Leu Glu Gly Met 85 90 95 Glu Thr Ser Leu Ile Glu Arg Ile Ile Ala Thr Asn Leu Thr Gly Val 100 105 110 Ile Lys Leu Thr Lys Tyr Ala Met Glu Ala Trp Phe Glu Arg Glu Asp 115 120 125 Ser Arg Thr Lys Ala Gln Gly Asn Gly Val Val Ile Asn Ile Ser Ser 130 135 140 Ile Leu Gly Leu Arg Ala Val Met Pro Ala Leu Thr Val Tyr Ser Ala 145 150 155 160 Ser Lys Gly Gly Ile Ile Met Phe Thr Lys Ala Leu Ala Ile Glu Gly 165 170 175 Gly Ala His Ala Ile Arg Ala Asn Ala Ile Cys Pro Gly Tyr Val Arg 180 185 190 Thr Ala Met Ser Glu Phe Met Glu Leu Pro Glu Pro Ser Pro Phe Gln 195 200 205 Glu Lys Asp Asp Asn Gly Asp Val Asp Lys Glu Ser Ile Ala Gly Ala 210 215 220 Ala Tyr Tyr Phe Ala Thr Asn Leu Gln Val Ser Gly Ala Ile Leu Ser 225 230 235 240 Val Asp Lys Ala Met Ala Ala Leu 245 <210> 8 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of Bm6 <400> 8 atgggtgaat tgtctggtaa aactgctgtt gtttctggtg gttctggtgg tattggtttc 60 gctatttgta ctaagttcgc tcaaaacggt gctaaggtta ttattttaga ctacaacaag 120 gaaactttgg atgaaatgtt gccaaagttg gttgctccag aaggtcaaaa gcatgaaggt 180 cacttctacg atgttactaa gaatgataga ccaccagttg atttttctaa ggttgatatt 240 ttgttaaacg gtgctggtat ttgtaacggt ggtttcttgg aaggtatgga aacttctttg 300 attgaaagaa ttattgctac taacttaact ggtgttatta agttgactaa atacgctatg 360 gaagcttggt ttgaaagaga agattctaga actaaggctc aaggtaatgg tgttgttatt 420 aacatttcct ctattttggg tttaagagct gttatgccag ctttgactgt ttattctgca 480 tctaagggtg gtattattat gtttactaag gcattggcta tcgaaggtgg tgctcatgct 540 attagagcta acgctatttg tccaggttac gttagaactg ctatgtctga attcatggaa 600 ttgcctgaac catctccatt ccaagaaaag gatgataacg gtgacgttga taaggaatct 660 attgctggtg ctgcttacta cttcgctact aacttgcaag tttccggtgc tattttgtct gttgataagg ctatggctgc tttgtaa 747 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> promoter‐F <400> 9 tttacagtcg acaatgctag tattttggag at <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> terminator‐R <400> 10 tttacactgc agctatataa cagttgaaat t

Claims

1. Valencia ene oxidase HPO mutant, characterized by: The HPO mutant is obtained by any one of the following mutations of SEQ ID No. 1: HPO_V482A, HPO_V482C, HPO_G302G-V482A-A484A, HPO_G302G-V482C-A484A, HPO_G302G-P365A-V482A-A484A, HPO_G302G-V480A-V482A-A484A, or HPO_G302G-P365A-V480A-V482A-A484A; wherein G302G and A484A are synonymous mutations, G302G refers to the nucleotide sequence of amino acid 302 changing from GGC to GGG, and A484A refers to the nucleotide sequence of amino acid 484 changing from GCT to GCG.

2. The gene encoding the Valencia ene oxidase HPO mutant of claim 1.

3. The gene of claim 2, wherein: The nucleotide sequence of the gene encoding the mutant HPO_G302G-V480A-V482A-A484A is shown in SEQ ID No.

5.

4. A recombinant expression vector containing the gene described in claim 2 or 3.

5. A recombinant expression cell containing the gene of claim 2 or 3.

6. The recombinant expression cell according to claim 5, characterized in that: The host bacteria of the recombinant expression cells are Saccharomyces cerevisiae, Pichia pastoris, Yeastra salviae, or Escherichia coli.

7. The recombinant expression cell according to claim 6, characterized in that: The brewing yeast is brewing yeast strain CEN.PK2-1Ca.

8. Use of the Valenciaene oxidase HPO mutant according to claim 1, the gene according to any one of claims 2 to 3, the recombinant expression vector according to claim 4 or the recombinant expression cell according to any one of claims 5 to 7 for the biosynthesis of cirsimaritin or cirsimarin, characterized in that, The catalytic substrate for the biosynthesis of terpineol or terpineone is Valenciaene.

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