Use of oxidoreductases and their mutants in the biosynthesis of nootkatone.

Abstract

The present invention provides a use of an oxidoreductase and a mutant thereof in the biosynthesis of nootkatone. The oxidoreductase is derived from Wickerhamomyces anomalus M15 and has a high conversion ability for nootkatoll, and a more capable oxidoreductase mutant can be obtained by site-directed mutagenesis. Compared with existing oxidoreductases capable of catalyzing nootkatoll, the oxidoreductase and a mutant thereof have excellent substrate tolerance, high conversion rate, and high salt tolerance, providing conditions for in vitro enzyme-catalyzed synthesis of nootkatone, and realizing an environmentally friendly and efficient method for catalyzing the synthesis of nootkatone product from nootkatoll.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of biotechnology, and in particular relates to the use of oxidoreductases and mutants thereof in the biosynthesis of nootkatone. [Background technology]

[0002] Nootkatone (also known as nootkatone) is a sesquiterpenoid with a pomelo-like aroma and slightly bitter taste. It was originally extracted from Alaskan cypress and is found in grapefruit, citrus fruits, and other plants. Pure nootkatone is a white to slightly yellow crystal and is approved by the FDA and EPA to blend grapefruit, orange, tropical fruit, and other food and tobacco flavors. Nootkatone sprays are effective insecticides against lone star ticks and deer ticks, and also provide satisfactory control of mosquitoes, bedbugs, and lice. Nootkatone is considered an environmentally friendly insecticide due to its high volatility and non-toxicity to humans. In 2014, the CDC officially approved two companies to produce nootkatone-based insecticides. Furthermore, recent research has shown that nootkatone inhibits cancer cell proliferation and has potential therapeutic effects against neuroinflammation and Alzheimer's disease. Its research and development has attracted the attention of the pharmaceutical industry, and its future applications are promising.

[0003] Nootkatone, a sesquiterpenoid, has a complex chemical structure, making its large-scale industrial production difficult. Currently, three main production methods for nootkatone exist: physical extraction, chemical synthesis, and biocatalytic conversion. Nootkatone is isolated from grapefruit through processes such as distillation and extraction, but its active concentration is low, the separation and purification process is complex, and it is susceptible to seasonal and climate changes, making it unable to meet industrial needs. The most commonly used industrial method is the chemical synthesis of nootkatone using the relatively inexpensive precursor valencene. However, the oxidation reaction requires catalysts such as chromium trioxide, cobalt acetylacetonate, and other heavy metal salts, which generates a large amount of toxic waste and violates the concept of environmentally friendly development. To meet the growing market demand for nootkatone, the use of biocatalytic conversion offers significant advantages, as it is not limited by raw materials and avoids problems such as high energy consumption, low yield, and environmental pollution caused by the plant extraction process.

[0004] In recent years, significant progress has been made in the metabolic engineering of microorganisms. By constructing efficient microbial cell factories and improving their physiological performance, it is expected that inexpensive raw substrates can be efficiently converted into high-value-added target products, significantly reducing the production costs of microbial fermentation. The production of nootkatone by heterologous expression of related enzymes in microorganisms such as yeast and Escherichia coli has attracted much attention. Researchers have isolated the valencene synthase gene from C. sinensis, which can be efficiently and functionally expressed in E. coli and subsequently used for the production of nootkatone (Chappell J. et al. Novel sesquiterpene synthase gene and protein: US, US20120196340 A1[P]. 2006). Furthermore, we found that valencene dioxygenase (ValOx) derived from P. sapidus was expressed in the cytoplasm of Escherichia coli and converted valencene to nootkatol and nootkatone via the intermediate hydrogen peroxide (Zelena K. et al. Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli [J]. Bio Tec, 2012, 108:231-239. (Non-patent document 1)).The model organism yeast is currently one of the most commonly used gene expression hosts, boasting simple culture conditions, a well-defined genetic background, and ease of genetic modification. Marine yeasts, in particular, are significantly more tolerant than terrestrial yeasts to inhibitory compounds such as acetic acid, formic acid, furfural, vanillin, and salt. Among these, the most tolerant is Wickerhamomyces anomalus M15, whose salt tolerance is 1.6-fold higher than that of Saccharomyces cerevisiae NCYC2592 (Greetham D. et al., Exploring the tolerance of marine yeast to inhibitory compounds for improving bioethanol production[J]. Sustainable Energy & Fuels, 2019, 3(3). (Non-Patent Document 2)). Therefore, further development of highly efficient and environmentally tolerant nootkatol dehydrogenases using genetic engineering remains an urgent need in this field.

[0005] NCBI contains a hypothetical protein from Wickerhamomyces anomalus (NRRL Y-366-8), initially named WICANDRAFT_92107 (NCBI accession number: XP_019039214.1). This hypothetical protein contains a total of 264 amino acids, and the nucleotide sequence of its encoding gene (NCBI accession number: XM_019186339.1) contains a total of 795 nucleotides. To date, there are no published reports on the actual function and use of this protein. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 20120196340 [Non-patent literature]

[0007] [Non-Patent Document 1] Zelena K. et al. Functional expression of a valencene dioxygenase from Pleurotussapidus in E. coli[J]. Bio Tec, 2012, 108:231-239 [Non-patent document 2] Greetham D. et al. Exploring the tolerance of marine yeast to inhibitory compounds for improving bioethanol production[J]. Sustainable Energy & Fuels, 2019, 3(3). Summary of the Invention [Problem to be solved by the invention]

[0008] In order to overcome the drawbacks and deficiencies of the prior art, the object of the present invention is to provide the use of oxidoreductases and mutants thereof in the biosynthesis of nootkatone to solve the problems of existing alcohol dehydrogenases in the biosynthesis of nootkatone, such as low substrate concentration tolerance, low conversion rate, low salt tolerance, and being unsuitable for industrial production.

[0009] In the present invention, an oxidoreductase with high conversion ability for nootkatol was isolated from Wickerhamomyces anomalus M15 for the first time, and an oxidoreductase mutant with higher conversion ability was obtained by site-directed mutagenesis. [Means for solving the problem]

[0010] The object of the present invention is achieved by the following technical solutions.

[0011] The present invention provides the use of oxidoreductase enzymes and / or mutants thereof in the biosynthesis of nootkatone.

[0012] In the present invention, the oxidoreductase is NRRL derived from Wickerhamomycesanomalus M15, the amino acid sequence of which is shown in SEQ ID NO: 2, and contains a total of 264 amino acids.

[0013] In the present invention, the nucleotide sequence of the coding gene for the oxidoreductase NRRL is shown in SEQ ID NO: 1 and contains a total of 795 nucleotides.

[0014] Furthermore, the nucleotide sequence of the oxidoreductase NRRL-encoding gene after codon optimization is shown in SEQ ID NO:3.

[0015] The present invention also relates to an oxidoreductase mutant whose amino acid sequence is mutated at one or both of the amino acids at positions 69 and 136 of SEQ ID NO: 2, and further wherein the amino acid at position 69 is mutated from lysine K to threonine T, and the amino acid at position 136 is mutated from glycine G to arginine R or alanine A.

[0016] Furthermore, the amino acid sequence of the oxidoreductase mutant is such that the amino acid at position 69 of SEQ ID NO: 2 is mutated from lysine K to threonine T, and the amino acid at position 136 is mutated from glycine G to arginine R, and the specific amino acid sequence is shown in SEQ ID NO: 4.

[0017] Preferably, in the oxidoreductase mutant, the gene sequence encoding the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:3.

[0018] In another aspect of the present invention, the amino acid sequence SEQ ID NO: 2 is an amino acid sequence obtained by modifying, deleting or adding one or more amino acids, and sequences with only 90% homology are also within the scope of protection of the present invention.

[0019] The present invention provides a recombinant expression vector containing the gene encoding the oxidoreductase and its mutants, and preferably, the recombinant vector is obtained by recombining the oxidoreductase and its mutants with the 2μ high-copy plasmid YEp352.

[0020] The present invention provides a recombinant expression cell of a recombinant expression vector containing a gene encoding the oxidoreductase or its mutant described in the present invention. Preferably, the recombinant expression cell is obtained by transforming a host cell with a recombinant expression vector obtained by recombining the oxidoreductase or its mutant with a vector. In the present invention, the host cell is preferably Saccharomyces cerevisiae, more preferably the Saccharomyces cerevisiae CEN. PK2-1 Ca strain.

[0021] The present invention also relates to a method for producing nootkatone.

[0022] The present invention provides two methods for biosynthesizing nootkatone using recombinant expression cells carrying recombinant expression vectors containing genes encoding the oxidoreductases and mutants thereof described in the present invention.

[0023] In a whole-cell in vitro catalytic method, nootkatone is obtained by exogenously adding a precursor substance, nootkatol, and then performing in vitro conversion using the recombinant expression cells described in the present invention. The reaction substrate is nootkatol.

[0024] The conversion efficiency described in the present invention is expressed as the rate at which the precursor substance nootkatol is converted to the target product nootkatone within a certain period of time.

[0025] Alternatively, nootkatone can be produced by biological fermentation (i.e., culturing recombinant cells expressing the relevant enzymes in a reactor containing a medium).

[0026] In a preferred embodiment, the host cell is a eukaryotic cell, particularly selected from the strain Saccharomyces cerevisiae PK2-1 Ca (Saccharomyces cerevisiae CEN. PK2-1 Ca). Using the technology described in Chinese Patent Application No. 201910271558.6, genome modifications are made to the host cell, including knocking out the limiting factor rox1 in the mevalonate pathway and downregulating the expression level of erg9, an enzyme involved in the downstream branch pathway of the sesquiterpenoid precursor FPP, to increase the supply of the precursor substance FPP. Then, FPP is converted to valencene, the precursor substance of nootkatone, using valencene synthase ValC from Chamaecyparis nootkatensis, described in International Patent Application No. PCT / NL2010 / 050848. Valencene is then further oxidized to nootkatol by the cytochrome P450 monooxygenase (CYP450) HPO from Hyoscyamus muticus (PCT International Publication No. WO2006 / 079020) and the cytochrome P450 reductase AtCPR from Arabidopsis thaliana (Urban, P., et al., 1997, Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 1997, 272, 19176-19186.). Additionally, the oxidoreductase enzymes or mutants thereof described in the present invention are utilized to convert nootkatol to the desired end product nootkatone.

[0027] The present invention also covers the use of said oxidoreductase and mutants thereof as highly salt-tolerant nootkatone dehydrogenases in the biosynthesis of nootkatone. [Effects of the Invention]

[0028] The present invention has the following advantages and effects compared to the prior art.

[0029] (1) The present invention provides an oxidoreductase NRRL derived from Wickerhamomyces anomalus M15. This enzyme is the first oxidoreductase derived from Wickerhamomyces anomalus to be discovered, which uses nootkatol as a substrate. Furthermore, the present invention is the first to disclose, both domestically and internationally, the use of the oxidoreductase NRRL and its mutants in catalyzing the synthesis of nootkatone from nootkatol.

[0030] (2) The present invention provides conditions for the in vitro enzyme-catalyzed synthesis of nootkatone, realizing an environmentally friendly and efficient method for catalyzing the synthesis of the corresponding nootkatone from nootkatol, overcoming the drawbacks of low yield, low efficiency, complicated operations, serious environmental pollution, and high cost when obtaining nootkatone by physical extraction or chemical synthesis.

[0031] (3) Compared with existing oxidoreductases capable of catalyzing nootkatol, NRRL and its mutants have excellent substrate tolerance, high conversion rates, and high salt tolerance. Their effectiveness is significantly superior to that of the previously discovered nootkatol dehydrogenase ABA2 (NCBI accession number: HM036684.1) derived from citrus fruits. The results show that, under the same conditions, the conversion rates of the substrates to the desired product obtained by using the oxidoreductases described in the present invention are higher, approximately 3.8 times higher than those of citrus ABA2. Furthermore, some of the nootkatol dehydrogenase mutants of the present invention have a nootkatol conversion rate that is approximately 2.4 times higher than that of the oxidoreductase shown in SEQ ID NO: 2 under the same conditions, have better substrate tolerance, and exhibit improved tolerance to NaCl at various concentrations (6%, 9%, 12%, 15%, and 18% (W / V)).

[0032] (4) The present invention provides an important tool enzyme for the synthesis of nootkatone, which will bring great economic benefits to the nootkatone synthesis industry. [Brief explanation of the drawings]

[0033] [Figure 1] Schematic diagram of plasmid construction and reactions. [Figure 2] Graph showing gas phase detection of whole cell catalysis with related alcohol dehydrogenases. [Figure 3] GC-MS mass spectrum of nootkatone. [Figure 4] Comparison of the catalytic performance of expression strains before and after codon optimization of NRRL gene sequences. [Figure 5] Substrate conversion efficiency of oxidoreductase mutants. [Figure 6] Effect of different concentrations of NaCl (W / V) on the conversion rate of oxidoreductase or oxidoreductase mutants. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be described in more detail below with reference to examples and drawings, but the embodiments of the present invention are not limited to these.

[0035] The following examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make modifications and improvements based on the present invention, such as changing the type of expression vector, changing the method of constructing the expression vector, and changing the type of host cell, without departing from the concept of the present invention. All of these modifications and improvements fall within the scope of the present invention.

[0036] The S. cerevisiae CEN. PK2-1 Ca and S. c. PK2-M used in the examples are both disclosed in "CN201910271558 - Valencene-producing engineered Saccharomyces cerevisiae strains and methods for constructing and using the same."

[0037] Wickerhamomycesanomalus M15 used in the examples is disclosed in Non-Patent Document 2.

[0038] Example 1 Cloning of oxidoreductase NRRL and construction of expression vector Wickerhamomyces anomalus M15 was inoculated into 5 mL of YPD (glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L) liquid medium and grown at 30°C until logarithmic growth phase. Total genomic DNA was extracted from Wickerhamomyces anomalus using the Yeast DNA Kit (purchased from Omega).

[0039] A pair of primers specific for the coding gene of a hypothetical protein derived from Wickerhamomyces anomalus NRRL Y-366-8 (designated WICANDRAFT_92107 (NCBI accession number: XP_019039214.1)), which contains a total of 264 amino acids, and its coding gene (NCBI accession number: XM_019186339.1) contains a total of 795 nucleotides), was designed. The YEp352 expression vector was constructed using homologous recombination cloning with a nucleotide sequence homologous to the vector at the 5' end of each primer. Using 0.5 μL (approximately 10 ng) of the total DNA solution as a template, PCR was performed using KOD FX (purchased from Toyobo Co., Ltd., Japan) to obtain the target gene fragment, i.e., the oxidoreductase NRRL gene fragment. The nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. Alignment of this sequence with NCBI revealed that it had 100% homology with the above hypothetical protein derived from Wickerhamomyces anomalus (NRRL Y-366-8).

[0040] Here, specific amplification primers are as follows (the underlined sequences are bases homologous to the vector sequence): [ka]

[0041] The KOD-FX PCR system and conditions are shown in Table 1 below. [Table 1]

[0042] After the PCR reaction was completed, the size of the gene fragment was confirmed by agarose gel electrophoresis, the amplified fragment was purified using an oligonucleotide purification kit (purchased from Omega), the concentration of the gene fragment was detected by a microspectrophotometer, and the target gene fragment, i.e., the oxidoreductase NRRL gene fragment, was obtained by amplification.

[0043] The genome of S. cerevisiae CEN. PK2-1 Ca was extracted using the Yeast DNA Kit. Using the genome as a template, a TDH3 promoter fragment was amplified with the TDH3-F / TDH3-R primer pair, and an ADH1 terminator fragment was amplified with the ADH1-F / ADH1-R primer pair.

[0044] The primer sequences used are as follows (underlined sequences are homologous bases): [ka]

[0045] The YEp352-F1 / YEp352-R1 primer pair was used to amplify the YEp352 plasmid vector fragment purchased from Invitrogen. [ka]

[0046] Using the ClonExpress II Recombinant Cloning Kit (purchased from Nanjing Nuoviplex Co., Ltd.), the obtained TDH3 promoter fragment, ADH1 terminator fragment, and YEp352 plasmid vector fragment were subjected to multi-fragment homologous recombination ligation to generate the vector YEp352-TDH3 promoter -ADH1 terminator (Abbreviated as YEp352-TDH3 p -ADH1 t ) was obtained.

[0047] YEp352-F2 (5′-AGCTTTGGACTTCTTCGCC-3′) and YEp352-R2 (5′-TTTGTTTGTTTATGTGTGTT-3′) were used to generate YEp352-TDH3 p -ADH1 t was amplified, and then, using the ClonExpress II recombination cloning kit (purchased from Nanjing Nuoveiss Co., Ltd.), homologous recombination ligation was carried out between the amplified vector fragment of YEp352-F2 / R2 and the target gene obtained above.

[0048] Next, 10 μL of the ligation product was transformed into E. coli DH5α competent cells using a chemical method, spread onto LB / Amp plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar, autoclaved at 121°C for 20 minutes, ampicillin added to a final concentration of 1000 mg / mL before use), and cultured at 37°C for 12 to 16 hours to generate the recombinant E. coli strain (YEp352-TDH3 p -NRRL-ADH1 t) was obtained. The constructed map is shown in Figure 1. Recombinant bacteria were selected and inoculated into 5 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, autoclaved at 121°C for 20 minutes. Ampicillin was added to a final concentration of 1000 mg / mL before use) containing 100 μg / mL ampicillin. After culturing at 37°C and 220 rpm for 12 hours, the plasmid was extracted using a rapid plasmid small-scale extraction kit (purchased from Tiangen (Beijing) Co., Ltd.) and sent to Engineering Biotechnology (Shanghai) Co., Ltd. for gene sequencing. The sequencing results were analyzed using Snapgene software, and the recombinant plasmid YEp352-TDH3 containing the oxidoreductase gene was identified. p -NRRL-ADH1 t obtained.

[0049] Example 2 Construction of recombinant expression cells The recombinant expression plasmid YEp352-TDH3 constructed in Example 1 was transformed using the yeast transformation kit Sc Easy Comp Transformation Kit (purchased from Invitrogen, USA). p -NRRL-ADH1 t The vector was transformed into Saccharomyces cerevisiae CEN. PK2-1 Ca competent cells.

[0050] 1) 500 ng of recombinant plasmid and 250 μL of Solution III (Transformation solution) were added to every 25 μL of competent cells. 2) The mixture was shaken uniformly using a vortex shaker and then left in a constant temperature incubator at 30°C for 15 minutes. 3) After taking it out, it was shaken evenly again and left to stand at 30°C for 15 minutes, and this operation was repeated twice. 4) Then, 200 μL of the recombinant yeast cells were uniformly spread on an auxotrophic plate SD / ΔUra (6.7 g / L YNB, 2 g / L amino acid mixture, 20 g / L glucose, 20 mg / L leucine, 20 mg / L tryptophan) and cultured at 30°C for 2 to 4 days. When a single clone grew on the plate, it was identified as a recombinant expressing cell line S. c. CEN. PK2-1 Ca(YEp352-TDH3 p -NRRL-ADH1 t ) was obtained.

[0051] Example 3 Cell-catalyzed in vitro production of nootkatone The recombinant expression cells S. c. CEN. PK2-1 Ca(YEp352-TDH3) obtained in Example 2 p -NRRL-ADH1 t ) was transferred to a test tube containing 5 mL of SD / ΔUra liquid medium and cultured at 30°C and 220 rpm for 24 hours. 600 The inoculum was transferred to a 250 mL shake flask containing 50 mL of SD / ΔUra liquid medium at an inoculum size of 0.05 and cultured at 30°C and 220 rpm for 24 hours. 600 The bacterial suspension was collected and centrifuged at 3000 rpm at 4°C for 5 minutes, and the supernatant was discarded. The recombinant expression cells were then resuspended in potassium phosphate buffer (50 mM, pH 7.4), diluted to 1 mL, and diluted to 50 OD. 600 20 μL of a 100 mM nootkatol solution (containing 1% (v / v) Triton-100 dissolved in dimethyl sulfoxide) was added to give a final substrate concentration of 2 mM, and the reaction was carried out at 25°C and 220 rpm for 24 hours.

[0052] The detection method for the product is as follows (the detection method for the product in the following examples is the same as in Example 3).

[0053] After the reaction was complete, the reaction mixture was transferred to a 2 mL centrifuge tube, 1 mL of ethyl acetate was added, and the mixture was shaken for 10 minutes for extraction. The mixture was then centrifuged at maximum speed for 5 minutes to separate the organic and aqueous layers. 500 μL of the upper layer of ethyl acetate was transferred to a 1.5 mL centrifuge tube, and an additional 500 μL of ethyl acetate was added. Finally, the mixture was filtered through a 0.22 μm organic filter membrane into a chromatography bottle for gas phase detection. The gas phase used was a Hewlett-Packard 5890II gas chromatograph. The chromatography column was a 30 m x 0.10 mm x 0.10 μm 5% Ph-Me siloxane column, the detector was a hydrogen flame detector (FID), and the carrier gas was N2.

[0054] The detection method was as follows: 1 μL of sample was split and injected at a split ratio of 15:1. The injection port temperature was 250°C, the detector temperature was 350°C, and the column temperature was kept at 100°C for 5 minutes, then increased to 200°C at 20°C / min and kept at 200°C for 5 minutes, for a total time of 15 minutes.

[0055] The gas phase used was a Hewlett-Packard 5890 II gas chromatograph, the chromatography column was a quartz capillary column with specifications of 30 m x 0.25 mm x 0.25 μm, and the detector was a mass selective detector (MSD). The procedure was the same as above. Mass scan method: ion scan was selected. Mass analysis conditions: electron impact (EI) ionization source, electron energy 70 eV.

[0056] When the standard was detected by GC-FID, the retention time of nootkatone was 19,540 minutes. The results of gas-phase detection of whole-cell catalysis by several candidate alcohol dehydrogenases are shown in Figure 2, and the results of further qualitative analysis of the substance structure by GC-MS are shown in Figure 3. When compared with the standard, the chromatograms and mass spectra of the samples were consistent with those of the standard, indicating that all of the constructed recombinant yeast strains were able to convert valencene to nootkatol and nootkatone.

[0057] Finally, recombinantly expressed cells of the oxidoreductase S. c. CEN. PK2-1 Ca(YEp352-TDH3 p -NRRL-ADH1 t The conversion rate of nootkatol by ) was 100%.

[0058] Similarly, the present invention relates to a method for producing recombinant citrus-derived oxidoreductase ABA2 (NCBI accession number: HM036684.1)-expressing cells S. c. CEN. PK2-1 Ca(YEp352-TDH3) according to the methods described in Examples 1 to 3. p -ABA2-ADH1 t ) were also constructed and their catalytic abilities were tested under the same conditions as above. As shown in Figure 2, the conversion efficiency was 26% of that described in Example 3. The recombinant expression cells of the oxidoreductases described in this invention, S. c. CEN. PK2-1 Ca(YEp352-TDH3 p -NRRL-ADH1 t ) under the same conditions is 3.8 times that of the recombinantly expressed cells corresponding to ABA2, indicating that the oxidoreductase described in the present invention has good conversion ability for nootkatol.

[0059] Example 4 Codon optimization of oxidoreductase NRRL The screened oxidoreductase NRRL sequences capable of catalyzing the conversion of nootkatol to nootkatone were evaluated according to the Saccharomyces cerevisiae codon adaptability index (CAI). Analysis was performed using Detaibio (http: / / www.detaibio.com / ) software, and the NRRL sequences of oxidoreductases with high catalytic activity and low CAI were sent to Shanghai Bioengineering Co., Ltd. for codon optimization and sequencing. Following the methods described in Examples 1 and 2, the codon-optimized NRRL (cp) sequences were cloned into YEp352-TDH3. p -ADH1 t The expression cassette was constructed and expressed as YEp352-TDH3 p -NRRL(cp)-ADH1 tThe plasmid was obtained and further transformed into recombinant expression cells S. c. CEN. PK2-1 Ca(YEp352-TDH3 p -NRRL(cp)-ADH1 t ) was obtained, where the nucleotide sequence of NRRL(cp) is shown in SEQ ID NO:3.

[0060] Following the method described in Example 3, whole-cell catalytic experiments were performed with the strains of the original sequence to evaluate the difference in catalytic performance before and after optimization. As shown in the results in Figure 4, the substrate conversion rates of all strains tended to decrease with increasing substrate concentration. In contrast, the codon-optimized NRRL(cp) strain showed a lower rate of decrease in substrate conversion rate, with a 1.26-fold increase in conversion rate at substrate concentrations of 4 and 6 mM compared to the original strain. This indicates that codon optimization can improve the expression level of NRRL(cp) in the host bacterium to some extent, thereby further improving its catalytic performance.

[0061] Example 5 Production of oxidoreductase NRRL mutants Simulation analysis of the protein spatial structure of the oxidoreductase NRRL revealed that the mutation sites were lysine K at position 69, which was mutated to threonine T, and glycine G at position 136, which was mutated to arginine R or alanine A.

[0062] Mutation primers for site-directed mutagenesis were designed according to the NRRL optimized sequence (NRRL(cp)) shown in SEQ ID NO: 3, and the oxidoreductase NRRL gene was mutated by site-directed mutagenesis to obtain a new oxidoreductase gene. p -NRRL(cp)-ADH1 t Use fast PCR technology with the following as the template, where the universal primers for the recombinant plasmid are: YEp352-F3:5′-GTGACCGTCTCCGGGAGCTGCATGTG-3′ YEp352-R3:5′-CCGGAGACGGTCACAGCTTGTCTGTA-3′

[0063] The primers for introducing a single mutation at position 69 are as follows (the underlined sequence is the mutated base): [ka]

[0064] The above primers were amplified with the universal primers YEp352-F3 / YEp352-R3, respectively, and the amplification system and conditions are shown in Table 2.

[0065] [Table 2]

[0066] After the PCR reaction was completed, the size of the gene fragments was confirmed by agarose gel electrophoresis, and the amplified fragments were purified using an oligonucleotide purification kit. The concentrations of the gene fragments were detected by a microspectrophotometer, and the NRRL(cp):K69T-F / R, NRRL(cp):G136R-F / R, and NRRL(cp):G136A-F / R DNA fragments were obtained.

[0067] Using the method of Example 1, three recombinant plasmids containing single point mutant genes of the oxidoreductase were prepared: YEp352-TDH3 p -NRRL(cp)-K69T / G136R / G136A-ADH1 t obtained.

[0068] Example 6 Construction of double-site mutants of the oxidoreductase NRRL Recombinant plasmid YEp352-TDH3 constructed in Example 5 p -NRRL(cp)-K69T-ADH1 t Using this as a template, a single mutation was introduced at position 136 to construct a double-site mutant. The primers are shown as NRRL(cp):G136R-F / R and NRRL(cp):G136A-F / R.

[0069] Using the methods of Examples 1 and 5, two recombinant plasmids containing double point mutations in the oxidoreductase were obtained: YEp352-TDH3 p -NRRL(cp)-K69T-G136R / K69T-G136A-ADH1 t obtained.

[0070] Example 7 Catalytic efficiency and substrate tolerance of oxidoreductase mutants Using the recombinant expression cell construction method described in Example 2, the five recombinant expression vectors constructed were transformed into Saccharomyces cerevisiae S. c. CEN. PK2-1 Ca-competent cells to obtain five recombinant expression cells. Next, the conversion efficiency of the corresponding oxidoreductase mutants was tested using the whole-cell in vitro catalytic method described in Example 3.

[0071] As shown in Figure 5, compared to the original oxidoreductase NRRL(cp), the oxidoreductase mutants achieved significantly improved in vitro conversion efficiency and substrate tolerance of nootkatol. The beneficial mutation sites were NRRL(cp):K69T, NRRL(cp):G136R, NRRL(cp):G136A, NRRL(cp):K69T-G136R, and NRRL(cp):K69T-G136A. When compared to the original oxidoreductase NRRL(cp) in the same experimental system, the conversion efficiencies of the beneficial mutant oxidoreductases ranged from 107% (e.g., NRRL(cp):G136R) to 247% (e.g., NRRL(cp):K69T-G136R). For many of the oxidoreductase mutants tested, this improvement in conversion efficiency often occurred in conjunction with improved substrate tolerance, as shown in Table 3, indicating that the improved conversion efficiency is not primarily due to improved expression of these mutants in yeast.

[0072] Table 3 shows a summary of the substrate tolerance and conversion efficiency of the oxidoreductases with modifications compared to SEQ ID NO. 2.

[0073] [Table 3]

[0074] a : Position of amino acid residues in the modified oxidoreductase; residue code starts from the N-terminal threonine residue (=Met-1) of SEQ ID NO:2. b The test method was the same as in Example 3, except that the concentration of the nootkatol substrate added was different. c : Conversion efficiency of the modified oxidoreductase (= sample) relative to the conversion efficiency obtained with the wild-type oxidoreductase (= control), i.e. {(Nootkatone [sample] / substrate concentration [sample]) / (Nootkatone [control] / substrate concentration [control]) × 100%. d : The allowable concentration of the enzyme that can maintain a conversion rate of 60% or more under test conditions e : Wild-type oxidoreductase (SEQ ID NO. 2). f : () indicates the codon used to mutate to the corresponding amino acid.

[0075] Example 8 Salt tolerance of oxidoreductase mutants Following the whole-cell in vitro catalysis method described in Example 3, 3%, 6%, 9%, 12%, 15%, and 18% NaCl (W / V, g / 100 mL) were added to the reaction system, and catalysis was carried out at 25°C and 220 rpm for 24 hours to examine the effect of the oxidoreductase mutants on the substrate conversion rate.

[0076] As shown in Figure 6, the substrate conversion rate of WT (unmutated NRRL) was unaffected when catalyzed for 24 h in a 3% NaCl (W / V) reaction system. However, when treated with 6%, 9%, 12%, 15%, or 18% NaCl (W / V) for 24 h, the substrate conversion rate of the five mutants with high substrate catalytic efficiency and tolerance (NRRL(cp):K69T, NRRL(cp):G136R, NRRL(cp):G136A, NRRL(cp):K69T-G136R, and NRRL(cp):K69T-G136A) tended to decrease with increasing NaCl (W / V) concentration, but the substrate conversion rates of the mutants were all higher than that of WT. This indicates that the mutants have improved salt tolerance compared to WT.

[0077] Based on the above results, the optimal mutant NRRL(cp):K69T-G136R, which is an oxidoreductase mutant described in the present invention, has higher substrate conversion efficiency, substrate tolerance, and salt tolerance than the wild-type oxidoreductase due to single or double point mutations, and is expected to have future industrial applications.

[0078] Example 9 Production of nootkatone using simple carbon sources by biological fermentation Using the technology described in Chinese Patent Application No. 201910271558.6, the host cell S. c. CEN. PK2-1 Ca was modified in the genome to increase the supply of the precursor substance FPP by knocking out the limiting factor rox1 in the mevalonate pathway and downregulating the expression level of erg9, an enzyme involved in the downstream branching pathway of the sesquiterpenoid precursor FPP. This ultimately resulted in the S. c. PK2-M strain. The valencene synthase ValC gene (NCBI accession number: JX040471) derived from Japanese cedar (Chamaecyparis nootkatensis) described in International Patent Application No. PCT / NL2010 / 050848 (the promoter in the expression cassette for this gene is PDC1 and the terminator is SAG1) and the Saccharomyces cerevisiae endogenous HMG-CoA reductase tHMG1 (the rate-limiting enzyme in the mevalonate pathway) gene (NCBI accession number: NM_001182434) with a truncated N-terminal regulatory region (the promoter in the expression cassette for this gene is TEF1 and the terminator is CYC1) were then inserted into the recombinant expression vector YEp352-TDH3 obtained in Example 4. p -NRRL(cp)-ADH1 tThe recombinant expression vector YEp352-ValC-tHMG1-NRRL(cp) was obtained. Furthermore, TEF1p-Cas9-CYC1t of the p414-TEF1p-Cas9-CYC1t vector (a commercially available plasmid from Addgene) was transfected with the cytochrome P450 monooxygenase HPO gene (NCBI accession number: EF569601) from Hyoscyamus muticus (described in Patent WO2006 / 079020) (the promoter in the expression cassette of this gene is HXT7 and the terminator is TPI1), and the cytochrome reductase AtCPR gene (NCBI accession number: NM_118585) from Arabidopsis thaliana (described in a 1997 paper by Urban, Pd et al.) (Urban, P., et al. Cloning, yeast expression, and characterization of the coupling of two distantly related The recombinant expression vector p414-HPO-AtCPR was then transfected with Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5 (J. Biol. Chem. 1997, 272, 19176-19186). The recombinant expression vectors YEp352-ValC-tHMG1-NRRL(cp) and p414-HPO-AtCPR were then transformed together into the engineered strain ScPK2-M, which had a high FPP production capacity, to obtain the recombinant expression cell line ScPK2-M (YEp352-ValC-tHMG1-NRRL(cp) and p414-HPO-AtCPR), which was abbreviated as "PK2-VHN(cp)-HA."

[0079] The PK2-VHN(cp)-HA strain was inoculated into a test tube containing 5 mL of SD / ΔTrp-Ura medium (6.7 g / L YNB, 2 g / L amino acid mixture, 20 g / L glucose, 20 mg / L leucine) and cultured at 30°C in a shaker at 220 rpm for approximately 20 hours. 600 When the fermentation temperature reached 1 to 3°C, the mixture was transferred to a 50 mL Erlenmeyer flask containing 10 mL of SD / ΔTrp-Ura medium, covered with 20% n-dodecane (2 mL) as an organic phase, and fermented in a shaker at 25°C and 220 rpm for 96 hours.

[0080] After the fermentation was completed, 500 μL of the upper n-dodecane organic phase was taken and mixed with an equal volume of ethyl acetate, and the product was detected according to the gas phase detection method described in Example 4.

[0081] The final fermentation results showed a nootkatone yield of 24 mg / L, accounting for 20% of the total terpene yield (valencene, nootkatol, and nootkatone combined). When the oxidoreductase used was replaced with ABA2 derived from citrus fruits, the resulting nootkatone yield was only 2.5 mg / L, accounting for 2.6% of the total terpene yield. Thus, the use of the oxidoreductase described in this invention can improve the fermentation yield of nootkatone by 9.6-fold and its purity by 12-fold. Using an oxidoreductase mutant such as NRRL(cp):K69T-G136R, the resulting nootkatone yield increased to 42 mg / L, 1.75-fold higher than that of the wild-type oxidoreductase. The oxidoreductase described in this invention has been shown to have superior catalytic performance and tolerate higher substrate concentrations than previously reported isozymes. Therefore, the use of this oxidoreductase and its mutants may result in a nootkatone product with higher fermentation yield and purity.

[0082] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Various other changes, modifications, substitutions, combinations, simplifications, etc. that are made without departing from the spirit and principles of the present invention are all equivalent replacement methods, and all of these are intended to be included in the protection scope of the present invention.

[0083] (Addendum) (Appendix 1) Use of an oxidoreductase and / or a mutant thereof in the biosynthesis of nootkatone, The oxidoreductase has the name NRRL and the amino acid sequence shown in SEQ ID NO: 2, The use, characterized in that the amino acid sequence of the oxidoreductase mutant is mutated at one or both of the amino acid positions 69 and 136 of SEQ ID NO: 2.

[0084] (Appendix 2) The use of the oxidoreductase mutant described in Appendix 1 in improving substrate tolerance, conversion rate, and salt tolerance, wherein the substrate is nootkatol.

[0085] (Appendix 3) The use described in Appendix 1, characterized in that the amino acid sequence of the oxidoreductase mutant is mutated at one or both of the amino acids at positions 69 and 136 of SEQ ID NO: 2, with the amino acid at position 69 being mutated from lysine K to threonine T, and the amino acid at position 136 being mutated from glycine G to arginine R or alanine A.

[0086] (Appendix 4) The use described in Appendix 3, characterized in that the amino acid sequence of the oxidoreductase mutant is such that the amino acid at position 69 of SEQ ID NO: 2 is mutated from lysine K to threonine T and the amino acid at position 136 is mutated from glycine G to arginine R, and the specific amino acid sequence is shown in SEQ ID NO: 4.

[0087] (Appendix 5) The use according to any one of Appendices 1 to 4, wherein in the oxidoreductase mutant, the gene sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 3.

[0088] (Appendix 6) 5. The use according to any one of appendices 1 to 4, wherein the nucleotide sequence of the gene encoding the oxidoreductase NRRL is set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

[0089] (Appendix 7) An oxidoreductase mutant according to any one of appendices 1 to 5.

[0090] (Appendix 8) A gene encoding an oxidoreductase mutant described in Appendix 7.

[0091] (Appendix 9) A recombinant expression vector or recombinant expression cell containing the gene described in Appendix 8.

[0092] (Appendix 10) 10. Use of a recombinant expression vector or recombinant expression cell containing a coding gene according to Appendix 6, or a recombinant expression vector or recombinant expression cell according to Appendix 9, in the biosynthesis of nootkatone.

Claims

1. Use of an oxidoreductase and / or a mutant thereof in the biosynthesis of nootkatone, The oxidoreductase has the name NRRL and the amino acid sequence shown in SEQ ID NO: 2; The amino acid sequence of the oxidoreductase mutant is characterized in that one or both of the amino acids at positions 69 and 136 of SEQ ID NO: 2 are mutated, the mutation at position 69 being from lysine K to threonine T, and the mutation at position 136 being from glycine G to arginine R or alanine A.

2. The use of the oxidoreductase mutant according to claim 1, characterized in that the substrate is nootkatol, and the use of the oxidoreductase mutant is in improving substrate tolerance, conversion rate, and salt tolerance.

3. The use described in claim 1, characterized in that the amino acid sequence of the oxidoreductase mutant is such that the amino acid at position 69 of SEQ ID NO: 2 is mutated from lysine K to threonine T and the amino acid at position 136 is mutated from glycine G to arginine R, and the specific amino acid sequence is shown in SEQ ID NO:

4.

4. The use described in any one of claims 1 to 3, characterized in that the nucleotide sequence of the coding gene for the oxidoreductase NRRL is shown in SEQ ID NO: 3, the nucleotide sequence of the oxidoreductase mutant is identical to SEQ ID NO: 3 except for mutations of the amino acids at positions 69 and 136 of SEQ ID NO: 2, the codon used when the amino acid at position 69 of SEQ ID NO: 2 is mutated to threonine T is ACG, the codon used when the amino acid at position 136 is mutated from glycine G to arginine R is CGA, and the codon used when the amino acid at position 136 is mutated to alanine A is GCA.

5. The use according to any one of claims 1 to 3, characterized in that the nucleotide sequence of the coding gene for the oxidoreductase NRRL is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

6. A mutant of the oxidoreductase named NRRL and having the amino acid sequence shown in SEQ ID NO: 2, (1) the amino acid at position 69 of SEQ ID NO:2 is mutated from lysine K to threonine T, and the amino acid at position 136 is mutated from glycine G to arginine R or alanine A, or (2) The amino acid at position 69 of SEQ ID NO:2 is mutated from lysine K to threonine T, and the amino acid at position 136 is mutated from glycine G to arginine R, and the specific amino acid sequence is shown in SEQ ID NO:

4. An oxidoreductase mutant characterized in that:

7. A gene encoding the oxidoreductase mutant of claim 6.

8. A recombinant expression vector or recombinant expression cell containing the gene according to claim 7.

9. Use of a recombinant expression vector or recombinant expression cell containing the coding gene of claim 5, or the recombinant expression vector or recombinant expression cell of claim 8, in the biosynthesis of nootkatone.

Images