Use of oxidoreductase as naringenin dehydrogenase in biosynthesis of naringin

Abstract

The present invention discloses the application of a redox enzyme as a naringenin alcohol dehydrogenase in the biosynthesis of naringenin chalcone, belonging to the technical field of bioengineering. The enzyme is a redox enzyme YlADH_Bm6 derived from Yarrowia lipolytica CLIB122 (the amino acid sequence thereof is shown in SEQ ID No. 2) and a corresponding modified redox enzyme (the amino acid sequence thereof is shown in SEQ ID No. 3). Using the redox enzyme and the modified redox enzyme, together with their recombinant expression cells, they have a high tolerance to substrate concentration, a high conversion rate and mild reaction conditions; they also have excellent performance in the cell fermentation synthesis of naringenin chalcone, and a high yield of naringenin chalcone is obtained. The method for preparing naringenin chalcone according to the present invention is environmentally friendly and easy to operate. Therefore, the redox enzyme and the modified redox enzyme described in the present invention have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to an oxidoreductase or a modified oxidoreductase with an amino acid sequence shown in SEQ ID No.2 in the sequence listing and its recombinant expression cell as a nootkatol dehydrogenase for preparing nootkatone, and relates to a nucleic acid sequence encoding the enzyme, a recombinant expression vector containing the encoding nucleic acid sequence, and a method for preparing nootkatone. Background Art

[0002] Nootkatone, also known as nocardone, etc., is a natural sesquiterpene substance existing in citrus essential oils. It can be added as a flavor and fragrance in the processing of foods, perfumes, cigarettes, etc. At the same time, it has various industrial and medical uses such as insecticidal and cancer cell inhibitory effects. In its chemical synthesis methods, toxic and harmful oxidizing and reducing agents and organic reagents are often used; while the plant extraction methods are also often troubled by problems such as low effective concentration in the original plants, complex processes such as separation and purification, and susceptibility to seasonal and climate changes. Nowadays, the method of using organisms to synthesize nootkatone is becoming more and more popular.

[0003] However, the one-step enzymatic methods reported currently, such as using cytochrome CYP450 enzyme (Rebecca J.et al.Biotransformation ofthe sesquiterpene(+)-valencene by cytochrome P450 and P450[J].Organic&Biomolecular Chemistry,2004,3(1):57-64.) or lipoxygenase (Sven Krügener,et al.Adioxygenase of Pleurotus sapidus transforms(+)-valencene regio-specificallyto(+)-nootkatone via a stereo-specific allylic hydroperoxidation[J].Bioresource Technology,2010,101(2):457-462.) etc., to catalyze the precursor valencene to be transformed into the intermediate nootkatol and then further transformed into nootkatone, face problems such as many by-products, low yield and low conversion efficiency. And due to the lack of highly efficient nootkatol dehydrogenase, a large amount of the intermediate nootkatol accumulates and cannot be further dehydrogenated into a ketone, resulting in a still low biosynthesis yield of nootkatone. Therefore, it is crucial to find a dehydrogenase that can efficiently transform nootkatol into nootkatone.

[0004] However, some researchers found that overexpression of the endogenous ADH-C3 (NCBI accession number: XM_002492172) in Pichia pastoris significantly enhanced the conversion of nootkatol to nootkatone, and the production of nootkatone increased by approximately 20-fold (Wriessnegger, T. et al. Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris [J]. Metabolic Engineering, 2014, 24: 18-29.). Subsequently, it was also found that when the short-chain dehydrogenase BMD_2094 from Bacillus megaterium (NCBI accession number: WP_013082965) was applied to the whole-cell catalysis experiment of Bacillus megaterium, 0.2 mM nootkatol could be efficiently and selectively converted to nootkatone within 40 minutes (Milhim M, Hartz P, Gerber A, et al. A novel short chain dehydrogenase from Bacillus megaterium for the conversion of the sesquiterpene nootkatol to (+)-nootkatone [J]. Journal of Biotechnology, 2019, 301: 52-55.). This demonstrated that it was feasible to use dehydrogenases to solve the problem of low nootkatone production caused by the accumulation of the intermediate nootkatol. ZSD1 from Zingiber zerumbet (NCBI accession number: AB480831) and csABA2 from Citrus sinensis (NCBI accession number: HM036684), the former was reported to catalyze the formation of zerumbone from sesquiterpenoid substances, and the latter could catalyze xanthoxin to produce the sesquiterpenoid abscisic acid (ABA). The authors attempted to use both of them in the reaction of catalyzing nootkatol to nootkatone.The results also showed that these two short-chain dehydrogenases from plants can indeed convert nootkatone into nootkatone, and the effect is slightly better than ADH-C3 from Pichia pastoris. The production of nootkatone reached 59.78 mg / L and 53.48 mg / L, respectively (Meng, X. et al. Metabolic engineering Saccharomyces cerevisiae for de novo production of thesesquiterpenoid (+)-nootkatone [J]. Microb Cell Fact, 2020, 19: 21.).

[0005] However, the concentration of naringenol that can be catalyzed by the currently reported naringenol dehydrogenase is still relatively low, and the highest biosynthetic yield of naringenone in situ is only 207 mg / L, which is not suitable for future industrial production. Therefore, it is still necessary to find naringenol dehydrogenase with better performance such as high substrate tolerance, strong specific conversion ability, and high conversion efficiency to meet the needs of large-scale industrial biosynthesis in the future.

[0006] NCBI has included an oxidoreductase YlADH_Bm6 from Yarrowia lipolytica CLIB122, formerly known as YALI0D05929p (NCBI accession number XP_502463), whose amino acid sequence is shown in SEQ ID No. 2, and contains 248 amino acids in total; the nucleotide sequence of its coding gene (NCBI accession number XM_502463) is shown in SEQ ID No. 1, and contains 747 nucleotides in total. So far, no literature has reported any actual function and application of this alcohol dehydrogenase. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, one of the objects of the present invention is to provide an oxidoreductase used as a grapefruit alcohol dehydrogenase in the biosynthesis of grapefruit ketone.

[0008] The technical problem to be solved by the present invention is to provide a novel oxidoreductase in view of the defects of low substrate concentration and low conversion rate that can be tolerated by the existing alcohol dehydrogenase in the current biosynthesis of nutmeg ketone, which leads to low biosynthesis yield of nutmeg ketone and is not suitable for future industrial production. In the process of catalyzing the conversion of nutmeg alcohol to nutmeg ketone, the novel oxidoreductase exhibits the advantages of high substrate tolerance, strong specific conversion ability and high conversion rate; and by modifying the enzyme with amino acids, the oxidoreductase with further improved catalytic performance is obtained.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides an application of an oxidoreductase in the biosynthesis of nootkatone; meanwhile, a modified oxidoreductase is also provided, which, as nootkatol dehydrogenase, has better catalytic performance in the application of the biosynthesis of nootkatone.

[0011] In the present invention, the oxidoreductase is YlADH_Bm6 derived from Yarrowia lipolytica CLIB122, and its encoding gene is described as YALI0D05929p, with the accession number XP_502463 in NCBI. Its amino acid sequence is as shown in SEQ ID No.2, and it contains a total of 248 amino acids.

[0012] In the present invention, the nucleotide sequence of the encoding gene of the oxidoreductase YlADH_Bm6 (with the accession number XM_502463 in NCBI) is as shown in SEQ ID No.1, and it contains a total of 747 nucleotides.

[0013] The present invention also provides a modified oxidoreductase, whose amino acid sequence is as shown in SEQ ID No.3, wherein at least one position marked as "X" in SEQ ID No.3 is different from the amino acid residue at the corresponding position of SEQ ID No.2; this modified oxidoreductase has higher conversion efficiency and substrate tolerance;

[0014] Preferably, the amino acid sequence of the modified oxidoreductase has an amino acid sequence with 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.2.

[0015] In the present invention, a recombinant expression vector containing the above-mentioned oxidoreductase or the encoding gene of the modified oxidoreductase is provided.

[0016] In the present invention, a recombinant expression transformant containing the recombinant expression vector of the oxidoreductase or the modified oxidoreductase gene of the present invention is provided.

[0017] In the present invention, a method for preparing nootkatone is also involved.

[0018] The conversion efficiency described in the present invention is expressed as the ratio of converting the precursor nootkatol into the target product nootkatone within a certain period of time.

[0019] According to the present invention, it is found that the oxidoreductase shown in SEQ ID No.2 has good efficiency in converting nomilinolide to nomilin. Compared with the two nomilinolide dehydrogenases, namely ADH-C3 from Pichia pastoris (NCBI accession number XM_002492172) or BMD_2094 from Bacillus megaterium (NCBI accession number WP_013082965), which have been discovered, the effect is significantly better. Particularly, such a difference can be more evident at around neutral pH or in the in vitro determination of the conversion of nomilinolide to nomilin in recombinant expression cells. The results show that under the same conditions, the target conversion rate of the substrate obtained by using the oxidoreductase of the present invention is higher, which is nearly 1 time and 3 times that of ADH-C3 from Pichia pastoris or BMD_2094 from Bacillus megaterium, respectively.

[0020] In addition, the modified oxidoreductase provided by the present invention can have an efficiency of converting nomilinolide that is at least 1.04 times, preferably 2 to 10 times, particularly 2 to 5 times that of the oxidoreductase shown in SEQ ID No.2 of the sequence listing under the same conditions.

[0021] The term "modification" or "mutation" is used to indicate that at least one nucleotide or amino acid in the gene or amino acid sequence is different from the wild-type sequence. Specifically, it can be that one nucleotide or amino acid is respectively replaced, deleted, or inserted by a different nucleotide or amino acid. The modification or mutation of the enzyme can be achieved by conventional methods in the art, such as error-prone PCR, site-directed mutagenesis, mutagenesis and other technical means.

[0022] Compared with the oxidoreductase shown in SEQ ID No.2 of the sequence listing, the modified oxidoreductase provided by the present invention has better substrate tolerance.

[0023] Substrate tolerance indicates that in the presence of a high concentration of substrate, the enzyme can still maintain good activity and a conversion efficiency of more than 60%. The modified oxidoreductase provided by the present invention can have a substrate tolerance to nomilinolide that is at least 1.5 times, preferably 2 to 10 times, without an upper limit, that of the oxidoreductase shown in SEQ ID No.2 of the sequence listing under the same conditions. In a preferred embodiment, the substrate tolerance of the provided modified oxidoreductase is increased by 5 times.

[0024] The inventors have discovered modified oxidoreductases of the present invention that are different from the oxidoreductase shown in SEQ ID No. 2 and have higher conversion efficiency, substrate tolerance, etc. The modified oxidoreductases of the present invention can differ in that it contains a modification at only one amino acid position or in that it contains two or more modifications, and one or more of the modifications can particularly be one or more substitutions, and may also be the absence of one or more amino acids. For example, one or more amino acids at positions 1-15 or positions 1-16 of SEQ ID No. 2 may be absent.

[0025] It has been particularly found that mutating at at least one of the following positions in SEQ ID No. 2 is beneficial for obtaining oxidoreductases with improved conversion efficiency, substrate tolerance, etc.: 88, 89, 143, 152, 155, 157, 159, 161, 196, and 210.

[0026] In a preferred embodiment, the modified oxidoreductase of the present invention comprises the amino acid sequence shown in Sequence Listing SEQ ID No. 3. Here, the positions marked with "X" can in principle contain any amino acid residue, provided that preferably at least one amino acid residue is different from the corresponding amino acid residue in SEQ ID No. 2.

[0027] In a specific embodiment, the modified oxidoreductase with improved conversion efficiency contains a modification at one or more positions aligned with 88, 89, 143, 152, 155, 157, 159, 161, 196, and 210 as compared with SEQ ID No. 2.

[0028] In a preferred embodiment, the modified oxidoreductase with increased conversion efficiency contains a modification at one or more positions aligned with 88, 89, 152, 155, 157, 161, 196, and 210 as compared with SEQ ID No. 2.

[0029] In a particularly preferred embodiment, the modified oxidoreductase has one or more modifications selected from the following: 88A, 88C, 88T, 89G, 89V, 143V, 143L, 152A, 152V, 152M, 152P, 155M, 157G, 157L, 159C, 161Y, 161R, 196S, 196N, 196G, 196Y, 210R, and 210L.

[0030] Specifically, good results have been achieved with one or more modified oxidoreductases selected from the following: 88A, 88C, 88T, 89G, 89V, 143V, 143L, 152A, 152V, 152M, 152P, 155M, 157G, 157L, 159C, 161Y, 161R, 196S, 196N, 196G, 196Y, 210R and 210L.

[0031] Particularly high conversion efficiencies have been observed for oxidoreductases having at least one modification selected from the following: 88A, 88C, 89V, 152A, 152V, 152M, 155M, 196S and 196N.

[0032] Compared to SEQ ID No. 2, preferred examples of oxidoreductases comprising at least two modifications are those having at least two modifications selected from 88A, 88C, 89V, 152A, 152V, 152M, 155M, 157G, 196S, 196N and 210R. Specifically, good results have been achieved with oxidoreductases comprising at least two modifications compared to SEQ ID No. 2, wherein the at least two modifications are selected from modifications at at least two positions corresponding to 88, 89, 152, 155, 157, 196, 210, and these modifications can particularly be selected from the substitutions indicated as excellent above.

[0033] Although good results have been obtained with oxidoreductases having only one or two modifications compared to SEQ ID No. 2, the modified oxidoreductases of the present invention can comprise more modifications, particularly 3 or more. In principle, there is no limit to the number of modifications, provided that the enzyme retains sufficient catalytic performance as a yuzu alcohol dehydrogenase.

[0034] As used herein, the term "vector" refers to a structure composed of genetic material that is involved in directly transforming the targeted cells. The vector contains a plurality of genetic elements that are oriented in position and order, i.e., operably linked to other necessary elements, so that the nucleic acid in the nucleic acid cassette can be transcribed and, when needed, translated in the transformed cells.

[0035] In the present invention, a recombinant expression vector comprising the nucleic acid of the invention is provided. It can be constructed by connecting the nucleotide sequence of the oxidoreductase or modified oxidoreductase encoding gene of the present invention to various suitable vectors by conventional methods in the art. The vector can be various conventional vectors in the art, such as commercially available plasmids, cosmids, phage or viral vectors. The alcohol dehydrogenase gene can be ligated and expressed with a suitable regulatory sequence to achieve constitutive or inducible expression of the alcohol dehydrogenase. The present invention preferably uses the 2μ type high-copy plasmid YEp352.

[0036] In the present invention, a recombinant expression cell containing the vector of the present invention is also provided. The recombinant expression cell can be prepared by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated and passed on, and the gene carried by it can be effectively expressed. The host cell can be from any organism, and specifically can be selected from prokaryotic cells, eukaryotic cells, archaea, protists, plant cells (including algae), cells derived from animals. Saccharomyces cerevisiae is preferably used in the present invention, and Saccharomyces cerevisiae CEN.PK2-1 Ca strain is more preferably used.

[0037] In the present invention, two methods are provided for realizing the biosynthesis of nootkatone by using recombinant expression cells containing the recombinant expression vector of the oxidoreductase gene of the present invention.

[0038] One is the whole-cell in vitro catalysis method, that is, by adding the precursor nootkatol externally and using the recombinant expression cells of the present invention for in vitro transformation to obtain nootkatone. The reaction substrate is nootkatol, and nootkatol can be obtained by chemical synthesis, plant extraction or biosynthesis. Specific reaction conditions such as substrate concentration, pH, buffer composition, dosage of recombinant expression transformant, etc. can be selected according to the conventional conditions of such reactions in the art. The biotransformation reaction can be carried out under shaking or stirring conditions. The time of the biotransformation reaction is preferably based on a conversion rate > 98%. After the reaction is completed, the nootkatone product can be extracted from the reaction mixture according to the conventional methods in the art. In a specific embodiment, it can be observed that the recombinant expression cells have good conversion efficiency for nootkatol, and the obtained nootkatone has a high yield and high purity.

[0039] In another method, nootkatone is prepared by bioconversion (that is, by culturing recombinant expression cells expressing related enzymes in a reactor containing a culture medium).

[0040] By utilizing the mevalonate pathway or another metabolic pathway (such as the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway) of the host cell itself, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are C5 isoprenoid structural units, can be obtained. So far, it is known that unless specific genes have been knocked out, known organisms all contain this pathway. Eukaryotes can usually naturally prepare IPP via the mevalonate pathway, and IPP is then isomerized to DMAPP under the action of isopentenyl diphosphate isomerase (Idi). Prokaryotes rely on the DXP pathway, which supplies IPP and DMAPP in a ratio of 5:1.

[0041] The host cell further utilizes the endogenous isoprenoid synthase (IspA) to continuously add IPP / DMAPP based on the active isopentenyl diphosphate (IPP) unit to form geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranyl farnesyl pyrophosphate (GFPP), etc., thereby obtaining precursor isoprenoids such as monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes. Subsequently, through the catalytic reaction of heterologous or exogenous terpenoid synthases (TS) or cyclases, the main terpene skeletons are generated, and then modified by different functional enzymes (such as hydroxylases, dehydrogenase reductases, and glycosyl, methyl, and acyl transferases, etc.) to produce thousands of derivatives with different structures, jointly constituting tens of thousands of terpene compounds with different functions.

[0042] The host cells used can be selected from prokaryotes, eukaryotes, plants, etc. In principle, as long as the host cell type can ensure the normal operation of the relevant functional proteins, it can be used for the cell fermentation preparation of nootkatone.

[0043] In principle, the reactors used are selected from various commonly used devices in this field that can ensure the growth of host cells and the production of products, such as small shake flasks, fermenters, and other devices.

[0044] The culture medium used is mainly the commonly used one in this field that is suitable for the growth of host cells.

[0045] In principle, it is preferred to select the fermentation temperature at which the relevant enzymes (in the cells) show good activity. In a specific embodiment, the preferred fermentation temperature is 25°C to reduce the volatilization of the nootkatone precursor valencene.

[0046] If necessary, the nootkatone or its precursor substances valencene, nootkatol, etc. produced in the method of the present invention can be extracted by liquid-liquid extraction with a liquid immiscible with the reaction solution or fermentation broth.

[0047] Specifically, it is suitable to use a liquid organic solvent, such as liquid hydrocarbons, for extraction (from an aqueous reaction medium). From the preliminary results, it is obvious that this method is also suitable for extracting nootkatone (or other products) from the cell fermentation broth of the present invention containing the product nootkatone without the need to lyse the cells to recover nootkatone (or other products). In particular, the organic solvent can be selected from liquid alkanes, liquid long-chain alcohols (alcohols having at least 12 carbon atoms), and liquid esters of long-chain fatty acids (acids having at least 12 carbon atoms). Suitable liquid alkanes particularly include C6-C 16 alkanes, such as hexane, octane, decane, dodecane, isododecane, and hexadecane. Suitable long-chain fatty alcohols particularly include C 12 -C 18 fatty alcohols, such as oleyl alcohol and palmityl alcohol. Suitable esters of long-chain fatty acids particularly include C 12 -C 18 C1-C4 alcohol esters of fatty acids, such as isopropyl myristate and ethyl oleate.

[0048] In a preferred embodiment, nootkatone (or other products) is produced in a reactor. The reactor contains a first liquid phase (reaction phase) and a second liquid phase (an organic phase that remains substantially phase-separated from the first phase when contacted). The first liquid phase contains the host cells of the present invention in which nootkatone (or other products) is produced; the second liquid phase is an extraction phase that has a higher affinity for the formed product. This method is beneficial for in-situ recovery of the product. In addition, it helps to prevent or at least reduce the potential toxic effects of nootkatone (or other products) on the cells, because under the extraction of the second liquid phase, the concentration of the product in the reaction phase can be maintained at a relatively low level. At the same time, nootkatone (or other products) has low solubility in water and is therefore easily volatilized from water, and the presence of the second liquid phase can avoid this loss.

[0049] In a preferred embodiment, the host cell is a eukaryotic cell, particularly selected from the Saccharomyces cerevisiae CEN.PK2-1 Ca strain (Saccharomyces cerevisiae CEN.PK2-1 Ca). The genome of this host cell has been modified using the technology described in patent application No. 201910271558.6, including knocking out the rox1, a limiting factor of the mevalonate pathway, and downregulating the expression intensity of erg9, an enzyme related to the downstream branch pathway of the sesquiterpene precursor FPP, to increase the supply of the precursor substance FPP. The valencene synthase ValC from Chamaecyparis nootkatensis described in international patent application No. PCT / NL2010 / 050848 was used to convert FPP into the valencene, a precursor substance of nootkatone. Subsequently, the cytochrome P450 monooxygenase (CYP450) HPO from Hyoscyamus muticus described in patent WO2006 / 079020 and the cytochrome reductase AtCPR from Arabidopsis thaliana described by Urban, Pd et al. in their 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 P450 CYP73A5. J. Biol. Chem. 1997, 272, 19176–19186.) were used to further oxidize valencene to nootkatol. Then, the nootkatol was converted into the final desired product nootkatone using the oxidoreductase or the modified oxidoreductase described in the present invention. In the final fermentation product, the yield and purity of nootkatone are both high.

[0050] The present invention has the following advantages and effects compared with the prior art:

[0051] (1) The present invention provides an oxidoreductase derived from Yarrowia lipolytica CLIB122 and the corresponding modified oxidoreductase. The enzyme or the recombinant expression cell containing its gene recombinant expression vector can efficiently catalyze the dehydrogenation of nootkatol to obtain a high-value nootkatone product.

[0052] (2) Compared with other nootkatol dehydrogenases, the use of the oxidoreductase and the modified oxidoreductase of the present invention, and their recombinant expression cells, can tolerate high substrate concentrations, have high conversion rates and mild reaction conditions; they also have excellent performance in the cell fermentation synthesis of nootkatone, and a relatively high nootkatone yield is obtained. The method for preparing nootkatone of the present invention is environmentally friendly and easy to operate. Therefore, the oxidoreductase and the modified oxidoreductase of the present invention have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the oxidoreductase or the modified oxidoreductase catalyzing nootkatol to produce nootkatone.

[0054] Figure 2 It is a construction pattern diagram of the oxidoreductase or the modified oxidoreductase gene recombinant plasmid in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0055] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0056] For the test methods without specific experimental conditions noted in the following examples, they are generally carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0057] The schematic diagram of the oxidoreductase or the modified oxidoreductase catalyzing nootkatol to produce nootkatone in the embodiment is as Figure 1 shown.

[0058] S.cerevisiae CEN.PK2-1 Ca and S.c.PK2-M used in the embodiments are both disclosed in "CN201910271558 - Saccharomyces cerevisiae engineering bacteria for producing valencene, and construction method and application thereof".

[0059] The oxidoreductase YlADH_Bm6 in the embodiment, originally named YALI0D05929p, has a GenBank accession number of XP_502463 in NCBI. Its amino acid sequence is as shown in SEQ ID No.2, and it contains a total of 248 amino acids. The nucleotide sequence of the encoding gene (GenBank accession number of XM_502463) is as shown in SEQ ID No.1, and it contains a total of 747 nucleotides.

[0060] Example 1: Obtaining of the oxidoreductase YlADH_Bm6 gene

[0061] The Yarrowia lipolytica CLIB122 strain was purchased from ATCC (The Global Bioresource Center), and its ATCC number is MYA-2613.

[0062] The Yarrowia lipolytica CLIB122 was inoculated into 5 mL of YPD liquid medium and cultured at 30 °C until the logarithmic growth phase. The total genomic DNA was extracted from Yarrowia lipolytica using a genomic DNA extraction kit.

[0063] Take 0.5 μL (about 10 ng) of the total DNA solution described above as a template, use the following oligonucleotide sequences as primers, and perform 32 cycles of PCR according to the PCR cycle parameters set below. The PCR enzyme used is KOD FX (purchased from TOYOBO, Japan) to amplify the target gene fragment, that is, the YlADH_Bm6 gene fragment of oxidoreductase.

[0064] The set PCR cycle parameters are as follows:

[0065] 95 °C, 5 min; 95 °C, 30 s; 55 °C, 15 s; 72 °C, 1 min; 72 °C, 5 min, 32 cycles; 4 °C, 2 h.

[0066] The primer sequences are as follows (the bold ones are homologous arm sequences):

[0067] Forward primer F1: 5’-

[0068] Reverse primer R1: 5’-

[0069] Example 2: Construction of recombinant expression vector

[0070] The genome of Saccharomyces cerevisiae CEN.PK2-1 Ca was extracted using the Yeast DNAKit kit (purchased from Omega). Using the genome as a template, the TDH3 promoter fragment was amplified with the TDH3-F / TDH3-R primer pair, and the ADH1 terminator fragment was amplified with the ADH1-F / ADH1-R primer pair.

[0071] The primer sequences used are as follows:

[0072] TDH3-F: 5’-TACGAATTCTTTACCGTCGACACAGTTTATTCCTGGCAT-3’;

[0073] TDH3-R: 5’-GAAGTCCAAAGCTCCCGGGTTGTTTGTTTATGTGTGTT-3’;

[0074] ADH1-F: 5’-AAACACACATAAACAAACAACCCGGGAGCTTTGGACT-3’;

[0075] ADH1-R: 5’-GCAGGTCGACTCTAGAGGATCCCATAGGGTAGGGGAA-3’;

[0076] The YEp352 plasmid vector fragment purchased from Invitrogen was amplified using the 352-F / 352-R primer pair.

[0077] 352-F: 5’-GATCCTCTAGAGTCGACCTGC-3’;

[0078] 352-R: 5’-GTCGACGGTAAAGAATTCGTAATC-3’;

[0079] The obtained TDH3 promoter fragment, ADH1 terminator fragment and YEp352 plasmid vector fragment were ligated by homologous recombination using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novoprotein Co., Ltd.) to obtain the vector YEp352-TDH3 promoter -ADH1 terminator , abbreviated as YEp352-TDH3 p -ADH1 t .

[0080] YEp352-TDH3 p -ADH1 t, and then the vector fragment amplified by the upstream primers F2 / R2 and the target gene obtained in Example 1 were ligated by homologous recombination using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novoprotein Co., Ltd.). Then, 10 μL of the ligation product was added to 20 μL of E. coli DH5α competent cells. After placing on ice for 30 minutes, it was heat-shocked at 42 °C in a water bath for 90 seconds, and then quickly placed on ice for 2 minutes. Then, 1 mL of LB liquid medium without ampicillin resistance was added, and it was incubated at 37 °C and 220 rpm for 1 hour. 200 μL of the bacterial solution was taken and spread on an LB plate containing 100 μg / mL ampicillin. After culturing at 37 °C for 12 to 16 hours, the recombinant bacterium E. coli (YEp352-TDH3 p -YlADH_Bm6-ADH1 t ) was obtained. The construction map is shown in Figure 2 . A single colony of the recombinant bacterium was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37 °C and 220 rpm for 12 hours. Then, the plasmid was extracted using a Quick Plasmid Mini Kit (purchased from Tiangen (Beijing) Co., Ltd.) to obtain the recombinant plasmid YEp352-TDH3 containing the oxidoreductase gene p -YlADH_Bm6-ADH1 t .

[0081] Example 3: Construction of Recombinant Expression Cells

[0082] Using the S.c EasyComp Transformation Kit (purchased from Invitrogen, USA), the recombinant expression vector YEp352-TDH3 obtained in Example 2 p -YlADH_Bm6-ADH1 t was transformed into the competent cells of Saccharomyces cerevisiae S.cerevisiae CEN.PK2-1 Ca. Then, 200 μL of the transformation solution was spread on an SD plate lacking uracil and cultured at 30 °C for 2 - 3 days to obtain the recombinant expression cell Saccharomyces cerevisiae S.c.CEN.PK2-1 Ca (YEp352-TDH3 p -YlADH_Bm6-ADH1 t ).

[0083] Example 4: Preparation of Nootkatone by Whole-Cell In Vitro Catalysis

[0084] A single colony of the recombinant expression cell Saccharomyces cerevisiae S.c.CEN.PK2-1 Ca (YEp352-TDH3 p -YlADH_Bm6-ADH1 t) to the SD liquid medium lacking uracil, and cultured at 30 °C and 220 rpm for 24 hours. Then, inoculate at an initial OD 600 = 0.05 into a 250 mL shake flask containing 50 mL of fresh culture medium, and culture at 30 °C and 220 rpm for 24 hours. Subsequently, collect the bacterial liquid with a total OD 600 of 50, centrifuge at 3000 rpm and 4 °C for 5 minutes, and discard the supernatant. Then, resuspend the recombinant expression cells with potassium phosphate buffer (50 mM, pH 7.4) and make the volume up to 1 mL to obtain a reaction solution of 50 OD 600 / mL. Add 20 μL of 100 mM naringenin solution (containing 1% (v / v) Triton-100, dissolved in dimethyl sulfoxide) to make the final concentration of the substrate 2 mM, and catalyze at 25 °C and 220 rpm for 24 hours.

[0085] The product detection method is as follows (the product detection method in the subsequent examples is the same as that in Example 4):

[0086] After the reaction is completed, transfer the reaction solution to a 2 mL centrifuge tube, add 1 mL of ethyl acetate and shake for extraction for 10 minutes, then centrifuge at the maximum speed for 5 minutes. After separating the organic layer and the aqueous layer, take 500 μL of the upper ethyl acetate layer into a 1.5 mL centrifuge tube, and then add 500 μL of ethyl acetate. Finally, filter through a 0.22 μm organic filter membrane into a chromatographic vial for gas phase detection. The gas phase used is Shimadzu, the chromatographic column is a 5% Ph-Me silicone column, with a specification of 30 m × 0.10 mm × 0.10 μm; the detector is a flame ionization detector (FID); the carrier gas is N2.

[0087] The detection method is as follows: Inject 1 μL of the sample in split injection, with a split ratio of 15:1; the injection port temperature is 250 °C; the detector temperature is 350 °C; the column temperature is maintained at 100 °C for 5 minutes, then heated to 200 °C at a rate of 20 °C / min, and maintained at 200 °C for 5 minutes, with a total time of 15 minutes.

[0088] Finally, the recombinant expression cells S.c.CEN.PK2-1 Ca(YEp352-TDH3 p -YlADH_Bm6-ADH1 t ) of the oxidoreductase has a conversion rate of 100% for naringenin.

[0089] Similarly, according to the methods described in Examples 1 to 4, the present invention also constructed recombinant expression cells S.c.CEN.PK2-1 Ca(YEp352-TDH3 p -ADH-C3-ADH1 t) and the recombinant expression cell S.c.CEN.PK2-1 Ca(YEp352-TDH3 of the pomelo alcohol dehydrogenase BMD_2094 (NCBI accession number: WP_013082965) derived from Bacillus megaterium p -BMD_2094-ADH1 t ), and their catalytic abilities were tested under the same conditions as above. The results were 50% and 31% of the conversion effects described in Example 4 respectively. The recombinant expression cell S.c.CEN.PK2-1 Ca(YEp352-TDH3 p -YlADH_Bm6-ADH1 t ) of the oxidoreductase of the present invention had catalytic efficiencies 2 times and 3.2 times those of the corresponding recombinant expression cells of ADH-C3 and BMD_2094 respectively under the same conditions, showing good pomelo alcohol conversion ability of the oxidoreductase of the present invention.

[0090] Example 5: Testing the catalytic efficiency and substrate tolerance of the modified oxidoreductase

[0091] To test the catalytic efficiency and substrate tolerance of the modified oxidoreductase, using the same methods as in Examples 1 to 4, after cloning the gene encoding the modified oxidoreductase into a recombinant expression vector, it was transformed into the competent cells of Saccharomyces cerevisiae S.c.CEN.PK2-1 Ca to obtain recombinant expression cells. Then, the whole-cell in vitro catalysis method of Example 4 was used to test the conversion efficiency and substrate tolerance of the corresponding modified oxidoreductase.

[0092] The in vitro test results of some oxidoreductases with single amino acid modifications are shown in Table 1.

[0093] Table 1: Summary of the catalytic abilities of oxidoreductases with single modifications compared with SEQ ID NO.2

[0094]

[0095] a : Position of the amino acid residue of the modified oxidoreductase; residue numbering starts from the N-terminal methionine residue (=Met-1) in SEQ ID NO.2.

[0096] b : The test method refers to Example 3 or 4, with the difference being the different concentrations of the pomelo alcohol substrate input.

[0097] c: The conversion efficiency of the modified oxidoreductase (= sample) relative to that of the wild-type oxidoreductase (= control), i.e., {(Nootkatone[sample] / substrate concentration[sample]) / (Nootkatone[control] / substrate concentration[control])×100%}.

[0098] d : The tolerance concentration at which the enzyme can still maintain a conversion rate of more than 60% under test conditions.

[0099] e : Wild-type oxidoreductase (SEQ ID NO.2).

[0100] f : () The codons used when mutated to the corresponding amino acids are shown in parentheses.

[0101] The results in Table 1 show that the oxidoreductases with single-point modifications achieved significantly improved in vitro nootkatone conversion efficiency and substrate tolerance. Compared with the wild-type oxidoreductase in this test system, the conversion efficiency of these mutant oxidoreductases was 104% (such as YlADH_Bm6: N89A) to 260% (such as YlADH_Bm6: T152M), and the substrate tolerance capacity ranged from 4 mM (such as YlADH_Bm6: N89A) to 6 mM (such as YlADH_Bm6: G88A). For many of the tested modified oxidoreductases, this increase in conversion efficiency often occurred together with an increase in substrate tolerance, thus indicating the fact that the increase in conversion efficiency is not mainly caused by the increased expression of these mutants in Saccharomyces cerevisiae.

[0102] Table 2: Summary of the catalytic capabilities of oxidoreductases with two-point modifications compared with SEQ ID NO.2 (The meaning of the title is shown in Table 1)

[0103]

[0104] The results in Table 2 demonstrate that the oxidoreductases with at least two-point modifications also achieved significantly improved in vitro nootkatone conversion efficiency and substrate tolerance. Compared with the wild-type oxidoreductase, the conversion efficiency of these modified oxidoreductases was 115% (YlADH_Bm6: N89V-V157G) to 312% (YlADH_Bm6: G88C-T152M), and the substrate tolerance capacity ranged from 4 mM (such as YlADH_Bm6: N89V-V157G) to 10 mM (YlADH_Bm6: G88C-T152M).

[0105] The present invention further provides several particularly preferred redox enzymes with multi-point modifications, including YlADH_Bm6: G88C-N89V-T152M (conversion efficiency 290%, substrate tolerance 8 mM), YlADH_Bm6: G88C-T152V-T196S (conversion efficiency 270%, substrate tolerance 8 mM), YlADH_Bm6: T152M-L155M-T196S (conversion efficiency 310%, substrate tolerance 8 mM), YlADH_Bm6: G88C-T152M-T196S (conversion efficiency 446%, substrate tolerance 10 mM), YlADH_Bm6: G88C-N89V-T152M-T196S (conversion efficiency 431%, substrate tolerance 10 mM), etc. Compared with the wild-type redox enzyme, the conversion efficiency of these modified redox enzymes ranges from 270% (YlADH_Bm6: G88C-T152V-T196S) to 446% (such as YlADH_Bm6: G88C-T152M-T196S), and the substrate tolerance ability ranges from 8 mM (such as YlADH_Bm6: G88C-N89V-T152M) to 10 mM (such as YlADH_Bm6: G88C-T152M-T196S).

[0106] The modified redox enzyme described in the present invention has obtained higher conversion efficiency and substrate tolerance compared with the wild-type redox enzyme through single-point or multi-point modification, and has good industrial application prospects.

[0107] Example 6: Preparation of nootkatone using a simple carbon source by biological fermentation

[0108] Using the technology described in Patent Application No. 201910271558.6, the genome of the host cell S.c. CEN.PK2-1 Ca was modified, that is, the limiting factor rox1 of the mevalonate pathway was knocked out, and the expression intensity of the enzyme erg9 related to the downstream branch pathway of the sesquiterpene precursor FPP was down-regulated to increase the supply of the precursor substance FPP, and finally the S.c. PK2-M strain was obtained. And the valencene synthase ValC gene (NCBI accession number JX040471) derived from Chamaecyparis nootkatensis described in International Patent Application No. PCT / NL2010 / 050848 (the promoter in the expression cassette of this gene is PDC1, and the terminator is SAG1) and the endogenous HMG-CoA reductase tHMG1 of Saccharomyces cerevisiae with the N-terminal regulatory region truncated (the rate-limiting step enzyme of the mevalonate pathway) gene (NCBI accession number NM_001182434) (the promoter in the expression cassette of this gene is TEF1, and the terminator is CYC1) were introduced into the recombinant expression vector YEp352-TDH3 obtained in Example 2 p-YlADH_Bm6-ADH1 t A recombinant expression vector YEp352-ValC-tHMG1-YlADH_Bm6 was obtained. In addition, TEF1p-Cas9-CYC1t in the p414-TEF1p-Cas9-CYC1t vector (a commercial plasmid from Addgene) was replaced with the cytochrome P450 monooxygenase HPO gene (NCBI accession number EF569601) from the pure Hyoscyamus muticus described in Patent WO 2006 / 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 by Urban, Pd et al. in the 1997 article (the promoter in the expression cassette of this gene is HXT7 and the terminator is TPI1) (Urban, P., et al. 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.). A recombinant expression vector p414-HPO-AtCPR was obtained. The recombinant expression vectors YEp352-ValC-tHMG1-YlADH_Bm6 and p414-HPO-AtCPR were co-transformed into the previously constructed high-yield FPP engineering strain S.c. PK2-M to obtain a recombinant expression cell S.c. PK2-M (YEp352-ValC-tHMG1-YlADH_Bm6 and p414-HPO-AtCPR), abbreviated as "PK2-6tV-HA".

[0109] The PK2-6tV-HA strain was inoculated into a test tube containing 5 mL of SD / ΔTrp-Ura medium (SD medium lacking tryptophan (Trp) and uracil (Ura)) and cultured on a shaker at 30 °C and 220 rpm for about 20 hours until the OD 600 reached 1 - 3, then transferred to a 50 mL conical flask containing 10 mL of SD / ΔTrp-Ura medium and covered with 20% n-dodecane (2 mL) organic phase, and fermented on a shaker at 25 °C and 220 rpm for 96 hours.

[0110] After the fermentation was completed, 500 μL of the upper-layer n-dodecane organic phase was taken, mixed with an equal volume of ethyl acetate, and the product was detected according to the gas-phase detection method described in Example 4.

[0111] In the final fermentation result, the yield of nootkatone was 35 mg / L, accounting for 40.1% of the total terpene (the sum of valencene, nootkatol, and nootkatone) yield. When the used oxidoreductase was replaced with ADH-C3 derived from Pichia pastoris, the obtained nootkatone yield was only 2.5 mg / L, accounting for 2.6% of the total terpene yield. That is to say, after using the oxidoreductase described in the present invention, the fermentation yield of nootkatone can be increased by 13 times and the purity can be increased by 15 times. By continuing to use the modified oxidoreductase, such as YlADH_Bm6:G88A, the obtained nootkatone yield was increased to 50 mg / L, which was 1.4 times that of using the wild-type oxidoreductase. It was proved that the catalytic performance of the oxidoreductase described in the present invention was better than that of the reported isoenzymes in the past, and the substrate concentration that it could tolerate was also higher. Using the oxidoreductase and its mutants described above, a higher fermentation yield of nootkatone and a product with higher purity can be obtained.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. Sequence Listing <110> South China University of Technology <120> Application of Oxidoreductase as Nootkatol Dehydrogenase in Biosynthesis of Nootkatone <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of Oxidoreductase YlADH_Bm6 Encoding Gene <400> 1 atgggagaac tcagcggaaa aacagcggtt gtttcgggcg gctcgggcgg cattggattt 60 gccatctgta ccaagtttgc ccaaaatggc gccaaagtga tcattctgga ctacaacaag 120 gaaacccttg atgaaatgct gcccaagctg gtggcgcccg agggccagaa gcacgagggc 180 cacttctacg atgtgaccaa gaacgaccga ccccccgtgg acttctccaa agtcgacatt 240 ctgctcaacg gagccggaat aggaaatgga ggcttcctgg agggcatgga aaccagcctg 300 attgagcgaa tcatcgccac caacctcacg ggcgtcatca agctcaccaa atacgccatg 360 gaggcgtggt tcgagcggga agactctcgg accaaggccc agggcaatgg tgtggtcatc 420 aacatctcgt ccattctggg actcagagca gttactccgg ccctcactgt gtactcggcg 480 tccaagggtg gaatcatcat gttcaccaaa gccctggcca tcgaaggagg agcccatgcc 540 atccgagcca acgccatctg ccccggatac gtcagaactg ccatgaccga gttcatggag 600 ctgccggaac catcgccgtt ccaggaaaag gatgacaacg gagacgtgga caaggaatcc 660 attgccggag ctgcctatta ctttgccacc aacctgcagg tctccggggc cattctctcc 720 gtcgacaagg ccatggctgc tttgtag 747 <210> 2 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of oxidoreductase YlADH_Bm6 <400> 2 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 Gly 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 Thr 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 Thr 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> 3 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a modified oxidoreductase <220> <222> (88)..(88) <223> Any amino acid <220> <222> (89)..(89) <223> Any amino acid <220> <222> (143)..(143) <223> Any amino acid <220> <222> (152)..(152) <223> Any amino acid <220> <222> (155)..(155) <223> Any amino acid <220> <222> (157)..(157) <223> Any amino acid <220> <222> (159)..(159) <223> Any amino acid <220> <222> (161)..(161) <223> Any amino acid <220> <222> (196)..(196) <223> Any amino acid <220> <222> (210)..(210) <223> Any amino acid <400> 3 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 Xaa Xaa 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 Xaa Ser 130 135 140 Ile Leu Gly Leu Arg Ala Val Xaa Pro Ala Xaa Thr Xaa Tyr Xaa Ala 145 150 155 160 Xaa 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 Xaa Glu Phe Met Glu Leu Pro Glu Pro Ser Pro Phe Gln 195 200 205 Glu Xaa 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> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Forward primer F1 <400> 4 cacataaaca aacaaagagc tcggatggga gaactcagcg gaaa 44 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer R1 <400> 5 aagtccaaag ctcccgggta ctacaaagca gccatggcct 40 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> TDH3-F <400> 6 tacgaattct ttaccgtcga cacagtttat tcctggcat 39 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TDH3-R <400> 7 gaagtccaaa gctcccgggt tgtttgttta tgtgtgtt 38 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> ADH1-F <400> 8 aaacacacat aaacaaacaa cccgggagct ttggact 37 <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> ADH1-R <400> 9 gcaggtcgac tctagaggat cccatagggt aggggaa 37 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 352-F <400> 10 gatcctctag agtcgacctg c 21 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 352-R <400> 11 gtcgacggta aagaattcgt aatc 24 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer F2 <400> 12 tacccgggag ctttggactt 20 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer R2 <400> 13 ccgagctctt tgtttgttta tgtgt 25

Claims

1. Use of an oxidoreductase and / or a modified oxidoreductase as yuzu alcohol dehydrogenase in the biosynthesis of yuzuketone, characterized in that: The oxidoreductase, named YlADH_Bm6, has an amino acid sequence as shown in SEQ ID No. 2; The modified oxidoreductase has an amino acid sequence as shown in SEQ ID No. 3, wherein at least one position labeled "X" in SEQ ID No. 3 is different from the amino acid residue at the corresponding position of SEQ ID No. 2; The modified oxidoreductase has: (1) One modification selected from the following: G88A, G88C, G88T, N89G, N89V, S143V, S143L, T152A, T152V, T152M, T152P, L155M, V157G, V157L, S159C, S161Y, S161R, T196S, T196N, T196G, T196Y, K210R or K210L; Or, (2) Multiple modifications selected from the following: G88A-N89V, G88A-T152A, G88A-T152M, G88A-L155M, G88A-V157G, G88A-T196S, G88C-N89V, G88C-T152M, G88C-T152V, G88C-T196S, G88C-K210R, N89V-V157G, T152M-L155M, T152M-V157G, T152M-T196N, T152M-T196S, T152M-K210R, T152V-T196N, T152V-T196S, G88C-N89V-T152M, G88C-T152V-T196S, T152M-L155M-T196S, G88C-T152M-T196S or G88C-N89V-T152M-T196S.

2. The use according to claim 1, characterized in that: The use of the modified oxidoreductase in improving substrate tolerance, and the substrate is yuzu alcohol.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding oxidoreductase YlADH_Bm6 is as shown in SEQ ID No.

1.

4. The modified oxidoreductase according to any one of claims 1 to 3.

5. A gene encoding the modified oxidoreductase according to claim 4.

6. A recombinant expression vector or recombinant expression cell containing the gene according to claim 5.

7. Use of a recombinant expression vector or recombinant expression cell containing the gene according to claim 3, the gene according to claim 5 or the recombinant expression vector or recombinant expression cell according to claim 6 in the biosynthesis of yuzuketone.

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