An alcohol dehydrogenase mutant and a method for synthesizing raspberry ketone thereof

Abstract

The application discloses an alcohol dehydrogenase mutant and a method for synthesizing raspberry ketone by using the same. The mutant is obtained by mutating any one or more of positions 144, 145, 190, 40, 17, 64 and 94 of an alcohol dehydrogenase LbADH from Lactobacillus brevis shown in the sequence of SEQ ID NO:1. The application first mutates and screens a substrate binding pocket of LbADH, successfully obtains a mutant with improved catalytic activity for R-dulcitol and S-dulcitol, and realizes efficient catalysis of a single enzyme on two enantiomers. The application also mutates amino acids around an NADP+ binding site of LbADH, enhances the catalytic oxidation activity of the LbADH under high pH conditions, and successfully obtains a combined mutant with high catalytic efficiency under a high pH environment. The application provides a new technical means and solution for the oxidative conversion of 'natural' raspberry ketone.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an alcohol dehydrogenase mutant and a method for synthesizing natural raspberry ketone from R-rubroside and S-rubroside by using the mutant. BACKGROUND

[0002] Raspberry ketone has a raspberry aroma and a sweet fruit flavor. Industrially, raspberry ketone is synthesized from chemical intermediates by various methods, which can make the cost as low as a few dollars per pound to one-fifth of the cost of natural products. In recent years, due to the increasing public awareness, there is an increasing demand for sustainable production of flavor compounds derived from natural sources, and the production of raspberry ketone using synthetic biology technology has become a focus of attention.

[0003] Due to the toxicity of raspberry ketone, microbial de novo synthesis can only provide a low yield of up to 5-100 mg / L, which cannot be applied to industrial production in the short term. According to the regulations of the European Union, flavor substances labeled as "natural" can only be obtained by physical processes (such as distillation and extraction from natural sources) or by enzymatic / microbial processes to convert precursor substances isolated from nature. Therefore, synthesizing raspberry ketone from precursor substances isolated from nature becomes a method for efficient synthesis of "natural" raspberry ketone.

[0004] In 2003, the University of Graz in Austria proposed using freeze-dried cells containing dehydrogenase to catalyze the oxidation of rhododendrol (obtained from natural sources, such as the bark of various birch trees) to produce "natural" raspberry ketone biologically. Rhodococcus equi IFO 3730 and Rhodococcus ruber DSM 44541 can catalyze the conversion of 20 g / L rhododendrol to raspberry ketone at a maximum of 52% under 10% acetone conditions, with the S-enantiomer being preferentially oxidized and the R-enantiomer remaining (Tetrahedron 59 (2003) 9517-9521). In 2021, Jiangnan University used whole-cell biocatalysts to catalyze the reduction of p-hydroxybenzylideneacetone to biosynthesize RK. The reductionase RiRZS1 of Bacillus sp. and the glucose dehydrogenase SyGDH of Acidothermus afermentans were expressed in Escherichia coli, and NADPH was regenerated for whole-cell catalytic reaction. By balancing the co-expression of the two enzymes in pRSFDuet-1, 9.89 g / L of RK was obtained from 10 g / L substrate, with a conversion rate of 98% and a space-time yield of 4.94 g / (L-h) (J. Agric. Food Chem. 2021, 69, 8, 2549-2556). The CN118109429A patent used the panda alcohol dehydrogenase mutant AmADH1E-D224A, the Lactobacillus kefiri alcohol dehydrogenase LK-ADH, and the raspberry ketone / jacarenthone synthase AiRZS of the unexpected small white oval fungus to catalyze the synthesis of raspberry ketone with racemic rhododendrol and 4-hydroxybenzylideneacetone as substrates. The innovation lies in the coenzyme preference modification of AmADH1E (D224A mutation), which constructs a coenzyme regeneration cycle system, and the final conversion rate reaches 92.3% (200ul, substrate concentration 80mg / L), which is still far from industrial application.

[0005] However, the above method still needs to be further optimized for industrial production. First, the content of rhododendrol, R-rhododendrol and S-rhododendrol extracted from different plants can also be significantly different: for example, in dwarf birch, the R-enantiomer accounts for 97%, while in yellow birch, the two enantiomers are almost equal, and in European birch, the glycoside with S-enantiomer is mainly found. The currently disclosed method for ADH catalytic synthesis of "natural" raspberry ketone needs multi-enzyme cooperation or multi-enzyme multi-step cooperation to completely oxidize rhododendrol, and the proportion of different stereoselective alcohol dehydrogenases needs to be adjusted according to the content of naturally extracted rhododendrol R- and S-rhododendrol. At present, there is no report that an enzyme can simultaneously catalyze the oxidation of different proportions of rhododendrol R- and S-rhododendrol. Second, alcohol dehydrogenase catalyzes oxidation reaction, preferably at pH 9 to 10, which is more thermodynamically favorable. However, the coenzyme NAD+ / NADP+ is easily degraded under high pH conditions. Based on this, the present patent designs a mutant alcohol dehydrogenase that can simultaneously catalyze R-rhododendrol and S-rhododendrol, and efficiently synthesize "natural" raspberry ketone from rhododendrol under high pH conditions. SUMMARY

[0006] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides an alcohol dehydrogenase mutant and a method for biosynthesizing "natural" raspberry ketone using the same. The alcohol dehydrogenase mutant can simultaneously and efficiently catalyze the oxidation of R-rhododendrol and S-rhododendrol to raspberry ketone.

[0007] The present application also provides an alcohol dehydrogenase mutant that can catalyze the oxidation of R-rhododendrol and S-rhododendrol to raspberry ketone under high pH conditions.

[0008] The present application further aims to provide a method for biosynthesizing "natural" raspberry ketone using the above-mentioned alcohol dehydrogenase mutant.

[0009] The object of the present application is achieved by the following technical solutions:

[0010] An alcohol dehydrogenase mutant, wherein the mutant is obtained by mutating any one or more of positions 144, 145, 190, 40, 17, 64, and 94 of an alcohol dehydrogenase LbADH having an amino acid sequence as shown in SEQ ID NO: 1.

[0011] More preferably, the mutant is an alcohol dehydrogenase LbADH with the amino acid sequence as shown in SEQ ID NO:1, specifically the following mutants: E145L, I144L\E145L, I144L\Y190A, I144L\E145L\Y190A, I144L\E145L\Y190A\H40R, I144L\E145L\Y190A\H40R\S64V, I144L\E145L\Y190A\H40R\L17Q.

[0012] It is obtained by any mutation from I144L, E145L, Y190A, H40R, L17Q, or S64V.

[0013] A mutant of alcohol dehydrogenase, I144L\E145L, is obtained by mutating the 145th amino acid of alcohol dehydrogenase LbADH, as shown in the amino acid sequence of SEQ ID NO:1, from glutamic acid (E) to leucine (L), and the 144th amino acid from isoleucine (I) to leucine (L).

[0014] A mutant of alcohol dehydrogenase I144L\Y190A is obtained by mutating the 190th amino acid of alcohol dehydrogenase LbADH, as shown in SEQ ID NO:1, from tyrosine (Y) to alanine (A) and the 144th amino acid from isoleucine (I) to leucine (L).

[0015] A mutant of alcohol dehydrogenase, E145L\Y190A\I144L, is obtained by mutating the amino acid at position 145 of the alcohol dehydrogenase LbADH (as shown in SEQ ID NO:1) from glutamic acid (E) to leucine (L), the amino acid at position 190 from tyrosine (Y) to alanine (A), and the amino acid at position 144 from isoleucine (I) to leucine (L).

[0016] The mutants I144L\E145L, I144L\Y190A, and E145L\Y190A\I144L not only improved the catalytic efficiency for the substrate, but also had a catalytic activity ratio of close to 1 for different isomers, achieving highly efficient catalytic conversion of the two enantiomers, R-rhododendronol and S-rhododendronol.

[0017] The application discloses an alcohol dehydrogenase mutant E145L\Y190A\I144L\H40R\S64V\L17Q, which is obtained by mutating the 145th amino acid of an alcohol dehydrogenase LbADH shown in the amino acid sequence of SEQ ID NO: 1 from glutamic acid (E) to leucine (L), mutating the 190th amino acid from tyrosine (Y) to alanine (A), mutating the 144th amino acid from isoleucine (I) to leucine (L), mutating the 40th amino acid from histidine (H) to arginine (R), mutating the 64th amino acid from serine (S) to valine (V), and mutating the 17th amino acid from leucine (L) to glutamine (Q), and the mutant further enhances the binding capacity of NADP+ and maintains the stability of NADP+ under high pH conditions.

[0018] The mutant E145L\Y190A\I144L\H40R\S64V\L17Q is applied to an enzyme catalytic oxidation reaction of natural extraction of rhododendrol, and the economics and practicability of the mutant in an industrial catalytic process are further improved.

[0019] A nucleic acid sequence encoding the alcohol dehydrogenase mutant.

[0020] A nucleic acid sequence encoding the alcohol dehydrogenase mutant E145L\Y190A\I144L\H40R\S64V\L17Q, and the sequence is shown in SEQ ID NO. 2.

[0021] A recombinant plasmid connected with the nucleic acid sequence of the alcohol dehydrogenase mutant.

[0022] A recombinant cell containing the recombinant plasmid or the nucleic acid sequence of the alcohol dehydrogenase mutant, and the recombinant cell is applied to catalytic synthesis of raspberry ketone.

[0023] The alcohol dehydrogenase mutant or the recombinant cell is used as a catalyst to catalytically synthesize raspberry ketone from rhododendrol as a substrate under the conditions of pH 7-10 and temperature 25-45 DEG C.

[0024] Further preferably, the reaction is carried out under the conditions of pH 9.5 and temperature 35-40 DEG C.

[0025] The rhododendrol includes any one or a mixture of both of R-rhododendrol and S-rhododendrol in any ratio.

[0026] A method for biosynthesizing "natural" raspberry ketone, which uses the alcohol dehydrogenase mutant or the recombinant cell to catalytically synthesize raspberry ketone from rhododendrol. The method specifically comprises the following steps:

[0027] 1) Cloning alcohol dehydrogenase mutant gene into a vector, transforming into a host to construct a genetically engineered bacterium;

[0028] 2) Culturing the genetically engineered bacterium constructed, collecting bacterial bodies after centrifugation, and using the bacterial bodies as a whole cell catalyst for catalyzing synthesis of raspberry ketone;

[0029] 3) Catalyzing synthesis of raspberry ketone with rhododendrol as a substrate.

[0030] In step 3), the rhododendrol includes any one of R-rhododendrol and S-rhododendrol or a mixture of the two in any ratio, and the concentration is 10-50 g / L; the whole cell catalyst is used in an amount of 1-10 g / L.

[0031] The catalytic reaction condition is: pH 7-10, temperature 25-45 DEG C.

[0032] Beneficial effects:

[0033] The application provides an alcohol dehydrogenase mutant, which not only improves catalytic efficiency on a substrate, but also has a catalytic activity ratio of different isomers close to 1, realizes efficient catalytic conversion on two enantiomers, further mutates to enhance the binding capacity of NADP+, and maintains the stability of NADP+ under high pH conditions. The mutant E145L\Y190A\I144L\H40R\S64V\L17Q obtained is applied to enzyme catalytic oxidation reaction of natural extraction rhododendrol, and further improves the economics and practicability in industrial catalytic process. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Synthetic pathway of LbADH mutant catalyzing oxidation of rhododendrol into raspberry ketone;

[0035] Figure 2 GC detection chromatograms of rhododendrol and raspberry ketone;

[0036] Figure 3 Conversion rates of M14 catalyzing R / S rhododendrol under different pH conditions;

[0037] Figure 4 LbADH_M18 pH optimization;

[0038] Figure 5 LbADH_M18 temperature optimization;

[0039] Figure 6 LbADH-M18 catalyzing raspberry ketone in different proportions. DETAILED DESCRIPTION

[0040] The application will be further described in conjunction with the following examples and drawings, but the embodiments of the application are not limited thereto.

[0041] Unless otherwise specified, the following examples were carried out under conventional experimental conditions or under experimental conditions recommended by the reagent companies. The materials, reagents, etc. used, if not specified, were reagents and materials obtained from commercial channels. Example 1 Mutation and screening of LbADH substrate binding pocket

[0042] 1.1 Selection of mutation sites by molecular docking

[0043] The LbADH (GenBank: MW808993, SEQ ID NO. 1) with the characteristics of high temperature resistance and high substrate concentration resistance was selected as the starting template for optimization. By mutating the substrate binding pocket, the stereoselectivity of the LbADH for different conformations of the substrate rhododendrol was destroyed, so that it could simultaneously and efficiently oxidize R-rhododendrol and S-rhododendrol. The “Docking with AutoDock Vina” module in the online tool SWISS-Dock (http: / / www.swissdock.ch / ) was used for molecular docking. The crystal structure (PDB: 1ZK4) of LbADH was a protein structure, and the coordinates of the position of the phenylacetone carbonyl carbon atom were the center of molecular docking (13.785, 7.756, 13.498). The SMILES input of the substrate rhododendrol was “CC(O)CCC1=CC=C(O)C=C1”. Glu145, Tyr190 had hydrogen bond interactions with the substrate. Tyr156, ILE144, Glu145, Tyr190 had hydrophobic interactions with the substrate. Tyr156 was a key amino acid for the function of LbADH, and was rationally maintained. ILE144, Glu145, Tyr190 were selected for mutation. Based on the selective preference of alcohol dehydrogenase regulated by the size of substrate binding, the amino acid A94 in the size pocket of LbADH was selected for mutation. ILE144, Glu145, Tyr190 and A94 were designed to be mutated into Ala\Leu\Phe residues representing small, medium and large volumes, respectively, and a total of 11 mutants were constructed.

[0044] 1.2 Cloning and expression of LbADH

[0045] The LbADH gene fragment synthesized from General Biotech (Anhui) Co., Ltd. (the sequence is shown in GenBank: MW808993, and the amino acid sequence is shown in SEQ ID NO. 1) was cloned into pET24a vector (purchased from General Biotech (Anhui) Co., Ltd.) by NdeI and XhoI, and the ligation product was transformed into E. coli Top10 competent cells purchased from Thermo Fisher, and was cultured in 5 mL medium with kanamycin resistance at 37℃ overnight. The plasmid was extracted and sent for sequencing, and the plasmid with correct sequencing was named as pET24a-LbADH.

[0046] 1.3 Construction of LbADH mutant and mutant library

[0047] A mutant library from LbADH was constructed by using a large primer method, and mixed primers F1 / R1 (Table 1) were used. The 50 μL reaction system contained 25 μL PrimeSTAR premix, 0.5 μL (50-100 ng) template DNA and 0.5 μL 100 μM primer mixture. The short fragment PCR conditions were: 95℃ pre-denaturation for 5 minutes, (95℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 20 seconds) for 25 cycles, and 72℃ extension for 10 minutes. The large fragment PCR conditions were: 95℃ pre-denaturation for 5 minutes, (95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 7 minutes) for 30 cycles, and 72℃ extension for 10 minutes. The PCR product was analyzed by 1% agarose gel electrophoresis, and was digested with DpnI at 37℃ for 3 hours. 1-2 μL of the digested PCR product was transformed into electrocompetent E. coli BL21 (DE3), and a single colony was selected for sequencing to determine the final mutation.

[0048] Table 1 Primers for constructing LbADH mutant

[0049]

[0050] 1.4 Construction of recombinant plasmid E. coli genetically engineered bacteria and culture method

[0051] The recombinant plasmid successfully constructed in Example 1 was chemically transformed into *E. coli* BL21(DE3) to obtain the engineered *E. coli* strain *E. coli* BL21(DE3) / pET24a-LbADH. The above engineered *E. coli* strain was streaked onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated into LB tubes containing 50 μg / mL kanamycin. The LB medium formulation was as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The pH of the medium was adjusted to 7.4 with NaOH. After incubation at 37°C for 10 h, the culture was inoculated at a rate of 1-2% (v / v). Add the following to 400 mL of TB medium: 12 g / L tryptone, 24 g / L yeast extract, 5 g / L glycerol, 16.43 g / L dipotassium hydrogen phosphate, and 2.33 g / L potassium dihydrogen phosphate. Adjust the pH of the medium to 7.4 with NaOH. Incubate at 37°C until OD600 = 0.5, then add IPTG to a final concentration of 0.1 mM, and continue induction at 15°C and 180 rpm for another 16 h.

[0052] 1.5 Preparation of whole-cell catalysts from engineered bacteria

[0053] The engineered *E. coli* bacteria induced in step 1.4 were collected by centrifugation, resuspended in 0.9% physiological saline, and then centrifuged again to collect the bacteria. This process was repeated once more to thoroughly remove culture medium components from the bacteria. The collected bacteria were pre-cooled at -80°C and then freeze-dried in a vacuum freeze dryer. The resulting freeze-dried bacterial powder can be used as a whole-cell catalyst for the catalytic synthesis of raspberry ketones.

[0054] 1.6 Determination of Catalytic Activity of Mutants

[0055] R-rhododendronol (CAS: 501-96-2, purchased from Wuhan Woxuan Technology Co., Ltd.) or S-rhododendronol (CAS:

[0056] 59092-94-3 (purchased from Wuhan Prof Biotechnology Co., Ltd.) 30 g / L (180 mM), 3 mM NADP+, and 10% acetone were mixed, along with 5 g / L of the whole-cell catalyst from Example 1.5. Buffer solutions of different pH values ​​were added (pH 7.0-pH 7.5: Na2HPO4 and NaH2PO4 buffer system; pH 8.0, pH 8.5, pH 9.0: Tris-HCl buffer system; pH 9.5, pH 10.0, pH 10.5: Na2CO3 and NaHCO3 buffer system) to a final volume of 5 mL. The reaction was carried out in a 30°C temperature-controlled shaker (JRA-IIA centrifuge tube shaker) for 24 hours, with BL21(DE3) / pET24a as a negative control. Samples were then taken, and the reaction conversion rate was detected by GC.

[0057] 1.7 GC detection method

[0058] The gas chromatography (Agilent 7090A) was used to analyze the rhododendrol and raspberry ketone in this paper. The Agilent HP-5 (30 m*0.25 mm ID*0.25 um) was selected as the chromatographic column. The temperature program was set as initial temperature 100℃ for 1 min, then increased to 170℃ at a rate of 15℃ / min and kept for 1 min, and then increased to 180℃ at a rate of 3℃ / min and kept for 1 min. Under this condition, the retention time of R-rhododendrol and S-rhododendrol was consistent at 9.4 min, and the retention time of raspberry ketone was 9.3 min. Figure 2

[0059] 1.8 Iterative mutation

[0060] The conversion rate of the mutants was determined according to the catalytic reaction conditions of 1.6, and the results are shown in Table 2. In the pH 7.0 buffer system, the conversion rates of M2 for R-rhododendrol and S-rhododendrol were both improved compared with the wild type LbADH. The Y190A and E145L mutants had a significant improvement in the conversion rate of S-rhododendrol. A94 located in the substrate binding pocket of LbADH is very important for the benzene ring of the substrate rhododendrol to enter the active center, and the related mutation significantly reduces the activity of LbADH. Therefore, three mutants I144L\E145L, I144L\Y190A, I144L\E145L\Y190A were selected to construct. The conversion rate ratio of R-rhododendrol and S-rhododendrol of mutant M14 was close to 1.1, and the conversion rate was increased to about 18.3%.

[0061] Table 2 Conversion rate of LbADH mutants

[0062]

[0063]

[0064] 1.9 Activity of M14 mutant under different pH conditions

[0065] Alcohol dehydrogenase catalyzes the oxidation of alcohol, and as the pH increases, the thermodynamic equilibrium will shift to the oxidation direction. Therefore, the activity of M14 mutant under different pH conditions was explored, and the reaction conditions were the same as 1.6, and the results are shown in Table 3. Figure 3 The enzyme activity was significantly improved when the pH 7.0 was increased to pH 8.0. When the pH was higher than 9.0, the enzyme activity decreased significantly, which may be related to the degradation of NADP cofactor under high pH conditions.

[0066] Example 2 Mutating amino acids around NADP+ binding site ​

[0067] 2.1 Mutant site determination

[0068] The expensive cofactor NADP+ is degraded under high pH conditions, which is an important reason for the decrease of catalytic activity of alcohol dehydrogenase under high pH conditions and the increase of production cost. Enhancing the binding of alcohol dehydrogenase and cofactor NADP+ will protect its stability under high pH conditions. Based on the crystal structure of LbADH (PDB: 1ZK4), the amino acid sites H40, L17, S64V, I144V interacting with NADP+ of LbADH were selected for virtual saturation mutation. Based on the size of binding energy and the number of hydrogen bonds, the mutants H40R, L17Q, S64V were selected for testing. The new mutants H40R, H40R\L17Q, H40R\S64V were constructed based on the combination mutant M14 in Example 1.

[0069] The mutants and strains were constructed according to the method of Example 1 with the upstream and downstream primers in Table 3.

[0070] Table 3 Primers for constructing LbADH second round mutants

[0071] Primer name Construct mutant Sequence H40R-F H40R ATGATCACGGGTCGTCGTAGCGACGTTGGCGAA (SEQ ID NO. 17) H40R-R H40R CCCAGACCTTTGTTTTTCATGCGCTGGA (SEQ ID NO. 18) L17Q-F L17Q ATCACGGGCGGTACTCAGGGGATCGGTCTTGCG (SEQ ID NO. 19) L17Q-R L17Q CCGTCCAGATTGACCGCCAGCAATTTAC (SEQ ID NO. 20) S64V-F S64V TTCTTCCAGCATGATGTGAGCGACGAAGATGGC (SEQ ID NO. 21) S64V-R S64V CGTTATACGCGCCCAGAGACGGATCACCGACA (SEQ ID NO. 22)

[0072] 2.2 Mutant activity determination and iteration

[0073] The activities of different mutants were determined under pH 9.0 conditions, and M14 mutant was used as a control. The results are shown in Table 4. M15, M16 and M17 significantly improved the catalytic activity of rhodionol oxidation under pH 9.0 conditions. The catalytic activity of combination mutant M18 reached 61.8%.

[0074] Table 4 Conversion rates of LbADH mutants

[0075]

[0076]

[0077] Example 3 Optimization of LbADH-M18 catalytic conditions

[0078] Reaction temperature and pH are key factors affecting the catalysis of LbADH_M18 on rhodionol oxidation. Therefore, the reaction pH and temperature of LbADH_M18 were optimized according to the reaction conditions in Example 1.6. Figure 4 , Figure 5 ) At pH 9.5, the conversion rates of R-rhodionol and S-rhodionol reached 75.90% and 74.30%, respectively. At 40°C, the reaction conversion rate was increased by about 20% compared with 30°C, reaching 95.7%.

[0079] Example 4: LbADH_M12, LbADH_M13, and LbADH_M14 catalyze different ratios of rhododendronol.

[0080] The conversion rates of different ratios of 30 g / L R-rhododendronol and S-rhododendronol (90%, 50%, and 10% R-rhododendronol, respectively) catalyzed in a 5 ml system containing 5 g / L LbADH_M12, LbADH_M13, and LbADH_M14 at 30 °C for 24 hours are shown in Table 5. Mutants M12, M13, and M14 were able to catalyze the biosynthesis of raspberry ketone from different R / S rhododendronol ratios.

[0081] Table 5. LbADH_M12, LbADH_M13, and LbADH_M14 catalyze different R / S ratios of rhododendron alcohol.

[0082] Seq. No. Mutant 90% R mix 50% R mix 10% R mix M12 I144L\E145L 17.9％ 15.7％ 15.5％ M13 I144L\Y190A 18.2％ 14.1％ 13.5％ M14 I144L\E145L\Y190A 19.1％ 16.9％ 16.3％

[0083] Example 5: LbADH_M18 catalyzes different ratios of rhododendronol

[0084] Catalysis was performed at 40°C, 10% acetone, 3 mM NADP+, and 8 g / L LbADH_M18 on a mixture of 30 g / L R-rhododendronol and S-rhododendronol in different proportions (90%, 50%, and 10% R-rhododendronol, respectively), with pH adjusted.

[0085] Add 9.5% Na₂CO₃ and NaHCO₃ buffer salts to a 50 mL reaction system. The results are as follows: Figure 6 As shown. After 24 hours of reaction, rhododendron alcohols of different proportions were almost completely converted into "natural" raspberry ketones. LbADH-M18 can catalyze the synthesis of raspberry ketones from different proportions of R / S-rhododendron alcohols under high pH conditions, thus enabling it to adapt to natural rhododendron alcohol substrates from different plant sources, significantly expanding its applicability in industrial applications.

[0086] SEQ ID NO.1:

[0087] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQ

[0088] HDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFG

[0089] TRLGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDV

[0090] RVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSE

[0091] FVVDGGYTAQ

[0092] SEQ ID NO.2:

[0093] ATGTCAAATAGGCTAGATGGAAAAGTAGCTATCATCACGGGCGGTACTcagGGGATCGGT

[0094] CTTGCGATTGCAACCAAGTTCGTGGAGGAGGGTGCTAAAGTTATGATCACGGGTCGTcg

[0095] tAGCGACGTTGGCGAAAAAGCGGCCAAGTCCGTGGGCACTCCAGATCAGATTCAATTC

[0096] TTCCAGCATGATgtgAGCGACGAAGATGGCTGGACCAAATTGTTTGATGCAACGGAAAA

[0097] GGCGTTTGGTCCGGTTAGCACCCTCGTCAACAACGCGGGTATTGCCGTGAACAAGTCT

[0098] GTAGAGGAGACAACCACCGCAGAGTGGCGTAAATTGCTGGCGGTCAATCTGGACGGC

[0099] GTTTTTTTCGGCACGCGTCTGGGTATCCAGCGCATGAAAAACAAAGGTCTGGGCGCGA

[0100] GCATTATCAATATGTCCTCCctgctgGGTTTTGTCGGTGATCCGTCTCTGGGCGCGTATAAC

[0101] GCCAGCAAGGGTGCGGTTCGTATTATGTCCAAGAGTGCGGCACTGGACTGTGCTTTGA

[0102] AAGATTATGATGTGCGCGTGAACACCGTGCACCCGGGT gcgATCAAAACCCCGCTGGTG

[0103] GACGACCTGCCGGGTGCTGAAGAGGCGATGTCGCAAAGAACCAAGACCCCGATGGGT

[0104] CATATTGGCGAGCCGAATGACATCGCATATATCTGCGTTTACCTGGCTTCTAATGAAAGC

[0105] AAGTTCGCCACCGGCAGCGAATTTGTTGTTGACGGAGGCTACACCGCGCAACTCGAGC

[0106] ACCACCACCACCACCACTGA.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The mutant is obtained by any one of the mutations in the amino acid sequence of the alcohol dehydrogenase LbADH, as shown in SEQ ID NO:1, namely I144L\E145L\Y190A\H40R, I144L\E145L\Y190A\H40R\S64V, I144L\E145L\Y190A\H40R\L17Q, and I144L\E145L\Y190A\H40R\L17Q\S64V.

2. The nucleic acid sequence encoding the alcohol dehydrogenase mutant of claim 1.

3. A recombinant plasmid having a nucleic acid sequence of the alcohol dehydrogenase mutant of claim 2 attached thereto.

4. A recombinant cell comprising the nucleic acid sequence of the recombinant plasmid of claim 3 or the alcohol dehydrogenase mutant of claim 2.

5. The application of the alcohol dehydrogenase mutant of claim 1 and the recombinant cells of claim 4 in the catalytic synthesis of raspberry ketone.

6. The application according to claim 5, characterized in that, Using rhododendron alcohol as a substrate and alcohol dehydrogenase mutants or recombinant cells as catalysts, raspberry ketones were synthesized under catalytic conditions of pH 7–10 and temperature 25–45°C.

7. The application according to claim 6, characterized in that, The rhododendron alcohol comprises any one or a mixture of two of R-rhododendron alcohol and S-rhododendron alcohol in any proportion.

8. A method for biosynthesizing raspberry ketone, characterized in that, The method uses the alcohol dehydrogenase mutant of claim 1 or the recombinant cell catalysis of rhododendronol to synthesize raspberry ketone as described in claim 4.

9. The method for biosynthesizing raspberry ketone according to claim 8, characterized in that, The catalytic reaction conditions are pH 7~10 and temperature 25~45℃.

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