Ketoacid decarboxylase mutant and application thereof in synthesis of phenethyl alcohol derivative

Abstract

The invention discloses a keto acid decarboxylase mutant and application thereof in synthesis of phenethyl alcohol derivatives, the keto acid decarboxylase mutant integrates and expresses an ipdC mutant derived from Klebsiellaapneumoniae and aldehyde reductase Cgl0331 through plasmid or genome, valine at the 467th site of the ipdC is mutated into tryptophan, valine at the 467th site of the aldehyde reductase Cgl0331 is mutated into tryptophan, valine at the 467th site of the aldehyde reductase Cgl0331 is mutated into tryptophan, valine at the 467th site of the aldehyde reductase Cgl0331, valine at the 467th site of the aldehyde reductase Cgl0331 the aldehyde reductase Cgl0331 is aldehyde reductase of a corynebacterium glutamicum endogenous source; according to the invention, the conversion rate of phenylpyruvic acid, hydroxyphenylpyruvic acid and 3, 4-dihydroxybenzophenone acid is improved; according to the invention, the aldehyde reductase coded by the Cgl0331 gene is overexpressed, so that the synthesis efficiency of 2-phenethyl alcohol, tyrosol and hydroxytyrosol is improved; the corynebacterium glutamicum whole-cell catalyst disclosed by the invention has higher tolerance to 2-phenethyl alcohol, tyrosol and hydroxytyrosol.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a ketoacid decarboxylase mutant and a method and application thereof for synthesizing 2-phenylethanol. Background Art

[0002] Aromatic natural products are a class of organic compounds with benzene ring structures from the biological world, and they are numerous and numerous. Among them, phenylethanol derivatives (including tyrosol, hydroxytyrosol, etc.) have strong antioxidant activity and good biological activity in regulating glucose and lipid metabolism, preventing and treating tumors, anti-thrombosis, alleviating arteriosclerosis, protecting cartilage, and preventing and treating osteoporosis. Since the beginning of the last century, microbial fermentation production has been applied to many industrial production fields such as food, medicine, and chemical industry. As an important organic compound, phenylethanol and its derivatives have also been produced by microbial fermentation.

[0003] At present, there are two main types of microbial cell factories that can synthesize phenylethanol: the first type is yeast; the other type is Escherichia coli modified by metabolic engineering. Because Escherichia coli grows quickly and has complete genetic modification tools, it is more widely used. Despite the application of various metabolic engineering strategies, the fermentation yield of phenylethanol and its derivatives is still at a low level. This is because high concentrations of aromatic alcohols inhibit the growth of strains. For example, 2g / L phenylethanol can completely inhibit the growth of Escherichia coli; 2.5g / L hydroxytyrosol completely inhibits the growth of Escherichia coli; 4g / L phenylethanol almost completely inhibits the growth of Saccharomyces cerevisiae. To this end, researchers use in situ product separation technology to relieve the inhibition of the product on the strain and improve the final concentration and conversion efficiency of phenylethanol and its derivatives. However, the separation of this technology product is complex, and industrial production is not conducive to cost control. Breeding or modifying to obtain high-yield cell factories with excellent tolerance is another effective way to reduce production costs, increase yields and efficiency, and selecting suitable microbial starting strains is an important prerequisite.

[0004] CN113493760A discloses a Corynebacterium glutamicum for biosynthesizing phenylethanol, a construction method and an application thereof. A mutant strain PE3 is constructed using Corynebacterium glutamicum, which has a significant advantage in tolerance to aromatic compounds, as a chassis cell. The strain can produce 2-phenylethanol by aerobic fermentation of glucose and straw hydrolyzate, with a fermentation yield of 5.3 g / L.

[0005] There are three main pathways for the biosynthesis of phenylethanol and its derivatives. Among them, the Ehrlich pathway is the most important pathway. Under nitrogen source limitation, L-phenylalanine (L-tyrosine) is converted into phenylethanol (tyrosol) through three-step enzyme-catalyzed reactions; the second pathway is the shikimate pathway, which uses carbohydrates such as glucose as substrates to synthesize phenylethanol (tyrosol) from scratch. However, this complex reaction pathway has a strong feedback inhibition effect, resulting in low efficiency in the production of phenylethanol (tyrosol); the last one is the phenylethylamine pathway, which is similar to the Ehrlich pathway and plays a more important role in plants. The Ehrlich pathway only requires three steps of reaction to synthesize phenylethanol (tyrosol), and the metabolic pathway is short. It is a key pathway to increase the yield of phenylethanol. In this pathway, L-phenylalanine (L-tyrosine) is first converted into phenylpyruvate by transaminase, then decarboxylated to phenylacetaldehyde (hydroxyphenylacetaldehyde) by ketoacid decarboxylase, and finally reduced to phenylethanol (tyrosol) by alcohol dehydrogenase. The ARO10 gene product from Saccharomyces cerevisiae is a thiamine diphosphate-dependent phenylpyruvate decarboxylase with broad substrate specificity. The enzyme is widely used in the biosynthesis of phenylethanol, tyrosol and hydroxytyrosol. However, the eukaryotic ARO10 is expressed at a low level in prokaryotes, especially Escherichia coli, and requires low temperature expression, which limits the synthesis efficiency of phenylethanol and its derivatives. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a ketoacid decarboxylase mutant.

[0009] To solve the above technical problems, the present invention provides the following technical solution: a ketoacid decarboxylase mutant, wherein the ketoacid decarboxylase mutant expresses an ipdC mutant gene derived from Klebsiella pneumoniae and overexpresses an aldehyde reductase gene Cgl0331 through a recombinant plasmid or genome integration;

[0010] Among them, the amino acid sequence of the ipdC mutant gene is shown in SEQ ID NO.2, the aldehyde reductase Cgl0331 is an endogenous aldehyde reductase of Corynebacterium glutamicum, and the nucleotide sequence of the aldehyde reductase Cgl0331 is shown in SEQ ID NO.5.

[0011] As a preferred embodiment of the mutant of the present invention, the recipient bacterium is Corynebacterium glutamicum ATCC13032.

[0012] As a preferred embodiment of the mutant of the present invention, the plasmid is pXMJ19.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for constructing a ketoacid decarboxylase mutant, comprising:

[0014] The genome of C. glutamicum ATCC 13032 was used as a template, Cgl0331F and Cgl0331R were used as primers, and FastPfu high-fidelity polymerase was used to amplify a fragment of 1062 bp; the amplified product and the recombinant plasmid containing the ipdC mutant gene were digested with SalI and BamHI respectively and connected to obtain a recombinant vector;

[0015] Transforming the recombinant vector into C. glutamicum ATCC 13032 by electroporation to obtain the ketoacid decarboxylase mutant;

[0016] Among them, the nucleotide sequence of primer Cgl0331F is shown as SEQ ID NO.6, and the nucleotide sequence of primer Cgl0331R is shown as SEQ ID NO.7.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for producing phenylethanol derivatives, comprising: activating the recombinant Corynebacterium glutamicum and transferring it to a fermentation medium at an inoculum amount of 1-3%, adding IPTG at a final concentration of 1 mM to induce protein expression when the OD600 is 5 and the temperature is 30°C, culturing at 30-37°C, 200 r / min, and a cycle of 72-80h.

[0018] As a preferred embodiment of the method of the present invention, the fermentation medium is 1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate, 10 mg / L calcium chloride. -1 , ferrous sulfate 10 mg L -1 , manganese sulfate 0.1 mg L -1 , zinc sulfate 1mg L -1 , copper sulfate 0.2 mg L -1 , nickel chloride 20 μg L -1 , biotin 0.4mg L -1 , 3-morpholinepropanesulfonic acid 28g / L, yeast extract 3g / L, vitamin B120mg / L, vitamin B620mg / L.

[0019] As a preferred embodiment of the method of the present invention, wherein: L-phenylalanine is used as a substrate to synthesize 2-phenylethanol by biological method.

[0020] As a preferred embodiment of the method of the present invention, wherein: L-tyrosine is used as a substrate and tyrosol is synthesized by a biological method.

[0021] As a preferred embodiment of the method of the present invention, wherein: L-DOPA is used as a substrate to synthesize hydroxytyrosol by biological method.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of ketoacid decarboxylase mutants in the preparation of phenylethanol derivatives.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention obtains a novel ketoacid decarboxylase mutant by expressing the ketoacid decarboxylase of prokaryotic organism K. pneumoniae and site-directed mutagenesis, thereby improving the conversion rate of phenylpyruvate, hydroxyphenylpyruvate and 3,4-dihydroxybenzoketoacid, which is more than twice that of the wild type.

[0025] 2 The present invention further improves the synthesis efficiency of 2-phenylethanol, tyrosol and hydroxytyrosol by overexpressing the aldehyde reductase encoded by the Cgl0331 gene.

[0026] 3. The present invention improves the tolerance of 2-phenylethanol, tyrosol and hydroxytyrosol through the whole cell catalyst of Corynebacterium glutamicum. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0028] Figure 1 The recombinant vector pXMJ19-ipdC in Example 2 of the present invention V467W 's graph.

[0029] Figure 2 The recombinant vector pXMJ19-ipdC in Example 3 of the present invention V467W -Map of Cgl0331. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention, so the present invention is not limited to the specific embodiments disclosed below. Secondly, the "one embodiment" or "embodiment" referred to herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

[0032] The raw materials and reagents used in the present invention were purchased from Chinese medicine.

[0033] The culture medium involved in the embodiment is as follows:

[0034] Seed culture medium (CGIII medium): tryptone (10 g / L), yeast extract (10 g / L), NaCl (2.5 g / L).

[0035] Seed culture medium (LB medium): tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L.

[0036] Fermentation medium: potassium dihydrogen phosphate (1 g / L), potassium dihydrogen phosphate (1 g / L), magnesium sulfate (0.25 g / L), calcium chloride (10 mg L -1 ), ferrous sulfate (10 mg L -1 ), manganese sulfate (0.1 mg L -1 ), zinc sulfate (1 mg L -1 ), copper sulfate (0.2 mg L -1 ), nickel chloride (20 μg L -1 ), biotin (0.4 mg L -1 ), 3-morpholinepropanesulfonic acid (28g / L), yeast extract (3g / L), vitamin B1 (20mg / L), vitamin B6 (20mg / L).

[0037] The detection methods involved in the following embodiments are as follows:

[0038] Quantitative detection method of L-phenylalanine, L-tyrosine, L-DOPA, 2-phenylethanol, tyrosol and hydroxytyrosol: high performance liquid chromatography (HPLC) was used for detection; the HPLC chromatographic conditions were as follows: the mobile phase was 40% methanol and 60% water, the flow rate was 1 mL / min, the injection volume was 10 uL, the detection wavelength was 258 nm, the column temperature was 40°C, and the column was Agilent ZORBAX C18.

[0039] Unless otherwise stated, the experimental methods disclosed in the present invention all adopt conventional techniques in molecular biology and related fields in the art.

[0040] Example 1

[0041] 1. Construction of recombinant vector pXMJ19-ipdC and whole-cell catalyst:

[0042] The nucleotide sequence of the ipdC gene (as shown in SEQ ID NO.3 and the amino acid sequence as shown in SEQ ID NO.1) was synthesized by Suzhou Jinweizhi Company. The synthesized gene was double-digested with restriction endonucleases HindIII and SalI, and then inserted into the plasmid vector pXMJ19 cut with the same restriction endonucleases to obtain the expression vector pXMJ19-ipdC.

[0043] pXMJ19-ipdC was transformed into C. glutamicum ATCC 13032 by electroporation to obtain a whole-cell catalyst containing the recombinant vector pXMJ19-ipdC, which was named: CGA1.

[0044] Example 2

[0045] 1. Recombinant vector pXMJ19-ipdC V467W And the construction of whole-cell catalysts:

[0046] The nucleotide sequence of the ipdC mutant gene (the mutation site is the mutation of valine at position 467 to tryptophan) (as shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.2) was synthesized by Suzhou Jinweizhi Company. The synthesized gene was double-digested with restriction endonucleases HindIII and SalI, and then inserted into the plasmid vector pXMJ19 cut with the same restriction endonucleases to obtain the expression vector pXMJ19-ipdC V467W ,like Figure 1 shown.

[0047] pXMJ19-ipdC V467W Electroporation was performed to transform C. glutamicum ATCC 13032 to obtain the recombinant vector pXMJ19-ipdC V467W The whole-cell catalyst is named: CGA2.

[0048] Example 3

[0049] 1. Recombinant vector pXMJ19-ipdC V467W - Construction of Cgl0331 and whole cell catalyst containing the carrier:

[0050] The genome of C. glutamicum ATCC 13032 was used as a template, Cgl0331F (nucleotide sequence as shown in SEQ ID NO.6) and Cgl0331R (nucleotide sequence as shown in SEQ ID NO.7) were used as primers, and FastPfu high-fidelity polymerase was used to amplify the Cgl0331 gene (nucleotide sequence as shown in SEQ ID NO.5) fragment with a size of 1062 bp. The amplified product was then mixed with pXMJ19-ipdC V467W The recombinant vector pXMJ19-ipdC was obtained by digestion with SalI and BamHI respectively and ligation. V467W -Cgl0331, such as Figure 2 shown.

[0051] pXMJ19-ipdC V467W -Cgl0331 was transformed into C. glutamicum ATCC 13032 by electroporation to obtain the recombinant vector pXMJ19-ipdC V467W -Cgl0331 whole cell catalyst, the whole cell catalyst is named: CGA3.

[0052] Example 4

[0053] 1. Whole-cell catalyst for the production of 2-phenylethanol

[0054] The logarithmic phase seed culture fluids (CGA1, CGA2 and CGA3) stored at -80°C were taken separately, thawed on ice and inoculated into 250 mL conical flasks containing 20 mL CGIII liquid culture medium, and cultured overnight at 30°C and 200 rpm.

[0055] The inoculation amount was 2% and inoculated into a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium supplemented with 80 g / L glucose and 10 g / L L-phenylalanine, and cultured at 30°C and 220 rpm for 72 h.

[0056] After 6 h of culture, 1 mM IPTG was added and culture was continued for 66 h until glucose was completely consumed. The 2-phenylethanol yields of the three whole-cell catalysts are shown in Table 1 .

[0057] 2. Production of tyrosol using whole-cell catalyst

[0058] The logarithmic seed culture fluid (CGA1, CGA2 and CGA3) stored at -80°C was taken separately, melted on ice and inoculated into a 250mL conical flask containing 20mL CGIII liquid medium, and cultured overnight at 30°C and 200rpm. A 2% inoculum was inoculated into a 500mL conical flask, which contained 50mL of fermentation medium with 80g / L glucose and 10g / L L-tyrosine, and cultured at 30°C and 220rpm for 72h. After 6h of culture, 1mM IPTG was added and cultured for 66h until glucose was completely consumed. The tyrosol yields of the three whole-cell catalysts are shown in Table 1.

[0059] 3. Whole-cell catalyst production of hydroxytyrosol

[0060] The logarithmic phase seed culture fluid (CGA1, CGA2 and CGA3) stored at -80°C was taken separately, melted on ice and inoculated into a 250mL conical flask containing 20mL CGIII liquid medium, and cultured overnight at 30°C and 200rpm. A 2% inoculum was inoculated into a 500mL conical flask containing 50mL fermentation medium with 80g / L glucose and 10g / L L-DOPA added, and cultured at 30°C and 220rpm for 72h. After 6h of culture, 1mM IPTG was added and cultured for 66h until glucose was completely consumed. The tyrosol yields of the three whole-cell catalysts are shown in Table 1.

[0061] The results showed that the ketoacid decarboxylase mutant IpdCV 467W Compared with wild-type IpdC, the synthesis efficiency of 2-phenylethanol, tyrosol and hydroxytyrosol increased by more than two times. On this basis, overexpression of endogenous aldehyde reductase Cgl0331 of Corynebacterium glutamicum further increased the synthesis efficiency of 2-phenylethanol, tyrosol and hydroxytyrosol.

[0062] Table 1

[0063]

[0064]

[0065] Example 5

[0066] Aromatic alcohols have strong cytotoxicity. Taking tyrosol as an example, the tolerance of common chassis cells to it was tested. C. glutamicum ATCC13032 was cultured in CGIII solid medium containing tyrosol at 30°C and 220 rpm for 24 h, and E. coli MG1655 was cultured in LB solid medium containing tyrosol at 37°C and 220 rpm for 24 h.

[0067] Under the condition of adding different concentrations of tyrosol, the growth of the bacteria is shown in Table 2. Under the condition of 6g / L tyrosol, Escherichia coli only has a small amount of growth, while Corynebacterium glutamicum grows better; under the condition of 8g / L tyrosol, Escherichia coli does not grow, while Corynebacterium glutamicum can still grow. Therefore, it can be seen that Corynebacterium glutamicum also shows better tolerance and is more suitable as a whole-cell catalyst for the production of aromatic alcohols.

[0068] Table 2

[0069] Tyrosol concentration (g / L) 0 2 4 6 8 Escherichia coli ++ + +- +- - Corynebacterium glutamicum ++ ++ ++ ++ +-

[0070] SEQ ID NO.1

[0071] Artificial sequence

[0072] MQPTYTIGDYLLDRLVDCGIDRLFGVPGDYNLQFLDRVIAHSALGWVGCAN

[0073] ELNAAYAADGYARIKGAGALLTTYGVGELSALNGIAGSYAEHIPVLHIVGAP

[0074] STGAQQRGELLHHTLGDGDFRHFARMSEQITCSQALLTAGNACHEIDRVLRD

[0075] MLTHHRPGYLMLPADVARAAAIAPAQRLLVEPAPADENQLAGFCEHASRLL

[0076] RGSRRISLLADFLAQRYGLQKTLREWVAKTPVAHATMLMGKGLFDEQQSGF

[0077] VGTYSGIASAPQTREAIENADTIICIGTRFTDTITAGFTQHLAREKTIEIQPFAV

[0078] RVGDHWFSGVPMDKALAALMTLSAPLAAEWATPQVVAPEAEEGAEGELTQ

[0079] KNFWATVQGALRPGDIILADQGTAAFGIAALKLPSEASLIVQPLWGSIGFTLP

[0080] AAYGAQTAAAERRVVLIVGDGAAQLTIQEMGSMLRDKQKPLILLLNNEGYT

[0081] VERAIHGPEQRYNDIALWDWQRLPEAFAPDVASRCRRVTQTSELREAMTESI

[0082] TSDTLTLVEVMLPKMDIPDFLRAVTQALEERNSRSEQ ID NO.2

[0083] MQPTYTIGDYLLDRLVDCGIDRLFGVPGDYNLQFLDRVIAHSALGWVGCAN

[0084] ELNAAYAADGYARIKGAGALLTTYGVGELSALNGIAGSYAEHIPVLHIVGAP

[0085] STGAQQRGELLHHTLGDGDFRHFARMSEQITCSQALLTAGNACHEIDRVLRD

[0086] MLTHHRPGYLMLPADVARAAAIAPAQRLLVEPAPADENQLAGFCEHASRLL

[0087] RGSRRISLLADFLAQRYGLQKTLREWVAKTPVAHATMLMGKGLFDEQQSGF

[0088] VGTYSGIASAPQTREAIENADTIICIGTRFTDTITAGFTQHLAREKTIEIQPFAV

[0089] RVGDHWFSGVPMDKALAALMTLSAPLAAEWATPQVVAPEAEEGAEGELTQ

[0090] KNFWATVQGALRPGDIILADQGTAAFGIAALKLPSEASLIVQPLWGSIGFTLP

[0091] AAYGAQTAAAERRVVLIVGDGAAQLTIQEMGSMLRDKQKPLILLLNNEGYT

[0092] WERAIHGPEQRYNDIALWDWQRLPEAFAPDVASRCRRVTQTSELREAMTESI

[0093] TSDTLTLVEVMLPKMDIPDFLRAVTQALEERNSRVSEQ ID NO.3

[0094] ATGCAGCCAACCTACACCATCGGCGATTACCTGCTGGATCGCCTGGTGGA

[0095] TTGCGGCATCGATCGCCTGTTCGGCGTGCCTGGCGATTACAACCTGCAGT

[0096] TCCTGGATCGCGTGATCGCACACTCCGCACTGGGCTGGGTGGGCTGCGCA

[0097] AACGAACTGAACGCAGCATACGCAGCAGATGGCTACGCACGCATCAAGG

[0098] GCGCCGGCGCCCTGCTGACCACCTACGGCGTGGGCGAACTGTCCGCACTG

[0099] AACGGCATCGCCGGCTCCTACGCAGAACACATCCCTGTGCTGCACATCGT

[0100] GGGCGCACCATCCACCGGCGCACAGCAGCGCGGCGAACTGCTGCACCAC

[0101] ACCCTCGGCGATGGCGATTTCCGCCACTTCGCACGCATGTCCGAACAGAT

[0102] CACCTGCTCCCAAGCACTGCTGACCGCCGGCAACGCATGCCACGAAATCG

[0103] ATCGCGTGCTCCGCGATATGCTGACCCACCACCGCCCTGGCTACCTGATG

[0104] CTGCCTGCAGATGTGGCACGCGCAGCCGCAATCGCACCTGCACAGCGCCT

[0105] GCTGGTGGAACCTGCACCTGCAGATGAAAATCAGCTGGCCGGCTTCTGCG

[0106] AACACGCATCCCGCCTGCTGCGCGGCTCCCGCCGCATCTCCCTGCTGGCA

[0107] GATTTCCTGGCACAGCGCTACGGCCTGCAGAAGACCCTGCGCGAATGGGT

[0108] GGCAAAGACCCCTGTGGCACACGCAACCATGCTGATGGGCAAGGGCCTG

[0109] TTCGATGAACAGCAGTCCGGCTTCGTGGGCACCTACTCCGGCATCGCATC

[0110] CGCACCACAGACCCGCGAAGCAATCGAAAACGCAGATACCATCATCTGC

[0111] ATCGGCACCCGCTTCACCGATACCATCACCGCCGGCTTCACTCAGCACCT

[0112] GGCACGCGAAAAGACCATCGAAATTCAGCCATTCGCAGTGCGCGTGGGC

[0113] GATCACTGGTTCTCCGGCGTGCCAATGGATAAGGCACTGGCAGCACTGAT

[0114] GACCCTGTCCGCACCACTGGCAGCAGAATGGGCAACCCCACAAGTGGTG

[0115] GCACCTGAAGCAGAAGAAGGCGCAGAAGGCGAACTGACTCAGAAGAAC

[0116] TTCTGGGCAACCGTGCAAGGCGCACTGCGCCCTGGCGATATCATCCTGGC

[0117] AGATCAAGGCACCGCAGCATTCGGCATCGCAGCACTGAAGCTGCCATCC

[0118] GAAGCATCCCTGATCGTGCAGCCACTGTGGGGCTCCATCGGCTTCACCCT

[0119] GCCTGCAGCATACGGCGCACAGACCGCAGCCGCAGAACGCCGCGTGGTG

[0120] CTGATCGTGGGCGATGGCGCCGCACAGCTGACCATCCAAGAAATGGGCT

[0121] CCATGCTGCGCGATAAGCAAAAGCCACTGATCCTGCTCCTGAACAACGA

[0122] AGGCTACACCGTGGAACGCGCAATCCACGGCCCTGAACAGCGCTACAAC

[0123] GATATCGCACTGTGGGATTGGCAGCGCCTGCCTGAAGCATTCGCACCTGA

[0124] TGTGGCATCCCGCTGCCGCCGCGTGACTCAGACCTCCGAACTGCGCGAAG

[0125] CAATGACCGAATCCATCACCTCCGATACCCTGACCCTGGTGGAAGTGATG

[0126] CTGCCAAAGATGGATATCCCTGATTTCCTGCGCGCAGTGACCCAAGCACT

[0127] GGAAGAACGCAACTCCCGCGTG

[0128] SEQ ID NO.4

[0129] ATGCAGCCAACCTACACCATCGGCGATTACCTGCTGGATCGCCTGGTGGA

[0130] TTGCGGCATCGATCGCCTGTTCGGCGTGCCTGGCGATTACAACCTGCAGT

[0131] TCCTGGATCGCGTGATCGCACACTCCGCACTGGGCTGGGTGGGCTGCGCA

[0132] AACGAACTGAACGCAGCATACGCAGCAGATGGCTACGCACGCATCAAGG

[0133] GCGCCGGCGCCCTGCTGACCACCTACGGCGTGGGCGAACTGTCCGCACTG

[0134] AACGGCATCGCCGGCTCCTACGCAGAACACATCCCTGTGCTGCACATCGT

[0135] GGGCGCACCATCCACCGGCGCACAGCAGCGCGGCGAACTGCTGCACCAC

[0136] ACCCTCGGCGATGGCGATTTCCGCCACTTCGCACGCATGTCCGAACAGAT

[0137] CACCTGCTCCCAAGCACTGCTGACCGCCGGCAACGCATGCCACGAAATCG

[0138] ATCGCGTGCTCCGCGATATGCTGACCCACCACCGCCCTGGCTACCTGATG

[0139] CTGCCTGCAGATGTGGCACGCGCAGCCGCAATCGCACCTGCACAGCGCCT

[0140] GCTGGTGGAACCTGCACCTGCAGATGAAAATCAGCTGGCCGGCTTCTGCG

[0141] AACACGCATCCCGCCTGCTGCGCGGCTCCCGCCGCATCTCCCTGCTGGCA

[0142] GATTTCCTGGCACAGCGCTACGGCCTGCAGAAGACCCTGCGCGAATGGGT

[0143] GGCAAAGACCCCTGTGGCACACGCAACCATGCTGATGGGCAAGGGCCTG

[0144] TTCGATGAACAGCAGTCCGGCTTCGTGGGCACCTACTCCGGCATCGCATC

[0145] CGCACCACAGACCCGCGAAGCAATCGAAAACGCAGATACCATCATCTGC

[0146] ATCGGCACCCGCTTCACCGATACCATCACCGCCGGCTTCACTCAGCACCT

[0147] GGCACGCGAAAAGACCATCGAAATTCAGCCATTCGCAGTGCGCGTGGGC

[0148] GATCACTGGTTCTCCGGCGTGCCAATGGATAAGGCACTGGCAGCACTGAT

[0149] GACCCTGTCCGCACCACTGGCAGCAGAATGGGCAACCCCACAAGTGGTG

[0150] GCACCTGAAGCAGAAGAAGGCGCAGAAGGCGAACTGACTCAGAAGAAC

[0151] TTCTGGGCAACCGTGCAAGGCGCACTGCGCCCTGGCGATATCATCCTGGC

[0152] AGATCAAGGCACCGCAGCATTCGGCATCGCAGCACTGAAGCTGCCATCC

[0153] GAAGCATCCCTGATCGTGCAGCCACTGTGGGGCTCCATCGGCTTCACCCT

[0154] GCCTGCAGCATACGGCGCACAGACCGCAGCCGCAGAACGCCGCGTGGTG

[0155] CTGATCGTGGGCGATGGCGCCGCACAGCTGACCATCCAAGAAATGGGCT

[0156] CCATGCTGCGCGATAAGCAAAAGCCACTGATCCTGCTCCTGAACAACGA

[0157] AGGCTACACCTGGGAACGCGCAATCCACGGCCCTGAACAGCGCTACAAC

[0158] GATATCGCACTGTGGGATTGGCAGCGCCTGCCTGAAGCATTCGCACCTGA

[0159] TGTGGCATCCCGCTGCCGCCGCGTGACTCAGACCTCCGAACTGCGCGAAG

[0160] CAATGACCGAATCCATCACCTCCGATACCCTGACCCTGGTGGAAGTGATG

[0161] CTGCCAAAGATGGATATCCCTGATTTCCTGCGCGCAGTGACCCAAGCACT

[0162] GGAAGAACGCAACTCCCGCGTG

[0163] SEQ ID NO.5

[0164] ATGAGTATCTCAGTAAAAGCACTACAAAAGTCCGGCCCAGAAGCACCTTT

[0165] CGAGGTCAAGATCATTGAACGCCGTGACCCACGCGCAGATGATGTGGTT

[0166] ATTGATATCAAAGCTGCGGGCATCTGCCACAGCGATATCCACACCATCCG

[0167] CAACGAATGGGGCGAGGCGCACTTCCCGCTCACCGTCGGCCACGAAATC

[0168] GCAGGCGTTGTCTCTGCGGTTGGATCCGATGTAACCAAATGGAAAGTCGG

[0169] CGACCGCGTGGGCGTCGGCTGCCTCGTTAACTCCTGCGGCGAATGCGAAC

[0170] AGTGCGTCGCAGGATTTGAAAACAACTGCCTTCGCGGAAACGTCGGAAC

[0171] CTACAACTCTAACGACGTCGACGGCACCATCACCCAAGGCGGCTACGCTG

[0172] AAAAGGTAGTGGTCAACGAACGTTTCCTGTGCAGCATCCCAGAGGAACTT

[0173] AACTTCGATGTCGCAGCACCACTGCTGTGCGCAGGCATCACCACCTACTC

[0174] CCCAATCGCTCGCTGGAACGTTAAAGAAGGCGACAAAGTAGCAGTCATG

[0175] GGCCTCGGCGGACTCGGACACATGGGTGTCCAGATCGCTGCAGCCAAGG

[0176] GTGCTGAGGTTACCGTTCTGTCCCGTTCCCTGCGCAAGGCAGAACTTGCC

[0177] AAGGAACTCGGCGAGCTCGCACGCTTGCACTTCTGATGAGGATTTCTT

[0178] CACCGAACACGCCGGTGAATTCGACTTCATCCTCCAACACCATTAGCGCAT

[0179] CCATCCCAGTCGACAAGTACCTGAGCCTTCTCAAGCCACACGGTGTCATG

[0180] GCTGTTGTCGGTCTGCCACCAGAGAAGCAGCCACTGAGCTTCGGTGCGCT

[0181] CATCGGCGGCGGAAAAGTCCTCACCGGATCCAACATTGGCGGCATCCCTG

[0182] AAACCCAGGAAATGCTCGACTTCTGTGCAAAACACGGCTCGGTGCGAT

[0183] GATCGAAACTGTCGGCGTCAACGATGTTGATGCAGCCTACGACCGTGTTG

[0184] TTGCCGGCGACGTTCAGTTCCGCGTGTCATTGATACTGCTTCGTTTGCTG

[0185] AGGTTGAGGCGGTTTAG

[0186] SEQ ID NO.6

[0187] ACCCGTCGACAAAGGAGGACCATGAGTATCTCAGTAAAAGCACTAC SEQ ID NO.7

[0188] CCATTGGATCCTTAAACCGCCTCAACCTCAGCAAAC

[0189] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A ketoacid decarboxylase mutant, characterized in that: The ketoacid decarboxylase mutant expresses the ipdC mutant gene from Klebsiellapneumoniae and overexpresses the aldehyde reductase gene Cgl0331 through a recombinant plasmid or genome integration; Among them, the amino acid sequence of the ipdC mutant gene is shown in SEQ ID NO.2, the aldehyde reductase Cgl0331 is an endogenous aldehyde reductase of Corynebacterium glutamicum, and the nucleotide sequence of the aldehyde reductase Cgl0331 is shown in SEQ ID NO.

5.

2. The ketoacid decarboxylase mutant according to claim 1, characterized in that: The recipient bacteria is Corynebacterium glutamicum ATCC 13032.

3. The ketoacid decarboxylase mutant according to claim 1, characterized in that: The plasmid is pXMJ19.

4. A method for constructing a ketoacid decarboxylase mutant, characterized in that: The genome of C. glutamicum ATCC 13032 was used as a template, Cgl0331F and Cgl0331R were used as primers, and FastPfu high-fidelity polymerase was used to amplify a fragment of 1062 bp; the amplified product and the recombinant plasmid containing the ipdC mutant gene were digested with SalI and BamHI respectively and connected to obtain a recombinant vector; Transforming the recombinant vector into C. glutamicum ATCC 13032 by electroporation to obtain the ketoacid decarboxylase mutant; Among them, the nucleotide sequence of primer Cgl0331F is shown as SEQ ID NO.6, and the nucleotide sequence of primer Cgl0331R is shown as SEQ ID NO.

7.

5. A method for producing a phenethyl alcohol derivative, characterized in that: The method comprises the following steps: activating the ketoacid decarboxylase mutant strain and transferring it to a fermentation medium at an inoculation amount of 1-3%, adding IPTG with a final concentration of 1 mM to induce protein expression when OD600 is 5 and the temperature is 30°C, culturing at 30-37°C, 200 r / min, and a cycle of 72-80 hours.

6. The method for producing a phenylethanol derivative according to claim 5, characterized in that: The fermentation medium is 1 g / L of dipotassium hydrogen phosphate, 1 g / L of potassium dihydrogen phosphate, 0.25 g / L of magnesium sulfate, and 10 mg / L of calcium chloride. -1 , ferrous sulfate 10 mg L -1 , manganese sulfate 0.1 mg L -1 , zinc sulfate 1mg L -1 , copper sulfate 0.2 mg L -1 , nickel chloride 20 μg L -1 , biotin 0.4mg L -1 , 3-morpholinepropanesulfonic acid 28g / L, yeast extract 3g / L, vitamin B120mg / L, vitamin B620mg / L.

7. The method for producing a phenethyl alcohol derivative according to claim 5, characterized in that: The method uses L-phenylalanine as a substrate and adopts a biological method to synthesize 2-phenylethanol.

8. The method for producing a phenethyl alcohol derivative according to claim 5, characterized in that: The method uses L-tyrosine as a substrate and adopts a biological method to synthesize tyrosol.

9. The method for producing a phenethyl alcohol derivative according to claim 5, characterized in that: The method uses L-DOPA as a substrate and adopts a biological method to synthesize hydroxytyrosol.

10. Use of the ketoacid decarboxylase mutant according to claim 1 in the preparation of phenylethanol derivatives.

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