CN114574458A - Chalcone synthetase mutant capable of increasing naringenin yield - Google Patents

Abstract

The invention discloses a chalcone synthase mutant and an application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The present invention provides a series of chalcone synthase mutants that contribute to the increased accumulation of naringenin. Under appropriate culture conditions, the yields of mutants A308K, A308R, S208N, S208C, E61K, E61R, E61A, E61T, E61C, A308K‑S208N, A308K‑E61K and S208N‑E61K chalcone synthases synthesized naringenin, respectively It is 1.41 times, 1.24 times, 1.44 times, 1.28 times, 1.30 times, 1.10 times, 1.17 times, 1.09 times, 1.11 times, 1.05 times, 1.23 times and 1.23 times of the wild enzyme.

Description

Chalcone synthase mutants that increase naringenin production

technical field

The invention relates to a chalcone synthase mutant capable of increasing the yield of naringenin, and belongs to the technical field of genetic engineering and enzyme engineering.

Background technique

Flavonoids are a class of plant secondary metabolites with two phenolic hydroxyl benzene rings linked by the central three carbons, and are widely present in the plant kingdom. Based on the C6-C3-C6 structure, these compounds are divided into the following categories according to the degree of oxidation of the three carbon bonds (C3) and the difference in conformation: flavonoids, flavonols, flavanones (dihydroflavones), flavanones Alcohols (dihydroflavonols), isoflavones, isoflavones (dihydroisoflavones), chalcones, dihydrochalcones, flavans, flavanols and other flavonoids. In plants, it is usually combined with sugars to form glycosides in the form of ligands, and a small part exists in the form of free aglycones. Flavonoids not only play an important role in the growth and development of plants, but also have pharmacological activities such as antibacterial, anti-oxidative free radicals, anti-aging, anti-cardiovascular and cerebrovascular diseases, and liver protection. Therefore, the study of flavonoids has always been a hot topic in the field of biomedicine at home and abroad.

In plants, the biosynthetic pathway of flavonoids begins with phenylalanine ammonia lyase (PAL) and cinnamic acid 4-hydroxylase (C4H) catalyzing the synthesis of p-coumaric acid from L-phenylalanine, or starting with Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid. Subsequently, 4-hydroxycinnamic acid-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) convert p-coumaric acid to naringenin. As a platform compound, naringenin can synthesize various high value-added flavonoids through a series of derivatization reactions, such as methoxylation, hydroxylation, glycosylation and prenylation. Among the four enzymes in the naringenin synthesis pathway, chalcone synthase, which catalyzes the synthesis of one molecule of chalcone naringenin by 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA, has been confirmed to be one of them. Critical speed-limiting steps. Increasing the copy number or expression intensity of chalcone synthase can significantly increase the production of naringenin.

For rate-limiting enzymes, increasing the copy number or expression level may increase the metabolic burden of host bacteria and thus affect the growth of bacteria and the synthesis of products. Improving the catalytic efficiency of enzymes by site-directed mutagenesis can improve the pathway efficiency without affecting the growth of bacteria. CaoWeijia et al. ("Enhanced pinocembrin production in Escherichiacoli by regulating cinnamic acid metabolism", published in 2016) increased the production of pinocinocin through site-directed mutation of chalcone synthase, but so far, through directed evolution of chalcone synthase to improve The yield of naringenin has not yet been reported.

SUMMARY OF THE INVENTION

In order to improve the yield of naringenin, the present invention utilizes the means of genetic engineering and enzyme engineering to replace one or more of the amino acids at the 61st, 208th and 308th positions of the chalcone synthase derived from Sophorajaponica. The mutant of naringenin was prepared, and the chalcone synthase mutant that could increase the production of naringenin was prepared, and the high-efficiency enzyme element was provided for the high production of naringenin by fermentation.

The present invention provides a chalcone synthase mutant, the mutant takes the chalcone synthase whose amino acid sequence is shown in SEQ ID NO. f) mutants:

(a) The mutants replace the alanine at position 308 with lysine or arginine, and the mutants are named A308K and A308R;

(b) the mutants replace serine at position 208 with asparagine or cysteine, and the mutants are named S208N and S208C;

(c) the mutant is to replace the glutamic acid at position 61 with lysine or arginine or alanine or threonine or cysteine, and the mutants are named as E61K, E61R, E61A, E61T and E61C;

(d) the mutant is to replace the alanine at the 308th position with lysine, and replace the serine at the 208th position with asparagine, and the mutant is named A308K-S208N;

(e) the mutant is to replace the alanine at the 308th position with lysine, and replace the glutamic acid at the 61st position with lysine, and the mutant is named A308K-E61K;

(f) In the mutant, serine at position 208 was substituted with asparagine, and glutamic acid at position 61 was substituted with lysine, and the mutant was named S208N-E61K.

In one embodiment, the chalcone synthase is derived from Sophora japonica.

In one embodiment, the nucleotide sequence of the gene encoding the chalcone synthase is shown in SEQ ID NO.2.

The present invention provides genes encoding the mutants.

The present invention provides a recombinant vector carrying the gene.

In one embodiment, the recombinant vectors include, but are not limited to, the pY26 series.

The present invention provides microbial cells expressing the mutant, or carrying the gene, or containing the recombinant vector.

In one embodiment, the microbial cells include prokaryotic and eukaryotic microorganisms.

In one embodiment, the starting strain of the microbial cell comprises Saccharomyces cerevisiae.

Preferably, Saccharomyces cerevisiae YJ1 is used as the starting strain, and the Saccharomyces cerevisiae YJ1 has knocked out the GAL80 gene in Saccharomyces cerevisiae CEN.PK2-1D, and integrated and expressed 4-coumaroyl-CoA ligase and chalcone in its genome Isomerase; the NCBI accession number of the 4-coumaroyl-CoA ligase is KF765780.1, the NCBI accession number of the chalcone isomerase is P28012.1; the Gene ID of the GAL80 gene: 854954 .

In one embodiment, the pY26-aro10UD-P _GAL1 -Pc4CL-P _GAL7 -MsCHI-TRP1 plasmid expressing 4-coumaroyl-CoA ligase and chalcone isomerase is integrated into a GAL80 knockout gene The aro10 locus of Saccharomyces cerevisiae.

In one embodiment, the pY26-aro10UD-P _GAL1 -Pc4CL-P _GAL7 -MsCHI-TRP1 plasmid is disclosed in the document "Improving(2S)-naringenin production by exploring native precursorpathways and screening higher-active chalcone synthases from plants rich inflavonoids.

In one embodiment, the aro10 has Gene ID: 851987.

The invention provides a method for producing naringenin, which utilizes the microbial cells to transform p-coumaric acid to produce naringenin.

In one embodiment, a single colony of the microbial cells is placed in a YNB medium without uracil, cultured at 220 rpm for 20 to 24 hours to obtain a bacterial liquid, and the bacterial liquid is adjusted according to 1 to 5% (v/v) The amount was transferred to YPD medium, and 200-250 mg/L p-coumaric acid was added to the YPD medium.

In one embodiment, the culture is carried out at 28-35° C. and 220 rpm for not less than 60 h.

Preferably, the culture is not less than 72h.

The present invention provides the application of the chalcone synthase mutant, or the gene, or the microbial cell in preparing naringenin, naringenin derivatives or products containing naringenin.

Beneficial effects of the present invention:

The invention uses chalcone synthase in Sophora japonica as the starting gene, changes the amino acid sequence of chalcone synthase through site-directed mutation technology, and finally obtains 12 chalcone synthase mutants that can improve the production of naringenin A308K, A308R, S208N, S208C, E61K, E61R, E61A, E61T, E61C, A308K-S208N, A308K-E61K and S208N-E61K. By fermenting strains expressing chalcone synthase mutants, the yields of naringenin reached 165.33mg/L, 145.11mg/L, 168.98mg/L, 149.87mg/L, 152.82mg/L, 129.24mg/L, 137.49mg/L, 128.75mg/L, 130.05mg/L, 123.92mg/L, 143.96mg/L and 143.71mg/L, respectively 1.41 times, 1.24 times and 1.44 times of the strains expressing wild-type chalcone synthase. , 1.28 times, 1.30 times, 1.10 times, 1.17 times, 1.09 times, 1.11 times, 1.05 times, 1.23 times and 1.23 times.

Description of drawings

Figure 1 is the construction diagram of the chalcone synthase expression vector (pY26-SjCHS1) transformed by site-directed mutagenesis.

Figure 2 is a graph comparing the accumulation of naringenin by chalcone synthase mutants A308K, A308R, S208N, S208C, E61K, E61R, E61A, E61T, E61C and wild-type chalcone synthase.

Figure 3 is a graph comparing naringenin accumulation of chalcone synthase mutants A308K-S208N, A308K-E61K and S208N-E61K with wild-type chalcone synthase.

Fig. 4 is a graph showing the production of naringenin accumulated by a chalcone synthase mutant synthase in which alanine at position 308 is mutated to other 17 amino acids.

Fig. 5 is a graph showing the production of naringenin accumulated by a chalcone synthase mutant synthase in which alanine at position 208 is mutated to other 17 amino acids.

Fig. 6 is a graph showing the production of naringenin accumulated by a chalcone synthase mutant synthase in which glutamic acid at position 61 is mutated to other 17 amino acids.

Detailed ways

1. Culture medium and reagents

LB medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L. Add 20g/L agar powder to prepare LB solid medium.

YPD medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L. Add 20g/L agar powder to prepare YPD solid medium.

YNB medium: Yeast nitrogen source basal medium 1.74g/L (without ammonium sulfate), ammonium sulfate 5g/L, glucose 20g/L, amino acids (5g/L uracil, 10g/L tryptophan, 10g/L Leucine, 10g/L histidine, appropriate amino acids to be deleted as needed).

2. Construction of chassis cell YJ1

Construction of GAL80 knockout chassis cells based on Saccharomyces cerevisiae CEN.PK2-1D (GAL80 is involved in the regulation of galactose metabolism, and the GAL7 promoter can be constitutively expressed after knockout without inducible expression); knockout using the principle of yeast homologous recombination GAL80 (Gene ID: 854954), with G418 as the selection marker, was constructed to obtain the chassis cell C800. For the specific construction process, see the document Promoter-Library-Based Pathway Optimization for Efficient(2S)-NaringeninProduction From p-Coumaric Acid in Saccharomyces cerevisiae, Song Gao , Hengruizhou, Jingwen Zhou, Jian chen. J Agric Food Chem, 2020 Jun 24.

Chassis cell YJ1 was constructed based on Saccharomyces cerevisiae C800; pY26-aro10UD-P _GAL1 -Pc4CL-P _GAL7 -MsCHI-TRP1 (《Improving(2S)-naringenin production by exploring native precursor pathways and screening higher-active chalcone synthases from plants rich in flavonoids", published in 2021) was integrated into the aro10 site of the Saccharomyces cerevisiae C800 genome, the Gene ID of the aro10: 851987; using TRP as the selection marker, the chassis cell YJ1 was constructed.

3. HPLC detection of naringenin

Using a chromatographic column (250×4.6mm, 5μm, Thermo-Fisher, MA, USA), the detection conditions were at 40°C with a SPD-20A detector from Shimadzu, Japan, and the mobile phase was an aqueous solution containing 0.1% trifluoroacetic acid (A ) and acetonitrile (B) containing 0.1% trifluoroacetic acid, elution at a flow rate of 1 mL/min, detection wavelength 290 nm, elution conditions 0-10 min, 90%-60% A; 10-15 min, 60%-40 %A; 15-17min, 40%-90%A; 17-25min, 90%A.

Example 1 : Construction and expression of recombinant plasmid pY26-SjCHS1

(1) Construction of recombinant plasmid pY26-SjCHS1

Chalcone synthase 1 from Sophora japonica has the ability to catalyze 3 molecules of p-coumaroyl-CoA and 1 molecule of malonyl-CoA to synthesize 1 molecule of chalcone-naringenin, and finally obtains nucleotides after codon optimization The sequence is shown in SEQID NO.2, constructed on the pY26 plasmid, named pY26-SjCHS1, and synthesized by Suzhou Jinweizhi Company.

(2) Expression of chalcone synthase

The correctly sequenced plasmid was transformed into Saccharomyces cerevisiae YJ1 by the lithium acetate chemical transformation method, and cultured on a YNB plate without uracil at 30 °C for 3 days until a single colony grew; In YNB medium, cultured at 220rpm for 24h; then transferred to YPD medium according to the amount of 1% (v/v), and added 250mg/L p-coumaric acid, cultured at 30°C for 72h and sampled the liquid phase for detection of pomelo peel The yield of naringenin was 74.36 mg/L.

Example 2: Preparation and expression of chalcone synthase single mutant mutant

(1) Preparation of mutants

According to the pY26-SjCHS1 plasmid sequence, primers introducing mutations A308K, A308R, S208N, S208C, E61K, E61R, E61A, E61T and E61C were designed and synthesized respectively, and the gene sequence of chalcone synthase was site-directed mutation, and sequenced to confirm the search. Check whether the coding gene of the chalcone synthase mutant is correct, and obtain a single mutant chalcone synthase; introduce the vector carrying the mutant gene into Saccharomyces cerevisiae YJ1 for expression.

PCR amplification of site-directed mutant encoding gene: using PCR technology, with the expression vector pY26-SjCHS1 plasmid that encodes the wild-type chalcone synthase gene as a template, PCR amplification is performed, and the primer pairs are as follows (capital letters are mutant bases) ):

The site-directed mutagenesis primer pair F1/R1 for introducing the A308K mutation is:

F1: gctcatccaggtggtcccAAAattctagatcaag,

R1:gggaccacctggatgagctacccag;

The site-directed mutagenesis primer pair F2/R2 for introducing the A308R mutation is:

F2: gtagctcatccaggtggtcccAGAattctagatcaagtcgaag,

R2: cttcgacttgatctagaatTCTgggaccacctggatgagctac;

The site-directed mutagenesis primer pair for introducing the S208N mutation is F3/R3:

F3: agtgatactcacttggatAATttggtgggccaag,

R3: tccaagtgagtatcactaggaccacgaaaggtcacc;

The site-directed mutagenesis primer pair for introducing the S208C mutation is F4/R4:

F4: agtgatactcacttggatTGTttggtgggccaagc,

R4: atccaagtgagtatcactaggaccacgaaag;

The site-directed mutagenesis primer pair for introducing E61K mutation is F5/R5:

F5: gaaaagttcaagcgtatgtgcAAAaaatctatgataaagaagag,

R5: ctcttctttatcatagatttTTTgcacatacgcttgaacttttc;

The primer pair for site-directed mutagenesis to introduce the E61R mutation is F6/R6:

F6: gaaaagttcaagcgtatgtgcAGAaaatctatgataaagaagag,

R6: ctcttctttatcatagatttTCTgcacatacgcttgaacttttc;

The primer pair for site-directed mutagenesis to introduce the E61A mutation is F7/R7:

F7: aagttcaagcgtatgtgcGCTaaatctatgataaag,

R7: gcacatacgcttgaacttttcctttaggtct;

The site-directed mutagenesis primer pair for introducing E61T mutation is F8/R8:

F8: aagttcaagcgtatgtgcACTaaatctatgataaag,

R8: gcacatacgcttgaacttttcctttaggtct;

The primer pair for site-directed mutagenesis to introduce the E61C mutation is F9/R9:

F9: aagttcaagcgtatgtgcTGTaaatctatgataaag,

R9: gcacatacgcttgaacttttcctttaggtct.

The PCR system was: 25 μL 2×Phanta Max Master Mix, 1 μL forward primer (10 μmol·L ⁻¹ ), 1 μL reverse primer (10 μmol·L ⁻¹ ), 1 μL template DNA, and distilled water was added to 50 μL. The chalcone synthase PCR amplification program was set as follows: first, pre-denaturation at 95°C for 3 min; then into 30 cycles: denaturation at 95°C for 30s, annealing at 56°C for 30s, extension at 72°C for 5min; final extension at 72°C for 5min, 4°C Insulation.

(2) Expression of mutants

The mutant plasmid with correct sequencing was transformed into Saccharomyces cerevisiae YJ1 by lithium acetate chemical transformation method, and cultured on YNB plate without uracil at 30 °C for 3 days until a single colony grew; pick a single colony without urine In the YNB medium of pyrimidine, cultivate at 220rpm for 24h to obtain activated bacterial liquid; transfer the activated bacterial liquid to YPD medium according to 1% (v/v), add 250mg/L p-coumaric acid, and sample the liquid phase for 72h Detection of naringenin production.

The results showed that the yields of naringenin from strains expressing plasmids containing mutants A308K, A308R, S208N, S208C, E61K, E61R, E61A, E61T and E61C chalcone synthase mutants were 165.33 mg/L, 145.11 mg/L, 168.98mg/L, 149.87mg/L, 152.82mg/L, 129.24mg/L, 137.49mg/L, 128.75mg/L and 130.05mg/L, the naringenin yields were respectively the strains expressing wild-type chalcone synthase. 1.41 times, 1.24 times, 1.44 times, 1.28 times, 1.30 times, 1.10 times, 1.17 times, 1.09 times and 1.11 times.

Example 3: Preparation and expression of chalcone synthase double mutant mutants

(1) Preparation of mutant A308K-S208N

The mutant A308K constructed in Example 2 was used as the initial template, and the S208N mutant primers designed in Example 2 were used to carry out PCR technology (method as described in Example 2) to carry the plasmid encoding the mutant A308K gene. Site-directed mutagenesis to construct mutant A308K-S208N. And confirmed by sequencing whether the chalcone synthase A308K-S208N mutant is correct.

(2) Preparation of mutant A308K-E61K

Using the mutant A308K constructed in Example 2 as the initial template, and using the E61K mutant primers designed in Example 2, using PCR technology (the method is as described in Example 2), the plasmid carrying the gene encoding the mutant A308K was carried out. Site-directed mutagenesis to construct mutant A308K-E61K. And confirmed by sequencing whether the chalcone synthase A308K-E61K mutant is correct.

(3) Preparation of mutant S208N-E61K

Using the mutant S208N constructed in Example 2 as the initial template, and using the E61K mutant primers designed in Example 2, using PCR technology (the method is as described in Example 2), the plasmid carrying the gene encoding the mutant S208N was carried out. Site-directed mutagenesis to construct mutant S208N-E61K. And confirmed by sequencing whether the chalcone synthase S208N-E61K mutant is correct.

(4) Expression of mutants

The mutant expression procedure was as described in Example 1 for the expression of chalcone synthase. The results showed that the yields of naringenin from strains expressing plasmids containing mutants A308K-S208N, A308K-E61K and S208N-E61K chalcone synthase mutants were 123.92 mg/L, 143.96 mg/L and 143.71 mg/L, respectively. The yields of corticosteroids were 1.17 times, 1.09 times and 1.11 times that of strains expressing wild-type chalcone synthase, respectively.

Comparative Example 1

For specific embodiments, see Example 2, the difference is that the alanine at position 308 was mutated to the remaining 17 amino acids, and the mutants A308N, A308D, A308C, A308Q, A308E, A308G, A308H, A308I, A308L, A308M were constructed respectively. , A308F, A308P, A308S, A308T, A308W, A308Y, A308V;

Similarly, the serine at position 208 was mutated to the remaining 17 amino acids to construct mutants S208N, A208D, A208R, A208Q, A208E, A208G, A208H, A208I, A208L, A208M, A208F, A208P, A208S, A208T, A208W , A208Y, A208V;

The glutamic acid at position 61 was mutated to the remaining 14 amino acids, and mutants E61N, A308D, E61Q, E61G, E61H, E61I, E61L, E61M, E61F, E61P, E61S, E61W, E61Y, and E61V were respectively constructed.

The mutant obtained by the above construction was expressed according to the method in Example 2, and the content of naringenin in the fermentation broth was detected at 72 h. The results are shown in Figure 4, Figure 5, and Figure 6. The element content is lower than the original enzyme.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> BAA220031A

<130> Chalcone synthase mutant that increases naringenin production

<160> 2

<170> PatentIn version 3.3

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<211> 391

<212> PRT

<213> Sophora japonica

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Met Val Thr Val Glu Glu Ile Arg Asn Ala Gln Arg Ser Gln Gly Pro

1 5 10 15

Ala Thr Ile Leu Ala Phe Gly Thr Ala Thr Pro Ser Asn Cys Ile Thr

20 25 30

Gln Ala Asp Tyr Pro Asp Tyr Tyr Phe Arg Ile Thr Asp Ser Glu His

35 40 45

Met Thr Asp Leu Lys Glu Lys Phe Lys Arg Met Cys Glu Lys Ser Met

50 55 60

Ile Lys Lys Arg Tyr Met His Val Thr Glu Glu Phe Leu Lys Glu Asn

65 70 75 80

Pro Asn Met Cys Ala Tyr Met Ala Pro Ser Leu Asp Val Arg Gln Asp

85 90 95

Ile Val Val Val Glu Val Pro Lys Leu Gly Lys Glu Ala Ala Ser Lys

100 105 110

Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His Leu Val

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Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp Tyr Gln Leu

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Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Leu Met Met Tyr

145 150 155 160

Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu Ala Lys Asp

165 170 175

Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val Cys Ser Glu

180 185 190

Ile Thr Ala Val Thr Phe Arg Gly Pro Ser Asp Thr His Leu Asp Ser

195 200 205

Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala Met Ile Ile

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Gly Ala Asp Pro Asp Thr Ala Val Glu Arg Pro Ile Phe Gln Leu Val

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Ser Ala Ala Gln Thr Ile Leu Pro Asp Ser Asp Gly Ala Ile Asp Gly

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His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys Asp Val Pro

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Gly Ile Ile Ser Lys Asn Ile Glu Lys Ser Leu Val Glu Ala Phe Ala

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Pro Val Gly Ile Ser Asp Trp Asn Ser Ile Phe Trp Val Ala His Pro

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Met Ser Ser Ala Cys Val Leu Phe Ile Leu Asp Glu Met Arg Lys Lys

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His Ser Val Pro Leu Gln Gly

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<213> Artificial sequences

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atggtcaccg ttgaagaaat taggaatgcc caacgttccc aaggacctgc caccattctt 60

gcgtttggca cggctacccc ctctaactgc attacccaag cagactatcc agattactat 120

tttcgtatta ccgattccga gcacatgaca gacctaaagg aaaagttcaa gcgtatgtgc 180

gagaaatcta tgataaagaa gagatatatg catgtaactg aggagtttct gaaggaaaac 240

cccaacatgt gtgcttacat ggctccttca ctagatgtta gacaagacat cgtagttgta 300

gaggtaccta agttgggtaa agaggcagcg agtaaggcaa taaaggagtg ggggcaaccc 360

aagtccaaga ttacacacct tgtttttttgc acaactagtg gggtagacat gccaggtgct 420

gattatcagc taaccaagtt gcttggtcta aggccgtcag taaagaggct tatgatgtat 480

caacaaggat gcttcgcagg aggaaccgtg cttaggcttg caaaagacct tgccgagaac 540

aacaagggtg cgagagtatt ggttgtttgt tcagagatca cggcggtgac ctttcgtggt 600

cctagtgata ctcacttgga tagcttggtg ggccaagccc tattcggaga tggagcagct 660

gcaatgataa ttggcgcgga cccggatacc gctgtcgaga ggcctatctt ccagttggtt 720

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gtcggactaa ccttccatct attgaaagac gtccctggaa ttatctcaaa gaacatagaa 840

aaatcactgg ttgaggcttt tgcgcccgtt ggaatttcag actggaacag tatattctgg 900

gtagctcatc caggtggtcc cgcgattcta gatcaagtcg aagccaaact aggactaaaa 960

gaggagaaac ttaggtcaac taggcacgta ctatcagaat atgggaatat gagtagcgcg 1020

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Claims (10)

1. chalcone synthase mutant, it is characterized in that, described mutant takes aminoacid sequence as the chalcone synthase shown in SEQ ID NO.1 as parent, and the following positions are replaced respectively to obtain (a) Mutants of ~(f): (a) the mutant replaces the alanine at position 308 with lysine or arginine; (b) the mutant replaces serine at position 208 with asparagine or cysteine; (c) the mutant replaces glutamic acid at position 61 with lysine or arginine or alanine or threonine or cysteine; (d) in the mutant, alanine at position 308 is substituted with lysine, and serine at position 208 is substituted with asparagine; (e) the mutant replaces alanine at position 308 with lysine, and replaces glutamic acid at position 61 with lysine; (f) In the mutant, serine at position 208 is substituted with asparagine, and glutamic acid at position 61 is substituted with lysine. 2. A gene encoding the mutant of claim 1. 3. A recombinant vector carrying the gene of claim 2. 4. A microbial cell expressing the mutant of claim 1, or carrying the gene of claim 2, or containing the recombinant vector of claim 3. 5. The microbial cell according to claim 4, wherein the microbial cell comprises a prokaryotic microorganism and a eukaryotic microorganism. 6. The microbial cell according to claim 4 or 5, wherein the starting strain of the microbial cell comprises Saccharomyces cerevisiae YJ1. 7. A method for producing naringenin, characterized in that naringenin is produced by transforming p-coumaric acid with the microbial cells described in any one of claims 4 to 6. 8 . The method according to claim 7 , wherein the bacterial solution is obtained by culturing a single colony of the microbial cells for 20 to 24 hours, and the bacterial solution is transferred to YPD in an amount of 1 to 5% (v/v). 9 . medium, and 200-250 mg/L p-coumaric acid was added to the YPD medium. 9 . The method according to claim 8 , wherein the culture is carried out at 28-35° C. for not less than 60 hours. 10 . 10. The chalcone synthase mutant of claim 1, or the gene of claim 2, or the microbial cell of any one of claims 4 to 6 in the preparation of naringenin, naringenin derivatives or Use in products containing naringenin.

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