CN113502278A - Enzyme composition and application thereof in naringenin biosynthesis - Google Patents

Enzyme composition and application thereof in naringenin biosynthesis Download PDF

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CN113502278A
CN113502278A CN202110913251.9A CN202110913251A CN113502278A CN 113502278 A CN113502278 A CN 113502278A CN 202110913251 A CN202110913251 A CN 202110913251A CN 113502278 A CN113502278 A CN 113502278A
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naringenin
nucleic acid
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ebchs
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赵广荣
李玲玲
刘雪
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Georgia Institute Of Technology Tianjin University Shenzhen
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Abstract

The invention relates to the technical field of biology, in particular to an enzyme composition and application thereof in naringenin biosynthesis. According to the invention, the enzyme composition capable of effectively promoting naringenin is obtained by researching the interaction relationship between the chalcone synthase CHSs and the chalcone isomerase-like proteins CHILs. The composition takes p-coumaric acid as a substrate, the synthesized naringenin level can reach 187.10mg/L, and the naringenin yield is improved by 25.95% compared with the situation without CHIL. Meanwhile, the content of CTAL of the derailment product is reduced by 69.80 percent. The combination has important significance for promoting the industrial production of naringenin products and lays a foundation for the high-efficiency synthesis of other flavone products catalyzed by CHS.

Description

Enzyme composition and application thereof in naringenin biosynthesis
Technical Field
The invention relates to the technical field of biology, in particular to an enzyme composition and application thereof in naringenin biosynthesis.
Background
Naringenin (Naringenin) is an aglycone of naringin, belongs to flavanone compounds, and is widely present in rutaceae plants. Naringenin is chemically named as 4', 5, 7-trihydroxyflavanone, and has multiple sites in its molecular structure for glycosylation, methylation, isopentenyl, etc. to form flavone derivatives with high added value, including genistein, naringin, apigenin, breviscapine, etc. Naringenin also has biological activities of resisting oxidation, resisting inflammation, regulating immune response, etc., and is a kind of medicine for treating bacterial infection, tranquilizing and resisting cancer.
Naringenin biosynthesis starts from p-coumaric acid, is converted into p-coumaroyl CoA under the action of p-coumaroyl CoA ligase (4-coumarate: coenzyme A ligase,4CL), and then Chalcone synthase (CHS) catalyzes 1 molecule of p-coumaroyl CoA and 3 molecules of malonyl CoA to perform claisen condensation reaction to form naringenin Chalcone, and is catalyzed by Chalcone isomerase (CHI) to perform stereoisomeric ring closure to form naringenin. Among them, CHS has non-specific substrate, and is easy to catalyze and condense different amounts of malonyl CoA to generate derailed byproducts such as CTAL, BNY and the like, which becomes the rate-limiting step in naringenin synthesis.
Chalcone isomerase-like proteins (CHILs), which are type IV CHIs, are widely present in terrestrial plants, and can interact with CHS of the same species through proteins, so that the catalytic efficiency of the CHS is enhanced, and the generation of a derailment product CTAL is reduced. PhCHIL from petunia has been identified to enhance the accumulation of plant anthocyanidins, but there has been no report of its use in the synthesis of naringenin.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an enzyme composition for reducing the production of derailed byproducts, and its application in naringenin biosynthesis.
The enzyme composition provided by the invention consists of chalcone synthetase and chalcone isomerase-like protein; wherein,
the chalcone synthetase is selected from at least one of soybean GmCHS, apple MdCHS, hypericum HaCHS, petunia PhCHS, sorghum SbCHS and erigeron breviscapus EbCHS;
the chalcone isomerase-like protein is at least one selected from apple MdCHIL, petunia PhCHIL, soybean GmCHIL and Arabidopsis AtCHIL.
In the examples of the present invention, the enzyme composition;
consists of GmCHS and PhCHIL;
or consists of GmCHS and MdCHIL;
or consists of GmCHS and GmCHIL;
or consists of GmCHS and AtCHIL;
or consisting of MdCHS and PhCHIL;
or consists of MdCHS and MdCHIL;
or consists of MdCHS and GmCHIL;
or consisting of MdCHS and AtCHIL;
or consisting of HaCHS and PhCHIL;
or consisting of HaCHS and MdCHIL;
or consisting of HaCHS and GmCHIL;
or consisting of HaCHS and AtCHIL;
or consisting of PhCHS and PhCHIL;
or consisting of PhCHS and MdCHIL;
or consisting of PhCHS and GmCHIL;
or consisting of PhCHS and AtCHIL;
or consisting of SbCHS and PhCHIL;
or consisting of SbCHS and MdCHIL;
or consisting of SbCHS and GmCHIL;
or consisting of SbCHS and AtCHIL;
or EbCHS and PhCHIL;
or consists of EbCHS and MdCHIL;
or EbCHS and GmCHIL;
or EbCHS and AtCHIL.
The prior art research shows that the chalcone isomerase-like proteins CHILs can not realize naringenin biosynthesis by using p-coumaric acid as a substrate. However, the CHILs can generate protein interaction with chalcone synthetase CHS of the same species, and the catalytic efficiency of the CHS is enhanced. However, the research of the application shows that some CHILs can promote the generation of naringenin and reduce the generation of derailed byproducts after being combined with CHS of other species. Wherein, the enzyme combination formed by combining MdCHS, HaCHS, PhCHS, SbCHS or EbCHS with CHILs can improve the enzyme activity of CHS to different degrees. Among them, the more significant effect promoted by CHILs is MdCHS, PhCHS or SbCHS. Wherein, the activity of PhCHS, MdCHS and SbCHS is obviously improved under the promotion action of MdCHIL.
Of all the compositions attempted in the present invention, the enzyme compositions that produced higher levels of naringenin were: PhCHS + PhCHIL, PhCHS + MdCHIL, PhCHS + GmCHIL, SbCHS + PhCHIL, SbCHS + MdCHIL, SbCHS + GmCHIL, SbCHS + AtCHIL, EbCHS + PhCHIL, EbCHS + MdCHIL, EbCHS + GmCHIL, and EbCHS + AtCHIL.
Among the enzyme compositions that produce higher levels of naringenin are PhCHS + MdCHIL, SbCHS + PhCHIL, SbCHS + MdCHIL, SbCHS + GmCHIL, EbCHS + PhCHIL, EbCHS + MdCHIL, EbCHS + GmCHIL, and EbCHS + AtCHIL.
Further, enzyme compositions that produce higher naringenin levels were SbCHS + MdCHIL, SbCHS + GmCHIL, EbCHS + PhCHIL, EbCHS + MdCHIL, EbCHS + GmCHIL, and EbCHS + AtCHIL.
Further, the enzyme composition that produced the highest naringenin levels was EbCHS + PhCHIL.
The invention also provides nucleic acids encoding chalcone synthase and/or chalcone isomerase-like proteins in the enzyme compositions.
In embodiments of the invention, the nucleic acid encoding the chalcone synthase or chalcone isomerase-like protein is codon optimized. So that the nucleic acid fragment is more suitable for being expressed in a recombinant strain compared with a wild type nucleic acid fragment.
The sequence of the nucleic acid for coding the soybean GmCHS is shown as SEQ ID NO. 3;
the sequence of the nucleic acid for coding the apple MdCHS is shown as SEQ ID NO. 4;
the sequence of the nucleic acid for coding the hypericum HaCHS is shown as SEQ ID NO. 5;
the sequence of the nucleic acid for coding petunia PhCHS is shown as SEQ ID NO. 6;
the sequence of the nucleic acid for coding the SbCHS of the sorghum is shown as SEQ ID NO. 7;
the sequence of the nucleic acid for coding the erigeron breviscapus EbCHS is shown as SEQ ID NO. 8;
the sequence of the nucleic acid for coding the apple MdCHIL is shown as SEQ ID NO. 9;
the sequence of the nucleic acid for coding petunia PhCHIL is shown as SEQ ID NO. 10;
the sequence of the nucleic acid for coding the soybean GmCHIL is shown as SEQ ID NO. 11;
the sequence of the nucleic acid encoding Arabidopsis AtCHIL is shown in SEQ ID NO. 12.
The invention also provides plasmid vectors comprising the nucleic acids of the invention.
The construction method of the plasmid vector comprises inserting a nucleic acid segment coding for chalcone synthetase and/or chalcone isomerase-like protein into a corresponding site of the skeleton vector.
The plasmid vector of the present invention contains only a nucleic acid fragment encoding a chalcone isomerase-like protein or only a nucleic acid fragment encoding a chalcone synthase. Or a nucleic acid fragment encoding a chalcone isomerase-like protein and a nucleic acid fragment encoding a chalcone synthase together. In the present embodiment, the plasmid vector contains only one of a nucleic acid fragment encoding a chalcone isomerase-like protein or a nucleic acid fragment encoding a chalcone synthase.
In the present invention, the backbone vector of the plasmid vector expressing chalcone synthase is pNR1 (fig. 5); the backbone vector of the plasmid vector expressing the chalcone isomerase-like protein was pNR8 (fig. 6).
The site of the chalcone synthase-like nucleic acid fragment inserted and expressed in the pNR1 vector is NdeI/XhoI, and the site of the chalcone isomerase-like protein nucleic acid fragment inserted and expressed in the pNR8 vector is NcoI/BamHI.
The invention also provides strains expressing the enzyme compositions of the invention.
The construction method of the strain is to introduce the plasmid vector into a host bacterium through transformation or transfection. The host of the strain is Escherichia coli.
In the present invention, the transformation method is electrotransformation. Coli BL21(DE 3).
The strain of the invention only expresses chalcone isomerase-like protein or only expresses chalcone synthetase. Or the strain expresses both the chalcone isomerase-like protein and the chalcone synthase. In the present examples, only chalcone isomerase-like proteins, or only chalcone synthase, are expressed in the strain.
The preparation method of the enzyme composition comprises the steps of culturing the strain, inducing protein expression and obtaining a culture solution containing the enzyme composition.
The enzyme composition, the nucleic acid, the plasmid vector, the strain or the culture of the strain are applied to naringenin biosynthesis.
The invention also provides a biosynthesis method of naringenin, which comprises the following steps: inoculating the strain in the culture solution containing p-coumaric acid, and obtaining a culture containing naringenin after induction and fermentation.
In the culture solution containing p-coumaric acid, the culture solution is an M9 fermentation culture medium, and the concentration of the p-coumaric acid is 200 mg/L.
The culture solution containing p-coumaric acid also contains IPTG, and the concentration of the IPTG is 0.1 mM.
The M9 fermentation medium further comprises double antibodies, wherein the double antibodies are ampicillin and streptomycin.
The induction and fermentation specifically comprise: fermenting at 30 ℃ and 220rpm for 48h with shaking.
The strain is activated prior to inoculation. The activation was performed in LB medium containing streptomycin and ampicillin. The activation conditions include 37 ℃, 220rpm, and 12h shaking culture.
The application of the culture containing naringenin in preparing products for resisting oxidation and inflammation and regulating immune response is disclosed.
According to the invention, the enzyme composition capable of effectively promoting naringenin is obtained by researching the interaction relationship between the CHSs and the CHILs. The composition takes p-coumaric acid as a substrate, the synthesized naringenin level can reach 187.10mg/L, and the naringenin yield is improved by 25.95% compared with the situation without CHIL. Meanwhile, the content of CTAL of the derailment product is reduced by 69.80 percent. The combination has important significance for promoting the industrial production of naringenin products and lays a foundation for the high-efficiency synthesis of other flavone products catalyzed by CHS.
Drawings
FIG. 1 shows a liquid chromatogram of fermentation of strain BNR1-BNR 6; wherein I-the product of the derailment CTAL; II-derailment product BNY;
FIG. 2 shows the effect of different CHS and different CHIL combinations on naringenin synthesis;
FIG. 3 shows the effect of combinations of expression of CHSs and CHILs on the derailment product CTAL;
FIG. 4 shows the effect of combinations of expression of CHSs and CHILs on the derailment product BNY;
FIG. 5 shows a map of vector pNR 1;
FIG. 6 shows a map of vector pNR 8.
Detailed Description
The invention provides an enzyme composition and application thereof in naringenin biosynthesis, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
LB liquid medium: 10g/L NaCl, 10g/L peptone and 5g/L yeast powder, and the balance of water, and sterilizing at a pressure of 0.1MPa and a temperature of 121 ℃ for 20 min. Solid culture medium can be prepared by adding 1.5g/100mL agar powder.
M9 liquid medium: anhydrous Na2HPO46.78 g/L，KH2PO43 g/L，NH4Cl 1g/L, NaCl 0.5g/L, yeast powder 1g/L, and the balance of water, and sterilizing at a pressure of 0.1MPa and a temperature of 121 ℃ for 20 min. Adding sterilized MgSO40.24 g/L，CaCl211.1 mg/L and glucose 5 g/L. The antibiotic concentrations are 100 mug/L ampicillin respectively; streptomycin 50. mu.g/L. Mother liquor 1M MgSO4、1MCaCl2And 20% glucose were autoclaved at 115 ℃ for 30 min.
The detection method involved in the examples:
after fermentation, 5mL of fermentation liquid is taken to be placed in a 15mL centrifuge tube, ethyl acetate with the same volume is added, the mixture is shaken for 30min on a MIX-2500 mini mixer, the mixture is centrifuged for 10min at 8000rpm, supernatant is taken to be subjected to rotary evaporation, and extraction and rotary evaporation are repeated once. Dissolving with 1mL of anhydrous ethanol, filtering with 0.22 μm organic microporous membrane, and detecting by HPLC. Detection conditions 4.6X 150mm C18 chromatographic column; the mobile phase composition is 30 percent of acetonitrile-70 percent of water-0.5 percent of acetic acid, and the flow rate is 1 mL/min; an ultraviolet detector for detecting the wavelength of 290 nm; the sample volume is 10 mu L; the column temperature was 30 ℃.
In the present invention, the fragments and sources are as follows:
sources of species Amino acid sequence Nucleic acid sequences
GmCHS Soybean (Glycinemax) GenbankNP_001340309.1 SEQIDNO:3
MdCHS Apple (Malusxdomestic) GenbankNP_001315914.1 SEQIDNO:4
HaCHS Hypericum (Hypericum androsaemum) GenbankAAG30295.1 SEQIDNO:5
PhCHS Petunia (Petuniaxhybrida) GenbankAAF60297.1 SEQIDNO:6
SbCHS Sorghum (Sorghumbicolor) GenbankXP_002450870.1 SEQIDNO:7
EbCHS Erigeron breviscapus (Erigerontnebrevcapus) SEQIDNO:1 SEQIDNO:8
MdCHIL Apple (Malusxdomestic) SEQIDNO:2 SEQIDNO:9
PhCHIL Petunia (Petuniahybrida) GenbankBAJ10400.1 SEQIDNO:10
GmCHIL Soybean (Glycinemax) GenbankNP_001236782.1 SEQIDNO:11
AtCHIL Arabidopsis thaliana (Arabidopsis thaliana) GenbankNP_568154.1 SEQIDNO:12
ErCHI Eubacterium gracillium (Eubacterium ramulus) Genbank4D06_A SEQIDNO:13
At4CL Arabidopsis thaliana (Arabidopsis thaliana) GenbankU18675 SEQIDNO:14
The invention is further illustrated by the following examples:
EXAMPLE 1 construction of expression vectors for CHSs
According to the amino acid sequence of chalcone isomerase ErCHI (4D06_ A) of Eubacterium ramulus (Eubacterium ramulus), codon preference of escherichia coli is combined, common enzyme cutting sites are removed, a full-length Erchi gene is designed, the Erchi gene is connected between BamHI/HindIII enzyme cutting sites of pETDuet-1 plasmid, and the vector plasmid pNR1 is obtained through total artificial synthesis.
According to the amino acid sequence of chalcone synthetase GmCHS (NP-001340309.1) of soybean (Glycine max), codon preference of escherichia coli is combined, common enzyme cutting sites are removed, a full-length Gmchs gene is designed, the Gmchs gene is connected between NdeI/XhoI enzyme cutting sites of a pNR1 plasmid, and the vector plasmid pNR2 is obtained through total artificial synthesis.
According to the amino acid sequence of chalcone synthetase MdCHS (NP-001315914.1) of apple (Malus x domestica), the preference of Escherichia coli to codon is combined, common enzyme cutting sites are removed, a full-length Mdchs gene is designed, the Mdchs gene is connected between NdeI/XhoI cutting sites of pNR1 plasmid, and the vector plasmid pNR3 is obtained through full-artificial synthesis.
According to the amino acid sequence of chalcone synthetase HaCHS (AAG30295.1) of Hypericum (Hypericum anseramum), the preference of escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length Hachs gene is designed, the Hachs gene is connected between NdeI/XhoI enzyme cutting sites of a pNR1 plasmid, and the vector plasmid pNR4 is obtained through full-artificial synthesis.
According to the amino acid sequence of chalcone synthetase PhCHS (AAF60297.1) of petunia (Petuniahybrida), the preference of Escherichia coli to codon is combined, common enzyme cutting sites are removed, a full-length Phchs gene is designed, the Phchs gene is connected between NdeI/XhoI cutting sites of a pNR1 plasmid, and the vector plasmid pNR5 is obtained through full-artificial synthesis.
According to the amino acid sequence of chalcone synthetase SbCHS (XP _002450870.1) of Sorghum (Sorghum bicolor), the preference of escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length Sbchs gene is designed, the Sbchs gene is connected between NdeI/XhoI cutting sites of a pNR1 plasmid, and the vector plasmid pNR6 is obtained through full-artificial synthesis.
According to the amino acid sequence of chalcone synthetase EbCHS (SEQ ID NO:1) of erigeron breviscapus (Erigeron brevicapus), the preference of escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length Ebchs gene is designed, the Ebchs gene is connected between NdeI/XhoI enzyme cutting sites of pNR1 plasmid, and the vector plasmid pNR7 is obtained through full-artificial synthesis.
EXAMPLE 2 construction of expression vectors for CHILs
According to the amino acid sequence of 4-coumaroyl coenzyme A ligase At4CL (U18675) of Arabidopsis thaliana (Arabidopsis thaliana), the preference of Escherichia coli to codons is combined, common enzyme cutting sites are removed, a full-length At4cl gene is designed, the At4cl gene is connected between NdeI/XhoI enzyme cutting sites of a pCDFDuet-1 plasmid, and the vector plasmid pNR8 is obtained through total artificial synthesis.
According to the amino acid sequence of chalcone isomerase-like protein PhCHIL (BAJ10400.1) of petunia, combining the codon preference of escherichia coli, removing common enzyme cutting sites, designing a full-length Phchil gene, connecting the Phchil gene between NcoI/BamHI enzyme cutting sites of pNR8 plasmid, and carrying out total artificial synthesis to obtain a vector plasmid pNR 9.
According to the amino acid sequence of apple chalcone isomerase-like protein MdCHIL (SEQ ID NO:2), combining the codon preference of Escherichia coli, removing common enzyme cutting sites, designing a full-length Mdchil gene, connecting the Mdchil gene between NcoI/BamHI enzyme cutting sites of pNR8 plasmid, and carrying out total artificial synthesis to obtain a vector plasmid pNR 10.
According to the amino acid sequence of chalcone isomerase-like protein GmCHIL (NP-001236782.1) of soybean, combining the codon preference of escherichia coli, removing common enzyme cutting sites, designing a full-length GmChil gene, connecting the GmChil gene between NcoI/BamHI enzyme cutting sites of pNR8 plasmid, and carrying out total artificial synthesis to obtain a vector plasmid pNR 11.
According to the amino acid sequence of chalcone isomerase-like protein AtCHIL (NP-568154.1) of Arabidopsis, combining the codon preference of Escherichia coli, removing common enzyme cutting sites, designing a full-length Atchil gene, connecting the Atchil gene between NcoI/BamHI enzyme cutting sites of pNR8 plasmid, and completely synthesizing to obtain a vector plasmid pNR 12.
Example 3 construction of recombinant strains expressing CHILs and CHSs
Plasmids pNR2-pNR7 and pNR8 are transformed into E.coli BL21(DE3) competence by electric shock at the same time, the competence is cultured on LB solid culture medium containing streptomyces and ampicillin overnight, positive clones are screened, and control strains BNR1-BNR6 are constructed and stored for later use.
The plasmids pNR2-pNR7 and pNR9-pNR12 are transformed into escherichia coli E.coli BL21(DE3) competence by electric shock at the same time, the competence is cultured on LB solid culture medium containing streptomyces and ampicillin overnight, positive clones are screened, and a fermentation strain BNR7-BNR30 is constructed and stored for later use.
Example 4 biosynthesis of naringenin by recombinant strains
Respectively inoculating the recombinant strain BNR1-BNR30 in an LB liquid culture medium according to the inoculation ratio of 1%, adding streptomyces and ampicillin, culturing at 37 ℃ and 220rpm for 12h by shaking, then transferring the recombinant strain BNR1-BNR30 into an M9 fermentation culture medium containing antibiotics, adding 0.1mM IPTG, adding 200mg/L p-coumaric acid substrate, fermenting at 30 ℃ and 220rpm for 48h by shaking, and then carrying out HPLC detection. The liquid phase detection is shown in FIG. 1, and the fermentation yield is shown in FIG. 2.
According to FIG. 1, naringenin was detected in the fermentation broth of strains BNR1-BNR6, indicating that all six CHSs have the ability to catalyze the synthesis of naringenin. Wherein, the GmCHS and the MdCHS have the weakest functions and respectively synthesize 3.88mg/L and 6.58mg/L naringenin. HaCHS, PhCHS and SbCHS have stronger activity, and naringenin yield is 22.69mg/L, 33.09mg/L and 97.11mg/L in sequence, which are respectively 4.84 times, 7.52 times and 24.03 times higher than GmCHS. The EbCHS has the most excellent performance, and the yield of naringenin is 148.54mg/L, which is 38.28 times of that of GmCHS. At the same time, two derailed products CTAL and BNY of CHS were also detected in the fermentation broth.
According to fig. 2, GmCHS had little effect on naringenin production when combined with different CHILs. For MdCHS, four CHILs all produced a positive effect, wherein MdCHS synthesized 20.18mg/L naringenin with MdCHIL of the same species, with a 2.07-fold increase in yield compared to the control. For HaCHS, the combination of HaCHS + MdCHIL and HaCHS + GmCHIL with the foreign species CHIL increased naringenin production by 42.35% and 27.32%, respectively, at 32.30mg/L and 28.89 mg/L. For PhCHS, the four CHILs also enhanced the catalytic efficiency of PhCHS, with the xenogeneic MdCHIL promoting naringenin to 126.29mg/L, a 2.82 fold increase. For SbCHS, CHILs of three species were effective except for the insignificant effect of AtCHIL, and the combination of SbCHS + MdCHIL increased naringenin production by 90.76% and 185.31 mg/L. As for EbCHS, different kinds of CHILs have promotion effect on the activity of EbCHS, the combination effect of EbCHS + PhCHIL is optimal, naringenin with the concentration of 187.10mg/L is synthesized, and the naringenin is improved by 25.95% compared with a control.
Example 5 analysis of fermentation derailed products of combination with increased naringenin levels
CTAL and BNY are the major derailed products produced by fermentation of recombinant strains. Since CTAL and BNY have no commercial standards, their relative amounts of production are indirectly expressed by peak area and the results are shown in fig. 3 and 4, respectively.
This embodiment mainly analyzes the combination of increased naringenin levels. Changes in CTAL derailment products were first analyzed. As shown in FIG. 3, the combination of MdCHS and different CHILs all showed a reduction in CTAL content, ranging from 46.98% to 79.37%. The combination HaCHS + MdCHIL, HaCHS + GmCHIL also showed a CTAL content decrease of 74.82% and 75.77%, respectively. The combination of PhCHS, SbCHS and EbCHS with CHILs production also all reduced CTAL production, with the combination PhCHS + MdCHIL, SbCHS + MdCHIL, EbCHS + PhCHIL with higher naringenin levels reducing CTAL content by 39.76%, 26.47%, 69.80%, respectively. It follows that CHILs enhances the catalytic efficiency of CHS by reducing the content of the derailment product CTAL.
For the change in BNY derailed product, according to FIG. 4, MdCHS + PhCHIL, MdCHS + GmCHIL, MdCHS + AtCHIL reduced BNY levels by 22.39%, 76.93%, 56.47%, respectively, while MdCHS + MdCHIL increased BNY by 1.06-fold in the combinations produced by MdCHS with different CHILs. The combination of HaCHS + MdCHIL, HaCHS + GmCHIL served to increase the BNY content by 87.06% and 60.31%, respectively. The BNY accumulation amounts of PhCHS + PhCHIL, PhCHS + MdCHIL and PhCHS + GmCHIL are respectively increased by 0.39 times, 5.03 times and 3.80 times, while the accumulation amount of PhCHS + AtCHIL is almost unchanged by BNY. The BNY content of SbCHS + MdCHIL and SbCHS + GmCHIL increased by 76.67% and 190.18%, respectively, while the BNY content of SbCHS + PhCHIL decreased by 37.19%. For the EbCHS in combination with the four CHILs, the BNY variation was relatively insignificant. It can be seen that combinations of CHSs with different species of CHILs had no regular effect on the accumulation of the derailed product BNY. And (4) conclusion: in 24 combinations generated by four CHILs and six CHSs, the combination effect of petunia PhCHIL and erigeron breviscapus EbCHS is optimal, 187.10mg/L naringenin is synthesized, the yield of the naringenin is improved by 25.95% compared with the situation without the CHIL, and meanwhile, the content of a fermentation derailment product CTAL is reduced by 69.80%, so that the method is suitable for biosynthesis of the naringenin.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Zongzhi Zhizhen college of Tianjin university
<120> enzyme composition and application thereof in naringenin biosynthesis
<130> MP21017999
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 398
<212> PRT
<213> Erigeron breviscapus (Erigeron breviscapus)
<400> 1
Met Ala Ser Ser Ile Asp Ile Ala Ala Ile Arg Glu Ala Gln Arg Ala
1 5 10 15
Gln Gly Pro Ala Thr Ile Leu Ala Ile Gly Thr Ala Thr Pro Ser Asn
20 25 30
Cys Val Tyr Gln Ala Asp Tyr Pro Asp Tyr Tyr Phe Arg Ile Thr Lys
35 40 45
Ser Glu His Met Val Asp Leu Lys Glu Lys Phe Lys Arg Met Cys Asp
50 55 60
Lys Ser Met Ile Arg Lys Arg Tyr Met His Leu Thr Glu Glu Tyr Leu
65 70 75 80
Lys Glu Asn Pro Ser Leu Cys Glu Tyr Met Ala Pro Ser Leu Asp Ala
85 90 95
Arg Gln Asp Val Val Val Val Glu Val Pro Lys Leu Gly Lys Glu Ala
100 105 110
Ala Thr Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr
115 120 125
His Leu Ile Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp
130 135 140
Tyr Gln Leu Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Phe
145 150 155 160
Met Met Tyr Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu
165 170 175
Ala Lys Asp Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val
180 185 190
Cys Ser Glu Ile Thr Ala Val Thr Phe Arg Gly Pro Asn Asp Thr His
195 200 205
Leu Asp Ser Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala
210 215 220
Val Ile Val Gly Ser Asp Pro Asp Leu Thr Thr Glu Arg Pro Leu Phe
225 230 235 240
Glu Met Ile Ser Ala Ala Gln Thr Ile Leu Pro Asp Ser Glu Gly Ala
245 250 255
Ile Asp Gly His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys
260 265 270
Asp Val Pro Gly Leu Ile Ser Lys Asn Ile Glu Lys Ala Leu Thr Gln
275 280 285
Ala Phe Ser Pro Leu Gly Ile Ser Asp Trp Asn Ser Leu Phe Trp Ile
290 295 300
Ala His Pro Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Leu Lys Leu
305 310 315 320
Gly Leu Lys Glu Glu Lys Met Arg Ala Thr Arg His Val Leu Ser Glu
325 330 335
Tyr Gly Asn Met Ser Ser Ala Cys Val Leu Phe Ile Ile Asp Glu Met
340 345 350
Arg Lys Lys Ser Ala Glu Asp Gly Ala Ala Thr Thr Gly Glu Gly Leu
355 360 365
Asp Trp Gly Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Val Glu Thr
370 375 380
Val Val Leu His Ser Leu Pro Thr Thr Thr Ala Ile Ala Thr
385 390 395
<210> 2
<211> 209
<212> PRT
<213> apple (Malus x domestica)
<400> 2
Met Gly Thr Glu Val Val Leu Val Asp Glu Val Pro Phe Pro Ser Lys
1 5 10 15
Ile Thr Thr Thr Lys Pro Leu Ser Leu Leu Gly Gln Gly Ile Thr Asp
20 25 30
Ile Glu Ile His Phe Leu Gln Ile Lys Phe Thr Ala Ile Gly Val Tyr
35 40 45
Leu Asp Ala Lys Ile Val Ser His Leu Gln Gln Trp Lys Ala Lys Lys
50 55 60
Ala Asn Glu Leu Ala Glu Asp Asp Asp Phe Phe Asp Ala Leu Val Ser
65 70 75 80
Ala Pro Val Glu Lys Phe Ile Arg Val Val Val Ile Lys Glu Ile Lys
85 90 95
Gly Ser Gln Tyr Gly Val Gln Leu Glu Ser Ala Val Arg Asp Arg Leu
100 105 110
Ala Ala Asp Asp Lys Tyr Glu Glu Glu Glu Glu Glu Ala Leu Glu Lys
115 120 125
Val Val Glu Phe Phe Gln Ser Lys Tyr Phe Lys Lys Asp Ser Val Ile
130 135 140
Thr Phe His Phe Pro Ala Thr Ser Asn Thr Ala Glu Ile Val Phe Ser
145 150 155 160
Thr Glu Gly Lys Glu Glu Ser Lys Ile Lys Val Glu Asn Ala Asn Val
165 170 175
Val Glu Asn Ile Lys Lys Trp Tyr Leu Gly Gly Thr Arg Gly Val Ser
180 185 190
Pro Ser Thr Ile Ser Ser Leu Ala Asn Thr Leu Ser Ala Glu Leu Thr
195 200 205
Lys
<210> 3
<211> 1170
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggttagcg tggcggagat tcgtcaggcg caacgtgcgg aaggtccggc gaccatcctg 60
gcgattggca ccgcgaaccc gccgaaccgt gttgaccaga gcacctaccc ggattactat 120
ttccgtatta ccaacagcga ccacatgacc gagctgaagg aaaaatttca acgtatgtgc 180
gataagagca tgatcaaaac ccgttacatg tatctgaacg aggaaattct gaaggagaac 240
ccgaacatgt gcgcgtatat ggcgccgagc ctggacgcgc gtcaggatat ggtggttgtt 300
gaggtgccga agctgggcaa agaagcggcg gtgaaggcga tcaaagaatg gggtcaaccg 360
aagagcaaaa tcacccacct gattttctgc accaccagcg gcgtggacat gccgggtgcg 420
gattaccagc tgaccaagca actgggcctg cgtccgtatg ttaaacgtta catgatgtat 480
cagcaaggtt gctttgcggg tggcaccgtg ctgcgtctgg cgaaggacct ggcggagaac 540
aacaaaggtg cgcgtgttct ggttgtgtgc agcgaaatta ccgcggtgac ctttcgtggt 600
ccgagcgaca cccacctgga tagcctggtt ggtcaggcgc tgtttggtga tggtgcggcg 660
gcggtgattg tgggtagcga tccgattccg caggttgaga agccgctgta cgaactggtg 720
tggaccgcgc aaaccattgc gccggacagc gagggtgcga ttgatggtca cctgcgtgaa 780
gtgggcctga ccttccacct gctgaaagac gttccgggta tcgtgagcaa gaacattgat 840
aaagcgctgt tcgaggcgtt taacccgctg aacatcagcg actacaacag catcttttgg 900
atcgcgcacc cgggtggccc ggcgattctg gatcaggttg agcaaaagct gggcctgaaa 960
ccggaaaaga tgaaagcgac ccgtgacgtt ctgagcgagt atggtaacat gagcagcgcg 1020
tgcgtgctgt tcatcctgga tgagatgcgt cgtaagagcg cggaaaacgg ccacaaaacc 1080
accggcgagg gcctggaatg gggtgttctg ttcggttttg gcccgggtct gaccatcgaa 1140
accgttgtgc tgcacagcgt ggcgatttaa 1170
<210> 4
<211> 1170
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggttaccg tggaggaagt tcgtaaagcg cagcgtgcgg agggtccggc gaccgtgctg 60
gcgattggca ccgcgacccc gagcaactgc gttgaccaag cgacctaccc ggattactat 120
ttccgtatta ccaacagcga acacaagacc gagctgaagg aaaaatttca acgtatgtgc 180
gacaaaagca tgatcaagaa acgttacatg tatctgaccg aggaaattct gaaggagaac 240
ccgaccgtgt gcgaatatat ggcgccgagc ctggacgcgc gtcaggatat ggtggttgtt 300
gaggtgccgc gtctgggcaa agaagcggcg accaaggcga tcaaagagtg gggtcaaccg 360
aagagcaaaa ttacccacct ggttttctgc accaccagcg gcgtggacat gccgggtgcg 420
gattaccagc tgaccaagct gctgggcctg cgtccgagcg tgaaacgtct gatgatgtat 480
cagcaaggtt gctttgcggg tggcaccgtt ctgcgtctgg cgaaggatct ggcggagaac 540
aacaaaggcg cgcgtgttct ggttgtgtgc agcgaaatta ccgcggtgac ctttcgtggt 600
ccgagcgaca cccacctgga tagcctggtg ggtcaagcgc tgtttggtga tggtgcggcg 660
gcggtgatca ttggtgcgga cccgctgccg gaagtggaaa aaccgctgtt tgagctggtg 720
agcgcggcgc agaccatcct gccggacagc gatggcgcga ttgacggtca cctgcgtgaa 780
gttggcctga cctttcacct gctgaaggat gtgccgggtc tgatcagcaa gaacattgag 840
aaaagcctga acgaagcgtt caaaccgatc ggtattagcg actggaacag cctgttttgg 900
attgcgcacc cgggtggccc ggcgattctg gatcaggtgg agagcaaact ggcgctgaag 960
ccggagaaac tggaagcgac ccgtcaagtt ctgagcaact acggtaacat gagcagcgcg 1020
tgcgttctgt tcatcctgga cgaagtgcgt cgtaagagca ccgaaaaagg cctgcgtacc 1080
accggcgagg gcctggaatg gggtgtgctg ttcggttttg gcccgggtct gaccgttgaa 1140
accgttgtgc tgcacagcgt ggcggcgtaa 1170
<210> 5
<211> 1176
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgatggtta ccgtggagga agttcgtaaa gcgcagcgtg cggaaggtcc ggcgaccgtg 60
atggcgattg gcaccgcggt gccgccgaac tgcgtggacc aagcgaccta cccggattac 120
tatttccgta tcaccaacag cgagcacaag gcggagctga aggaaaaatt tcagcgtatg 180
tgcgacaaga gccaaattaa gaaacgttac atgtatctga acgaggaagt tctgaaagaa 240
aacccgaaca tgtgcgcgta tatggcgccg agcctggacg cgcgtcagga tatcgtggtt 300
gttgaggtgc cgaagctggg caaagaagcg gcggtgaagg cgattaaaga atggggtcaa 360
ccgaagagca aaatcaccca cctggttttc tgcaccacca gcggcgtgga catgccgggt 420
gcggattacc agctgaccaa gctgctgggc ctgcgtccga gcgttaaacg tctgatgatg 480
tatcagcaag gttgctttgc gggtggcacc gtgctgcgtc tggcgaagga cctggcggag 540
aacaacaaag gcgcgcgtgt tctggttgtg tgcagcgaaa ttaccgcggt gacctttcgt 600
ggtccgaccg acacccacct ggatagcctg gttggtcaag cgctgtttgg tgacggcgct 660
gcggcgatca ttatcggtag cgatccgatc ccggaagtgg aaaaaccgct gtttgagctg 720
gtgagcgcgg cgcagaccat tctgccggac agcgagggcg cgatcgatgg tcacctgcgt 780
gaagtgggcc tgacctttca cctgctgaag gatgttccgg gtctgattag caagaacgtg 840
gagaaaagcc tgaccgaagc gttcaaaccg ctgggtatca gcgactggaa cagcctgttt 900
tggatcgcgc acccgggtgg cccggcgatc ctggatcaag ttgaggcgaa gctgagcctg 960
aagccggaaa aactgcgtgc gacccgtcac gttctgagcg agtacggtaa catgagcagc 1020
gcgtgcgtgc tgttcattct ggacgagatg cgtcgtaaga gcaaagaaga tggcctgaaa 1080
accaccggcg agggcatcga atggggtgtg ctgttcggtt ttggcccggg tctgaccgtt 1140
gaaaccgttg tgctgcacag cgtggcgatc aactaa 1176
<210> 6
<211> 1170
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggttaccg tggaagaata tcgtaaagca cagcgtgcag aaggtccggc aaccgttatg 60
gcaattggca ccgcaacccc gaccaattgt gttgatcaga gcacctatcc ggattattac 120
tttcgtatta ccaacagcga acacaagacc gatctgaaag aaaaattcaa acgcatgtgc 180
gaaaaaagca tgatcaaaaa acgctatatg cacctgaccg aagagattct taaagaaaat 240
ccgagcatgt gtgaatatat ggcaccgagc ctggatgcac gtcaggatat tgttgttgtt 300
gaagttccga aactgggtaa agaagcagca cagaaagcaa ttaaagaatg gggtcagccg 360
aaaagcaaaa ttacccatct ggttttttgt accaccagtg gtgttgatat gcctggttgt 420
gattatcagc tgaccaaact gctgggtctg cgtccgagcg ttaaacgtct gatgatgtat 480
cagcagggtt gttttgccgg tggcaccgtt ctgcgtctgg caaaagatct ggcagaaaat 540
aacaaaggtg cacgtgttct ggttgtttgc agcgaaatta ccgcagttac ctttcgtggt 600
ccgaatgata cacatctgga tagcctggtg ggtcaggcac tgtttggtga tggtgccggt 660
gcaattatca ttggtagcga tccgattccg ggtgttgaac gtccgctgtt tgaactggtt 720
agcgcagccc agaccctgct gccggatagt catggtgcaa ttgatggtca tctgcgtgaa 780
gttggtctga cctttcatct gctgaaagat gttccgggtc tgattagcaa aaacattgaa 840
aaaagcctgg aagaagcatt tcgtccgctg agcattagcg attggaatag cctgttttgg 900
attgcacatc cgggtggtcc tgcaattctg gatcaggttg aaattaaact gggtctgaaa 960
ccggaaaaac tgaaagcaac ccgtaatgtg ctgagcaatt atggtaatat gagcagcgca 1020
tgcgttctgt ttattctgga tgaaatgcgt aaagcgagcg caaaagaagg tctgggcacc 1080
accggtgaag gcctggaatg gggtgttctg tttggttttg gtccaggtct gaccgttgaa 1140
accgttgttc tgcatagcgt tgcaacctaa 1170
<210> 7
<211> 1206
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggctgcgg cgaccgttac cgtggaggaa gttcgtaaag cgcaacgtgc gaccggtccg 60
gcgaccgtgc tggcgattgg caccgcgacc ccggcgaact gcgttcacca agcggactac 120
ccggattact atttccgtat taccaagagc gagcacatga ccgagctgaa ggaaaaattt 180
aagcgtatgt gcgacaaaag ccagatccgt aagcgttaca tgcacctgac cgaggaatat 240
ctggcggaaa acccgaacat gtgcgcgtat atggcgccga gcctggacgc gcgtcaagat 300
attgtggttg ttgaggtgcc gaaactgggc aaggctgcgg cgcagaaagc gatcaaggaa 360
tggggtcaac cgaaaagcaa gattacccac ctggttttct gcaccaccag cggcgtggac 420
atgccgggtg cggattacca gctgaccaaa atgctgggcc tgcgtccgag cgttaagcgt 480
ctgatgatgt atcagcaagg ttgctttgcg ggtggcaccg ttctgcgtgt ggcgaaagac 540
ctggcggaga acaaccgtgg cgcgcgtgtt ctggttgtgt gcagcgagat taccgcggtg 600
accttccgtg gtccgagcga aagccacctg gacagcatgg tgggtcaagc gctgtttggt 660
gatggtgcgg cggcggtgat tgtgggtgcg gacccggatg agcgtgttga acgtccgctg 720
ttccagctgg tgtctgcgag ccaaaccatc ctgccggaca gcgagggcgc gattgatggt 780
cacctgcgtg aagttggcct gacctttcac ctgctgaaag atgtgccggg tctgatcagc 840
aagaacattg agcgtagcct ggaggaagcg ttcaaaccgc tgggtatcac cgactacaac 900
agcatcttct gggtggcgca cccgggtggc ccggcgatcc tggatcaggt tgaggcgaaa 960
gtgggcctga agaaagaacg tatgcgtgcg acccgtcacg tgctgagcga atatggtaac 1020
atgagcagcg cgtgcgttct gttcatcctg gacgagatgc gtaagcgtag cgcggaagat 1080
ggccaagcga ccaccggcga gggcctggat tggggtgttc tgttcggttt tggcccgggt 1140
ctgaccgttg aaaccgttgt gctgcatagc gtgccgatca ccaccggtgc ggcgattacc 1200
gcgtaa 1206
<210> 8
<211> 1197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggcgagca gcatcgacat tgcggcgatt cgtgaggcgc agcgtgcgca aggtccggcg 60
accatcctgg cgattggcac cgcgaccccg agcaactgcg tgtaccaagc ggactatccg 120
gattactatt tccgtatcac caaaagcgag cacatggttg acctgaagga aaaatttaag 180
cgtatgtgcg ataaaagcat gattcgtaag cgttacatgc acctgaccga ggaatatctg 240
aaggagaacc cgagcctgtg cgaatatatg gcgccgagcc tggacgcgcg tcaggatgtg 300
gttgtggttg aggtgccgaa actgggcaag gaagcggcga ccaaagcgat taaggaatgg 360
ggtcaaccga aaagcaagat cacccacctg attttctgca ccaccagcgg cgttgacatg 420
ccgggtgcgg attaccagct gaccaaactg ctgggcctgc gtccgagcgt gaagcgtttc 480
atgatgtatc agcaaggttg ctttgcgggt ggcaccgttc tgcgtctggc gaaagacctg 540
gcggagaaca acaagggcgc gcgtgttctg gtggtgtgca gcgaaatcac cgcggttacc 600
ttccgtggcc cgaacgacac ccacctggat agcctggtgg gtcaagcgct gtttggtgat 660
ggtgcggcgg cggtgattgt tggtagcgac ccggatctga ccaccgagcg tccgctgttc 720
gaaatgatca gcgcggcgca gaccattctg ccggacagcg agggcgcgat cgatggtcac 780
ctgcgtgaag tgggcctgac ctttcacctg ctgaaagacg ttccgggtct gatcagcaaa 840
aacattgaga aggcgctgac ccaagcgttc agcccgctgg gtattagcga ctggaacagc 900
ctgttttgga ttgcgcaccc gggtggcccg gcgattctgg atcaggttga gctgaagctg 960
ggcctgaaag aggaaaagat gcgtgcgacc cgtcacgtgc tgagcgaata tggtaacatg 1020
agcagcgcgt gcgttctgtt tatcattgac gagatgcgta agaaaagcgc ggaagatggt 1080
gcggcgacca ccggcgaggg cctggattgg ggtgtgctgt tcggttttgg cccgggtctg 1140
accgttgaaa ccgtggttct gcatagcctg ccgaccacca ccgcgattgc gacctaa 1197
<210> 9
<211> 630
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgggcaccg aggtggttct ggttgatgaa gtgccgttcc cgagcaagat caccaccacc 60
aaaccgctga gcctgctggg tcagggcatt accgatatcg agattcactt cctgcaaatc 120
aagtttaccg cgattggcgt ttacctggac gcgaagatcg tgagccacct gcaacaatgg 180
aaagcgaaga aagcgaacga gctggcggaa gacgatgact tctttgacgc gctggttagc 240
gcgccggtgg agaagtttat tcgtgtggtt gtgatcaagg aaattaaagg tagccagtac 300
ggcgttcaac tggaaagcgc ggtgcgtgat cgtctggcgg cggatgacaa atatgaggaa 360
gaggaagagg aagcgctgga gaaggttgtg gagttcttcc agagcaaata cttcaagaaa 420
gacagcgtta tcaccttcca ctttccggcg accagcaaca ccgcggagat tgtttttagc 480
accgagggta aagaggagag caagatcaaa gtggagaacg cgaacgttgt ggaaaacatt 540
aagaaatggt atctgggtgg cacccgtggc gtgagcccga gcaccatcag cagcctggcg 600
aacaccctga gcgcggagct gaccaaataa 630
<210> 10
<211> 633
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggcaaga acgaggttat ggtggacgaa atcccgttcc cgagccagtt tatgatgacc 60
accaaaccgc tgccgctgat gggtcacggc attaccgaca tcgagattca cttcctgcaa 120
atcaagttta ccgcgattgg cgtttacctg gacccggaaa tcgtgaccca cctgcaacaa 180
tggaagggta aaagcggcgc ggagctgatc gaaaacgacg agttctttga agcgattgtt 240
aacgcgccgg tggataagtt cctgcgtgtg gttgtgatca aggagattaa aggtagccag 300
tacggcgttc aactggagag cgcggtgcgt gaccgtctgg cggaagttga taaatatgag 360
gaagaggaag aggaagcgct ggagaagatc gtggagttct tccagagcaa atacttcaag 420
aaagacagcg ttgtgaccta tagctttccg gcgaccagcg gtaacgttaa gattagcttt 480
gcgaccgagg gcaaagaaga tagcgagatc gaagttcaaa acgcgaacgt ggcgggcgag 540
attaagaaat ggtatctggg tggcagccgt ggcctgagcc cgaccaccat tagcagcctg 600
gcgaacaccc tgagcgcgga actgagcaaa taa 633
<210> 11
<211> 630
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atggcgaccg aggaagtgct ggttgacgaa atcacctacc cgaccaaaat caccaccacc 60
aaaccgctga gcctgctggg ccacggcatc accgacatgg agatccactt cattcacgtg 120
aagttttaca gcattggtgt ttatctggag ccggaagtgg ttggtcacct ggaccagttc 180
aagggcaaaa gcgcgaagga gctggaagat aacgaggagt tcttcaacgc gctgatcagc 240
gcgccggtgg agaaatttat tcgtctggtg gttatcaagg aaattaaagg tgcgcagtac 300
ggcgtgcaaa tcgaaaccgc ggtgcgtgac cgtctggcgg cggaagataa atatgaggaa 360
gaggaagagg aagcgctgga gaaagtgatt gagttcttcc aaagcaaata cttcaagaaa 420
ctgagcgtta tcacctatca ctttccggcg aacagcgcga ccgcggaaat tgtggttagc 480
ctggagggca aggaagatag caaatacgtt atcgagaacg cgaacgtggt tgaagcgatt 540
aagaaatggt atctgggtgg cagcagcgcg gttagcagca gcaccattca gagcctggcg 600
agcaccttta gccaagaact gagcaaataa 630
<210> 12
<211> 630
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggcaccg aaatggttat ggttcacgag gttccgttcc cgccgcaaat catcaccagc 60
aagccgctga gcctgctggg tcaaggtatc accgatatcg agattcactt cctgcaagtg 120
aagtttaccg cgattggcgt gtacctggac ccgagcgacg ttaagaccca cctggataac 180
tggaagggta aaaccggcaa agaactggcg ggtgacgatg acttctttga cgcgctggcg 240
agcgcggaga tggaaaaagt gatccgtgtg gttgtgatca aggagattaa aggtgcgcag 300
tacggcgttc aactggaaaa caccgtgcgt gatcgtctgg cggaggaaga caagtatgag 360
gaagaggaag aaaccgagct ggaaaaagtt gtgggcttct ttcagagcaa gtacttcaaa 420
gcgaacagcg tgattaccta tcactttagc gcgaaggacg gtatctgcga gattggcttc 480
gaaaccgagg gtaaagaaga ggaaaagctg aaagttgaaa acgcgaacgt tgtgggcatg 540
atgcaacgtt ggtatctgag cggtagccgt ggcgtgagcc cgagcaccat tgtgagcatt 600
gcggatagca tcagcgcggt tctgacctaa 630
<210> 13
<211> 852
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atggccgatt ttaagtttga accgatgcgt agcctgattt atgttgattg tgttagcgaa 60
gattaccgtc cgaaactgca gcgttggatc tataaagttc atattccgga tagcatcagc 120
cagtttgagc cgtatgttac caaatatgca ttttatccga gctttccgat tccgcctcag 180
ggtgatcgtt ttggttatgc acgtatgcag ctgaccgaac atcattggct ggttagcgat 240
ttagatccgc gtctggaaat taaagcaatt gcagaaacct ttccgatgga tgttctggtt 300
tggcagggtc agattccggc agcagcacat accgatgcac agattgatag tgatggtgat 360
gcaggtaatg cagcacgtaa aagcaataat gcagaaggca atccgtttat ctttgcattt 420
ctgccgatgt ggtgggagaa agatctgaaa ggtaaaggtc gtaccattga agatggtgcc 480
aattatcgct ttaatatgac cattggtttt ccggaaggtg tggataaagc cgaaggtgaa 540
aaatggctgt ttgaaaaagt tgttccgatt ctgcaggcag caccggaatg tacccgtgtt 600
ctggcaagcg cagttaaaaa agatattaat ggttgcgtga tggactgggt gctggaaatt 660
tggtttgaaa atcagagcgg ttggtataag gttatggtgg atgatatgaa agcactggaa 720
aaaccgagct gggcacagca ggatgcattt ccgtttctga aaccgtatca taatgtttgt 780
agcgcagcag ttgcagatta taccccgagc aataatctgg caaattatcg cggttatatc 840
accatgcgct aa 852
<210> 14
<211> 1686
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atggcgccac aagaacaagc agtttctcag gtgatggaga aacagagcaa caacaacaac 60
agtgacgtca ttttccgatc aaagttactg gatatttaca tcccgaacca cctatctctc 120
cacgactaca tcttccaaaa catctccgaa ttcgccacta agccttgcct aatcaacgga 180
ccaaccggcc acgtgtacac ttactccgac gtccacgtca tctcccgcca aatcgccgcc 240
aattttcaca aactcggcgt taaccaaaac gacgtcgtca tgctcctcct cccaaactgt 300
cccgaattcg tcctctcttt cctcgccgcc tccttccgcg gcgcaaccgc caccgccgca 360
aaccctttct tcactccggc ggagatagct aaacaagcca aagcctccaa caccaaactc 420
ataatcaccg aagctcgtta cgtcgacaaa atcaaaccac ttcaaaacga cgacggagta 480
gtcatcgtct gcatcgacga caacgaatcc gtgccaatcc ctgaaggctg cctccgcttc 540
accgagttga ctcagtcgac aaccgaggca tcagaagtca tcgactcggt ggagatttca 600
ccggacgacg tggtggcact accttactcc tctggcacga cgggattacc aaaaggagtg 660
atgctgactc acaagggact agtcacgagc gttgctcagc aagtcgacgg cgagaacccg 720
aatctttatt tccacagcga tgacgtcata ctctgtgttt tgcccatgtt tcatatctac 780
gctttgaact cgatcatgtt gtgtggtctt agagttggtg cggcgattct gataatgccg 840
aagtttgaga tcaatctgct attggagctg atccagaggt gtaaagtgac ggtggctccg 900
atggttccgc cgattgtgtt ggccattgcg aagtcttcgg agacggagaa gtatgatttg 960
agctcgataa gagtggtgaa atctggtgct gctcctcttg gtaaagaact tgaagatgcc 1020
gttaatgcca agtttcctaa tgccaaactc ggtcagggat acggaatgac ggaagcaggt 1080
ccagtgctag caatgtcgtt aggttttgca aaggaacctt ttccggttaa gtcaggagct 1140
tgtggtactg ttgtaagaaa tgctgagatg aaaatagttg atccagacac cggagattct 1200
ctttcgagga atcaacccgg tgagatttgt attcgtggtc accagatcat gaaaggttac 1260
ctcaacaatc cggcagctac agcagagacc attgataaag acggttggct tcatactgga 1320
gatattggat tgatcgatga cgatgacgag cttttcatcg ttgatcgatt gaaagaactt 1380
atcaagtata aaggttttca ggtagctccg gctgagctag aggctttgct catcggtcat 1440
cctgacatta ctgatgttgc tgttgtcgca atgaaagaag aagcagctgg tgaagttcct 1500
gttgcatttg tggtgaaatc gaaggattcg gagttatcag aagatgatgt gaagcaattc 1560
gtgtcgaaac aggttgtgtt ttacaagaga atcaacaaag tgttcttcac tgaatccatt 1620
cctaaagctc catcagggaa gatattgagg aaagatctga ggacaaaact agcaaatgga 1680
ttgtaa 1686

Claims (9)

1. An enzyme composition consisting of a chalcone synthase and a chalcone isomerase-like protein; wherein,
the chalcone synthetase is selected from at least one of soybean GmCHS, apple MdCHS, hypericum HaCHS, petunia PhCHS, sorghum SbCHS and erigeron breviscapus EbCHS;
the chalcone isomerase-like protein is at least one selected from apple MdCHIL, petunia PhCHIL, soybean GmCHIL and Arabidopsis AtCHIL.
2. The enzyme composition according to claim 1,
consisting of PhCHS and PhCHIL;
or consisting of PhCHS and MdCHIL;
or consisting of PhCHS and GmCHIL;
or consisting of SbCHS and PhCHIL;
or consisting of SbCHS and MdCHIL;
or consisting of SbCHS and GmCHIL;
or EbCHS and PhCHIL;
or consists of EbCHS and MdCHIL;
or EbCHS and GmCHIL;
or EbCHS and AtCHIL.
3. A strain expressing the enzyme composition of claim 1 or 2.
4. The strain of claim 3, wherein the host is Escherichia coli.
5. A strain according to claim 3, wherein a plasmid vector containing a nucleic acid encoding the chalcone synthase and/or chalcone isomerase-like protein of the enzyme composition according to claim 1 or 2 is transformed;
the sequence of the nucleic acid for coding the soybean GmCHS is shown as SEQ ID NO. 3;
the sequence of the nucleic acid for coding the apple MdCHS is shown as SEQ ID NO. 4;
the sequence of the nucleic acid for coding the hypericum HaCHS is shown as SEQ ID NO. 5;
the sequence of the nucleic acid for coding petunia PhCHS is shown as SEQ ID NO. 6;
the sequence of the nucleic acid for coding the SbCHS of the sorghum is shown as SEQ ID NO. 7;
the sequence of the nucleic acid for coding the erigeron breviscapus EbCHS is shown as SEQ ID NO. 8;
the sequence of the nucleic acid for coding the apple MdCHIL is shown as SEQ ID NO. 9;
the sequence of the nucleic acid for coding petunia PhCHIL is shown as SEQ ID NO. 10;
the sequence of the nucleic acid for coding the soybean GmCHIL is shown as SEQ ID NO. 11;
the sequence of the nucleic acid encoding Arabidopsis AtCHIL is shown in SEQ ID NO. 12.
6. Use of the enzyme composition of claim 1 or 2, the strain of any one of claims 3 to 5, or a culture of the strain of any one of claims 3 to 5 for naringenin biosynthesis.
7. A method for the biosynthesis of naringenin comprising: inoculating the strain of any one of claims 3 to 5 into a culture solution containing p-coumaric acid, inducing, and fermenting to obtain a culture containing naringenin.
8. The biosynthesis method according to claim 8, wherein the culture solution containing p-coumaric acid is M9 fermentation medium, and the concentration of p-coumaric acid is 200 mg/L.
9. The biosynthesis method according to claim 8, wherein the culture solution containing p-coumaric acid further contains IPTG at a concentration of 0.1 mM.
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CN114990086A (en) * 2022-06-16 2022-09-02 天津大学佐治亚理工深圳学院 Enzyme composition and application thereof

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