CN114134060B - Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method - Google Patents
Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method Download PDFInfo
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Abstract
The invention relates to an engineering saccharomycete capable of efficiently synthesizing an intermediate product or a final product of a ginsenoside Ro synthesis path and a method thereof, belonging to the fields of synthetic biology and metabolic engineering. The 4 strains of engineering saccharomycetes are respectively as follows: engineering saccharomycete CE-P1 with the preservation number of CGMCC No.23731 and capable of efficiently synthesizing calendula officinalis E, engineering saccharomycete IVA-P1 with the preservation number of CGMCC No.23732 and capable of efficiently synthesizing panax japonicus saponin IVa, engineering saccharomycete R1-P1 with the preservation number of CGMCC No.23733 and capable of efficiently synthesizing Jiang Zhuang panax notoginseng glycoside R1, and engineering saccharomycete Ro-P1 with the preservation number of CGMCC No.23734 and capable of efficiently synthesizing ginsenoside Ro. The engineering saccharomycete has the advantages of stability, short growth period, mild culture condition, low culture cost and high target product yield, and realizes the efficient de novo synthesis of target compounds.
Description
Technical Field
The invention belongs to the fields of synthetic biology and metabolic engineering, and particularly relates to 4 engineering yeasts and a method capable of efficiently synthesizing intermediate products or end products of a ginsenoside Ro synthesis path.
Background
Ginsenoside (ginsenosides) is a steroid compound, also known as triterpenoid saponin. Mainly in ginseng medicinal materials. Ginsenoside is regarded as the main active ingredient in ginseng, and has various pharmacological and physiological activities, including: anti-tumor, central nerve excitation, fatigue resistance, memory and learning improvement, DNA and RNA synthesis promotion, fatigue relief, aging delay, platelet aggregation inhibition, shock resistance, myocardial ischemia and anoxia improvement, coronary heart disease treatment and prevention, myocardial function enhancement, human body autoimmune system protection and the like, so that various ginsenosides are widely used for body health care and tumor, and treatment of myocardial contractility failure, liver diseases and the like caused by various different causes.
Ginsenosides all have similar basic structures and all contain a stanol core of 30 carbon atoms. They are divided into two groups according to the glycosidic architecture: dammarane type and oleanane type. Among them, dammarane type is the main configuration of ginsenoside, and has been the hot spot of research in recent years, and microbial synthesis of dammarane type ginsenoside is also increasing. While oleanane-type ginsenoside has few rare ginsenoside Ro under natural conditions, such as low content (about 0.4%) in plants, so that the study is few, and the ginsenoside Ro has the effects of diminishing inflammation, detoxification, antithrombotic, inhibiting acid system platelet coagulation and resisting hepatitis and activating macrophages, has high research value, but the natural synthesis route is unknown (two-step glycosyl modifying enzyme just reported in the present year), and the complexity of glycosyl modification (two and three) causes the de novo synthesis difficulty. Ginsenoside Ro and its precursor compounds of ginsenoside IVa and Jiang Zhuang, namely, ginsenoside R1, have not been reported to be synthesized de novo by microorganisms.
Disclosure of Invention
In order to fill the blank in the field, the invention provides 4 strains of engineering saccharomycetes which can efficiently participate in the ginsenoside synthesis way, and can realize the de novo synthesis of three oleanane-type rare ginsenoside such as ginsenoside Ro, panax japonicus saponin IVa, jiang Zhuang sanchinoside R1 and calendula glycoside E without adding a heterologous precursor or a substrate only by culturing microorganisms.
The technical scheme adopted for solving the technical problems is as follows:
an engineering saccharomycete CE-P1 capable of efficiently synthesizing calendula glycoside E, which is characterized in that the preservation number is CGMCC No.23731.
A method for efficiently synthesizing calendula extract E is characterized in that engineering saccharomycete CE-P1 with a preservation number of CGMCC No.23731 is cultivated; preferably, the culturing means that the engineering yeast CE-P1 is inoculated with YPD medium for 3-7 days, preferably 5 days; preferably, the inoculation means will OD 600 2-5 percent of engineering saccharomycete CE-P1 seed solution is inoculated into YPD culture medium according to the volume ratio of 5-15 percent, preferably 10 percent; preferably, the culture conditions are 28-30 ℃.
An engineering yeast IVA-P1 capable of efficiently synthesizing panax japonicus saponin IVa is characterized in that the preservation number is CGMCC No.23732.
A method for efficiently synthesizing a panax japonicus saponin IVa is characterized in that engineering saccharomycete IVA-P1 with a preservation number of CGMCC No.23732 is cultivated; preferably, the culturing means inoculating the engineering yeast IVA-P1 with YPD for 3-7 days, preferably 5 days; preferably, the inoculation means will OD 600 2-5 of engineering yeast IVA-P1 is inoculated into YPD culture medium according to the volume ratio of 5% -15%, preferably 10%; preferably, the culture conditions are 28-30 ℃.
An engineering microzyme R1-P1 capable of efficiently synthesizing Jiang Zhuang pseudo-ginseng glycoside R1 is characterized by having a preservation number of CGMCC No.23733.
A method for efficiently synthesizing Jiang Zhuang pseudo-ginseng glycoside R1 is characterized in that engineering saccharomycetes R1-P1 with the preservation number of CGMCC No.23733 are cultivated; preferably, the culture means that the engineering yeast R1-P1 and the substrate are added into YPD medium simultaneously for 3-7 days, preferably 5 days; preferably, the culture condition is 28-30 ℃, and the inoculation amount of the engineering saccharomycetes R1-P1 in the YPD culture medium is 5-15 percent by volume, preferably 10 percent; the inoculation means that the OD 600 2-5 engineering saccharomycete seed liquid is inoculated into liquid culture medium in certain volume ratio.
An engineering microzyme Ro-P1 capable of efficiently synthesizing ginsenoside Ro is characterized by having a preservation number of CGMCC No.23734.
A method for efficiently synthesizing ginsenoside Ro is characterized in that engineering saccharomycete Ro-P1 with the preservation number of CGMCC No.23734 is cultivated; preferably, the culturing means that engineering yeast Ro-P1 is inoculated into YPD medium for 3-7 days, preferably 5 days; preferably, the inoculation means will OD 600 2-5 of engineering yeast Ro-P1, and inoculating 5-15% of seed liquid, preferably 10% of seed liquid into a liquid culture medium according to the volume ratio; preferably, the culture conditions are 28-30 ℃.
The use of the enzyme shown in SEQ ID NO.1 for efficiently synthesizing calendula glycoside E.
A gene of an enzyme for efficiently synthesizing calendula E is characterized by having a nucleotide sequence shown as SEQ ID NO. 2.
The use of the enzyme shown in SEQ ID NO.3 in the efficient synthesis of Jiang Zhuang sanchinoside R1 and/or ginsenoside Ro.
An enzyme gene for efficiently synthesizing Jiang Zhuang sanchinoside R1 and/or ginsenoside Ro, which is characterized by having a nucleotide sequence shown as SEQ ID NO. 4.
The use of the enzyme shown in SEQ ID NO.5 in the efficient synthesis of the panax japonicus saponin IVa and/or the ginsenoside Ro.
The gene of the enzyme for efficiently synthesizing the panax japonicus saponin IVa and/or the ginsenoside Ro is characterized by having a nucleotide sequence shown as SEQ ID NO. 6.
The invention provides an engineering saccharomycete CE-P1, which solves the problem that Saccharomyces cerevisiae is insufficient in UDP-GlcA (uridine diphosphate-glucuronic acid) supply. Compared with the reported existing method, the engineering saccharomycete CE-P1 provided by the invention catalyzes oleanolic acid to generate calendula glycoside E through the cellulose-like synthase derived from liquorice and soybeans, but has lower activity, so that the bottleneck situation of a synthetic route is caused, and the strain CE-P1 provided by the invention solves the synthetic bottleneck from oleanolic acid to calendula glycoside E, and can efficiently synthesize calendula glycoside E.
The second aspect of the invention provides an engineering yeast IVA-P1, which realizes the synthesis of the panax japonicus saponin IVa.
The third aspect of the invention provides an engineering microzyme R1-P1, which realizes the efficient synthesis of Jiang Zhuang pseudo-ginseng glycoside R1 by using the engineering microzyme.
The fourth aspect of the invention provides an engineering yeast Ro-P1, solves the problem of low modification efficiency of a third glycosyl group, and realizes efficient synthesis of ginsenoside Ro by using engineering yeast.
The invention achieves the following effects: the required substrate or precursor is synthesized without the need of heterogenous addition of any link in the ginsenoside Ro synthesis pathway, the engineering yeasts are cultured by using a common YPD (glucose as carbon source input) culture medium, and the culture solution is separated and purified after 5-7 days, so that intermediate products and final products of all links in the ginsenoside Ro synthesis pathway can be efficiently obtained: comprises oleanane type rare ginsenoside such as calendula glycoside E, ginsenoside Ro and precursor compounds thereof as panax japonicus saponin IVa, jiang Zhuang ginsenoside R1 and the like. The invention has the advantages of stable host, short growth period, mild culture condition and low culture cost, and realizes the efficient de novo synthesis of the target compound.
Name of strain preservation: CE-P1
Deposit number: CGMCC No.23731
Classification naming: saccharomyces cerevisiae
Latin name: saccharomyces cerevisiae
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 11, 5
Name of strain preservation: IVA-P1
Deposit number: CGMCC No.23732
Classification naming: saccharomyces cerevisiae
Latin name: saccharomyces cerevisiae
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 11, 5
Name of strain preservation: R1-P1
Deposit number: CGMCC No.23733
Classification naming: saccharomyces cerevisiae
Latin name: saccharomyces cerevisiae
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 11, 5
Name of strain preservation: ro-P1
Deposit number: CGMCC No.23734
Classification naming: saccharomyces cerevisiae
Latin name: saccharomyces cerevisiae
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 11, 5
Drawings
FIG. 1 shows the results of liquid chromatography-mass spectrometry combined detection of the synthesis of calendula E from engineered yeast CE-P1.
FIG. 2 is a comparison of the results of liquid chromatography detection of engineered yeast CE-P1 with human UGDH, reported cellulose synthase for the synthesis of calendula E.
FIG. 3 shows the result of liquid chromatography-mass spectrometry combined detection of the synthetic panax japonicus saponin IVa of the engineering yeast IVA-P1.
FIG. 4 shows the result of liquid chromatography-mass spectrometry combined detection of the bamboo Jiang Zhuang notoginsenoside R1 synthesized by the engineering yeast R1-P1.
FIG. 5 shows the result of liquid chromatography-mass spectrometry combined detection of ginsenoside Ro synthesized by engineering yeast Ro-P1.
FIG. 6 is a graph showing the yields of the compounds synthesized by engineering yeasts CE-P1, IVA-P1, R1-P1 and Ro-P1.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Group 1 example, engineering yeasts CE-P1 of the invention
The embodiment of the group provides an engineering saccharomycete CE-P1 capable of efficiently synthesizing calendula E. The preservation number of the engineering saccharomycete CE-P1 is CGMCC No.23731.
In a specific embodiment, the gene expression system in the engineering yeast CE-P1 contains a nucleotide sequence shown as SEQ ID NO.2 or the engineering yeast CE-P1 can express an amino acid sequence shown as SEQ ID NO. 1.
Group 2 example, method of the invention for Synthesis of calendoside E
The embodiment provides a method for efficiently synthesizing calendula glycoside E. The method is as follows: the engineering saccharomycete CE-P1 with the culture preservation number of CGMCC No.23731.
In a preferred embodiment of this group, the culturing means that the engineering yeast CE-P1 is inoculated with YPD medium for 3-7 days, preferably 5 days;
preferably, the inoculation means will OD 600 2-5 percent of engineering saccharomycete CE-P1 seed solution is inoculated into YPD culture medium according to the volume ratio of 5-15 percent, preferably 10 percent;
preferably, the culture conditions are 28-30 ℃.
Group 3 example, engineering yeasts IVA-P1 of the invention
The embodiment of the group provides an engineering saccharomycete IVA-P1 capable of efficiently synthesizing the ginsenoside IVa. The preservation number of the engineering saccharomycete IVA-P1 is CGMCC No.23732.
In specific examples, the engineered yeast IVA-P1 can express a gene sequence shown as SEQ ID NO.6 or an amino acid sequence shown as SEQ ID NO. 5.
Group 4 example, method of synthesizing Panax japonicus saponin IVa of the present invention
The embodiment of the group provides a method for efficiently synthesizing the panax japonicus saponin IVa. The method is as follows: culturing engineering saccharomycete IVA-P1 with a preservation number of CGMCC No. 23732;
engineering yeast IVA-P1
Preferably, the culturing means that the engineering yeast IVA-P1 and the substrate are added into YPD simultaneously for 3-7 days, preferably 5 days;
preferably, the culture condition is 28-30 ℃, and the inoculation amount of the engineering yeast IVA-P1 in the YPD culture medium is 5-15% by volume, preferably 10%;
preferably, the inoculation refers to inoculating an engineering yeast seed solution with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
Group 5 example, engineering yeasts of the invention R1-P1
The embodiment of the group provides engineering saccharomycetes R1-P1 capable of efficiently synthesizing Jiang Zhuang notoginsenoside R1. The preservation number of the engineering saccharomycetes R1-P1 is CGMCC No.23733.
In a specific embodiment, the gene expression system in the engineering yeast R1-P1 contains a nucleotide sequence shown as SEQ ID NO.4 or the engineering yeast R1-P1 can express an amino acid sequence shown as SEQ ID NO. 3.
Group 6 example, method of the invention for synthesizing zingiberesin R1
The embodiment provides a method for efficiently synthesizing Jiang Zhuang sanchinoside R1. The method is as follows: culturing engineering saccharomycete R1-P1 with a preservation number of CGMCC No. 23733;
preferably, the culture means that the engineering yeast R1-P1 and the substrate are added into YPD medium simultaneously for 3-7 days, preferably 5 days;
preferably, the culture condition is 28-30 ℃, and the inoculation amount of the engineering saccharomycetes R1-P1 in the YPD culture medium is 5-15 percent by volume, preferably 10 percent;
preferably, the inoculation refers to inoculating an engineering yeast seed solution with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
Group 7 example, engineering yeasts of the invention Ro-P1
The embodiment of the group provides an engineering saccharomycete Ro-P1 capable of efficiently synthesizing ginsenoside Ro. The preservation number of the engineering microzyme Ro-P1 is CGMCC No.23734.
Group 8 example, method of the invention for synthesizing ginsenoside Ro
The present embodiment provides a method for efficiently synthesizing ginsenoside Ro. The method is as follows: culturing engineering microzyme Ro-P1 with a preservation number of CGMCC No. 23734;
preferably, the culturing means that the engineering yeast Ro-P1 and the substrate are added into YPD medium simultaneously for 3-7 days, preferably 5 days;
preferably, the culture condition is 28-30 ℃, and the inoculation amount of the engineering yeast Ro-P1 in the YPD culture medium is 5-15% by volume, preferably 10%;
preferably, the inoculation refers to inoculating an engineering yeast seed solution with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
The actions of the engineering yeasts CE-P1 in the embodiment of the 1 st group and IVA-P1 in the embodiment of the 3 rd group and R1-P1 in the embodiment of the 5 th group and 1 strain or 2 strain or 3 strain or 4 strain engineering yeasts in the engineering yeasts Ro-P1 in the embodiment of the 7 th group are all within the protection scope of the invention.
Group 9 example, enzyme highly efficient in the Synthesis of calendoside E and Gene thereof
This group of examples provides the use of the enzyme shown in SEQ ID NO.1 for the efficient synthesis of calendula glycoside E.
Some embodiments provide a gene of an enzyme for efficiently synthesizing calendula E, which is characterized by having a nucleotide sequence as shown in SEQ ID NO. 2.
Enzyme for efficiently synthesizing Jiang Zhuang sanchinoside R1 and/or ginsenoside Ro and genes thereof in example 10
The present example provides the use of the enzyme shown in SEQ ID No.3 for efficient synthesis of Jiang Zhuang notoginsenoside R1 and/or ginsenoside Ro.
Other embodiments of the present group provide an enzyme gene for efficiently synthesizing Jiang Zhuang notoginsenoside R1 and/or ginsenoside Ro, which is characterized by having a nucleotide sequence shown as SEQ ID NO. 4.
Enzyme for highly efficient Synthesis of Panax japonicus saponin IVa and/or ginsenoside Ro and Gene thereof in group 11 example
The present group of examples provides the use of the enzyme shown in SEQ ID No.5 for efficient synthesis of panax japonicus saponin IVa and/or ginsenoside Ro.
Other embodiments of the present group provide a gene of an enzyme for efficiently synthesizing panax japonicus IVa and/or ginsenoside Ro, which is characterized by having a nucleotide sequence as shown in SEQ ID NO. 6.
Experimental example, experimental operation of the invention
YPD medium: about 10g/L yeast extract, 20g/L glucose, 20g/L peptone, and 20g/L agar powder.
The culture was carried out using a 100mL flask and a constant temperature shaker at 30℃and 200rpm, and generally for about 5 days.
The engineering yeast CE-P1 is adopted, and is cultured for 5 days by using YPD culture medium, as shown in figures 1 and 2, the purpose of synthesizing calendula glycoside E (CE) from the head by using the engineering yeast CE-P1 is realized, and compared, the effect of AtUGDH is better than that of human HsUGDH, the activity of Pn022859 is far higher than that of GmSyCSl3 and other cellulose synthase from soybeans, and the bottleneck problem of CE synthesis is solved.
The engineering yeast IVA-P1 is adopted, and is cultured for 5 days by using YPD culture medium, as shown in figure 3, the purpose of de novo synthesis of the panax japonicus saponin IVa by using the engineering yeast IVA-P1 is realized, and no report of synthesizing the compound by using yeast exists at present.
The engineering yeast R1-P1 is adopted, and is cultured for 5 days by using YPD culture medium, as shown in figure 4, the purpose of synthesizing Jiang Zhuang pseudo-ginseng glycoside R1 from the engineering yeast R1-P1 is realized, and no report on synthesizing the compound by using yeast exists at present.
The engineering yeast Ro-P1 is adopted, and is cultured for 5 days by using YPD culture medium, as shown in figure 5, the purpose of synthesizing the trisaccharide compound ginsenoside Ro from the head by using the engineering yeast Ro-P1 is realized, and no report on synthesizing the compound by using the yeast exists at present.
SEQUENCE LISTING
<110> university of Beijing technology
<120> 4 strain engineering saccharomycete capable of efficiently synthesizing intermediate or final product of ginsenoside Ro synthesis path and method
<130> P210709/BLG
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 721
<212> PRT
<213> Artificial Sequence
<220>
<223> enzyme for highly efficient Synthesis of calendoside E
<400> 1
Met Ala Ser Thr Thr Ser Leu Leu His Thr Ala Thr Val His Lys Leu
1 5 10 15
His Ala Thr Met Asn Arg Thr His Ile Leu Phe His Phe Leu Leu Ile
20 25 30
Leu Ser Val Phe Tyr Tyr Arg Leu Phe Ser Leu Leu Asn Ser Thr Pro
35 40 45
Asp His Asn Phe Pro Ala Thr Ser Trp Ile Phe Met Ile Ile Ala Glu
50 55 60
Leu Ile Leu Thr Phe Ala Trp Phe Phe Gly Gln Ser Phe Arg Trp Arg
65 70 75 80
Pro Val Ser Arg Ser Val His Pro Glu Arg Leu Pro Gly Asp Lys Ser
85 90 95
Leu Pro Gly Val Asp Val Phe Ile Cys Thr Ala Asp Pro Lys Lys Glu
100 105 110
Pro Thr Leu Glu Val Met Asn Thr Val Ile Ser Ala Met Cys Leu Asp
115 120 125
Tyr Pro Pro Glu Lys Leu Ala Val Tyr Leu Ser Asp Asp Gly Gly Ala
130 135 140
Ala Val Thr Leu Thr Gly Met Arg Glu Ala Ala Val Phe Ala Arg Trp
145 150 155 160
Trp Ile Pro Phe Cys Arg Lys Tyr Asn Ile Lys Thr Arg Cys Pro Gly
165 170 175
Ala Tyr Phe Ser Ala Leu Thr Asp Gly Asn Glu Gln Asp Ile Arg Gly
180 185 190
Gly Glu Phe Gln Val Glu Gln Asp Lys Ile Lys Leu Lys Tyr Glu His
195 200 205
Phe Lys Glu Val Val Glu Ser Gly Gly Asp Asp Ala Gln Ala Asp Asp
210 215 220
Met Asn Asn Ile Thr Gly Asp Gly Arg Ala Pro Leu Val Gln Val Ile
225 230 235 240
His Asp Ser Ser Arg Gly Asn Glu Asp Asp Glu Asn Asp Glu Asn Asp
245 250 255
Glu Gln Met Pro Leu Leu Val Tyr Val Ser Arg Glu Arg Arg Pro Ser
260 265 270
Tyr Pro His Arg Phe Lys Ala Gly Ala Leu Asn Ala Leu Leu Arg Val
275 280 285
Ser Gly Ile Ile Ser Asn Phe Pro Tyr Val Met Val Leu Asp Cys Asp
290 295 300
Met Tyr Cys Asn Asp Pro Thr Ser Ala Lys Gln Ala Met Cys Phe His
305 310 315 320
Leu Asp Pro Asn Met Ser Ser Glu Leu Ala Phe Val Gln Tyr Pro Gln
325 330 335
Val Phe Tyr Asn Val Thr Ser Asn Asp Ile Tyr Asp Gly Gln Ala Arg
340 345 350
Ser Ala Phe Lys Thr Lys Trp Gln Gly Met Asp Gly Leu Arg Gly Pro
355 360 365
Val Leu Thr Gly Thr Gly Tyr Tyr Leu Lys Arg Lys Ala Leu Phe Gly
370 375 380
Ser Pro Asn Asp Gln Glu Val Thr Tyr Leu His Gln Pro Ala Leu Ser
385 390 395 400
Phe Gly Asn Ser Asp Met Phe Ile Ala Ser Leu His Gly Asn Asn Gln
405 410 415
Lys His Ile Val Asn Asn Gly Glu Thr Thr Glu Glu Gln Gln Leu Lys
420 425 430
Glu Ala His Val Leu Ala Ser Cys Ser Phe Glu Lys Gly Thr Lys Trp
435 440 445
Gly Ser Glu Ile Gly Tyr Thr Tyr Glu Cys Leu Leu Glu Ser Thr Phe
450 455 460
Thr Gly Tyr Tyr Leu Gln Cys Lys Gly Trp Lys Ser Val Tyr Cys Tyr
465 470 475 480
Pro Lys Lys Pro Ala Phe Leu Gly Cys Ala Thr Ile Asp Met Lys Asp
485 490 495
Ala Leu Val Gln Leu Arg Lys Trp Asn Gly Asn Leu Leu Glu Leu Gly
500 505 510
Phe Ser Lys Phe Ser Pro Leu Thr Tyr Ala Ile Ser Arg Met Ser Phe
515 520 525
Leu His Ser Met Cys Tyr Ala Tyr Phe Ala Phe Gln Pro Leu Thr Ser
530 535 540
Leu Ser Leu Leu Ile Tyr Gly Ile Val Pro Gln Phe Cys Phe Phe Lys
545 550 555 560
Gly Ile Ala Leu Phe Pro Lys Val Thr Asp Pro Trp Ile Ala Ala Phe
565 570 575
Ala Phe Val Trp Val Ser Ser Leu Leu Gln His Leu Tyr Glu Val Leu
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Ser Ser Gly Gly Ser Leu Arg Thr Trp Trp Asn Glu Gln Arg Ile Trp
595 600 605
Val Ile Lys Val Val Ser Gly Ser Leu Phe Gly Cys Met Asp Cys Val
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Arg Lys Lys Ile Gly Ile Ser Lys Thr Val Phe Arg Leu Thr Asn Lys
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Ala Ile Glu Lys Glu Lys Leu Glu Asn Tyr Glu Lys Gly Lys Phe Asp
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Trp Asn Met Ala Cys Leu Ile Gly Gly Ile Trp Arg Met Phe Val Gly
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Gly Asn Ser Ser Glu Met Phe Gly Gln Leu Phe Leu Ser Ser Phe Leu
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Ser Leu Val Ser Tyr Pro Ile Ile Gly Gly Met Leu Thr Arg Lys Gly
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Asn
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<211> 2166
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene of enzyme for highly efficient Synthesis of calendoside E
<400> 2
atggcttcta ctacatcttt gttacatact gctactgttc ataaattgca tgctactatg 60
aacagaactc atattttgtt tcatttcttg ttgatcttat ctgtctttta ttacagattg 120
ttttctttgt tgaattctac tcctgatcat aattttcctg ctacttcttg gatttttatg 180
attattgctg aattgatttt gacttttgct tggttttttg gtcaatcttt tagatggaga 240
cctgtttcta gatctgttca tcctgaaaga ttgcctggtg ataaatcttt gcctggtgtt 300
gatgttttta tttgtactgc tgatccaaaa aaagaaccaa ctttggaagt tatgaatact 360
gttatttctg ctatgtgttt ggattatcca cctgaaaaat tggctgttta tttgtctgat 420
gatggtggtg ctgctgttac tttgactggt atgagagaag ctgctgtttt tgctaggtgg 480
tggattccat tttgcagaaa atacaatatt aaaactagat gtcctggtgc ttatttttct 540
gctttgactg atggtaatga acaagatatt agaggtggag aatttcaagt tgagcaagat 600
aagattaaat tgaaatacga acattttaaa gaagttgttg aatctggtgg tgatgatgct 660
caagctgatg atatgaataa tattactggt gatggtagag ctccattggt tcaagttatt 720
catgattctt ctagaggtaa tgaagacgac gaaaatgatg aaaatgatga acaaatgcca 780
ttgttggttt atgtttctag agaaagaaga ccatcttatc cacatagatt taaagctggt 840
gctttgaatg ctttgttgag agtttctggt attatctcta actttccata cgttatggtt 900
ttggattgtg atatgtattg taatgatcca acttctgcta aacaagctat gtgttttcat 960
ttagatccaa atatgtcatc tgaattggct tttgttcaat atccacaagt tttttacaat 1020
gttacatcta atgatattta tgatggtcaa gctagatctg cttttaaaac taaatggcaa 1080
ggtatggatg gtttgagagg tcctgttttg actggtactg gatattattt gaaaagaaaa 1140
gctttgttcg gatctccaaa tgatcaagaa gttacttatt tgcatcaacc tgctttgtct 1200
ttcggtaatt cagatatgtt catcgcttca ttgcatggta ataatcaaaa gcacattgtc 1260
aataacggtg aaactactga agaacaacaa ttgaaagaag ctcatgtttt ggcttcttgt 1320
tcttttgaaa aaggtactaa atggggttca gaaattggtt acacttatga atgtttgttg 1380
gagtctacat ttactggtta ttatttgcag tgcaaaggat ggaaatctgt ttattgttat 1440
ccaaaaaaac ctgctttttt gggttgtgct actattgata tgaaagatgc tttggttcaa 1500
ttgagaaaat ggaatggtaa tttgttggaa ttgggttttt ctaaattttc tccattgact 1560
tatgctattt ctagaatgtc ttttttgcat tctatgtgtt atgcttattt tgcttttcaa 1620
ccattgactt ctttgtcttt gttgatttat ggtatcgttc cacagttttg tttttttaaa 1680
ggtattgctt tgtttccaaa agttactgat ccatggattg ctgcttttgc ttttgtttgg 1740
gtttcttctt tgttgcaaca tttgtatgaa gttttgtctt ctggtggttc tttgagaact 1800
tggtggaatg aacaaagaat ttgggttatt aaagttgttt ctggttcttt gttcggttgt 1860
atggattgtg ttagaaaaaa aattggtatc tctaaaactg tttttagatt aacaaataaa 1920
gctattgaaa aagaaaagtt agaaaattat gaaaaaggta aatttgattt tcaaggtgct 1980
caaattttta tggttccatt ggttggtttg gttatttgga atatggcttg tttgattggt 2040
ggtatttgga gaatgtttgt tggaggaaat tcttctgaaa tgtttggtca attgtttttg 2100
tcttcttttt tgtctttggt ttcttatcca attattggtg gtatgttgac tagaaaaggt 2160
aattaa 2166
<210> 3
<211> 494
<212> PRT
<213> Artificial Sequence
<220>
<223> enzyme for highly efficient Synthesis of Jiang Zhuang Notoginseng radix glycoside R1 and/or ginsenoside Ro
<400> 3
Met Met Glu Ser Gln Gln Leu His Val Val Phe Val Pro Phe Pro Thr
1 5 10 15
Pro Gly His Met Leu Pro Met Ile Asp Thr Ala Arg Leu Phe Ala Lys
20 25 30
His Gly Val Asn Val Thr Ile Ile Thr Thr His Ser Asn Ala Ser Thr
35 40 45
Phe Gln Lys Ser Ile Asp Ser Asp Phe Asn Ser Gly Tyr Ser Ile Lys
50 55 60
Thr Gln Leu Ile Gln Phe Pro Ser Ala Gln Val Gly Leu Pro Asp Gly
65 70 75 80
Val Glu Asn Ile Lys Asp Gly Thr Ser Arg Glu Ile Leu Gly Lys Ile
85 90 95
Ser Arg Gly Ile Trp Met Leu Gln Asn Pro Ile Glu Ile Leu Phe Lys
100 105 110
Asp Leu Gln Pro Asp Cys Ile Val Thr Asp Met Met His Ala Trp Thr
115 120 125
Val Glu Ala Ala Ala Lys Leu Gly Ile Pro Arg Ile His Tyr Tyr Ser
130 135 140
Ser Ser Tyr Phe Ser Asn Cys Ala Tyr His Phe Ile Met Lys Tyr Arg
145 150 155 160
Pro His Asp Gly Leu Val Ser Asp Thr Gln Lys Phe Thr Ile Pro Gly
165 170 175
Phe Pro His Ser Ile Glu Met Thr Arg Leu Gln Ile Pro Asp Trp Leu
180 185 190
Arg Glu Lys Ser Ala Ala Thr Ala Tyr Phe Glu Pro Ile Tyr Gln Ser
195 200 205
Glu Lys Arg Ser Tyr Gly Thr Leu Tyr Asn Ser Phe His Glu Leu Glu
210 215 220
Ser Asp Tyr Glu Lys Leu Asn Arg Ser Thr Leu Gly Ile Lys Ser Trp
225 230 235 240
Ser Val Gly Pro Val Ser Ser Trp Val Asn Lys Asp Asp Glu Lys Lys
245 250 255
Ala Asn Arg Gly His Met Glu Glu His Gly Lys Glu Ala Glu Trp Leu
260 265 270
Asn Trp Leu Asn Ser Lys Glu Asn Glu Ser Val Leu Tyr Val Ser Phe
275 280 285
Gly Ser Leu Thr Arg Leu Ala His Ala Gln Leu Val Glu Ile Ala His
290 295 300
Gly Leu Glu Asn Ser Gly Arg Asn Phe Ile Trp Val Val Arg Lys Asn
305 310 315 320
Asp Arg Asp Glu Gly Glu Asn Ser Phe Leu Gln Asp Phe Glu Gln Arg
325 330 335
Met Lys Glu Ser Lys Lys Gly Tyr Ile Ile Trp Asn Trp Ala Pro Gln
340 345 350
Leu Leu Ile Leu Asp His Pro Ala Thr Gly Gly Ile Val Thr His Cys
355 360 365
Gly Trp Asn Ser Ile Leu Glu Ser Leu Asn Ala Gly Leu Pro Met Ile
370 375 380
Ala Trp Pro Met Phe Ala Asp Gln Phe Tyr Asn Glu Lys Leu Leu Val
385 390 395 400
Asp Val Leu Lys Ile Gly Val Pro Val Gly Ala Lys Glu Asn Lys Leu
405 410 415
Trp Asn Ser Val Ser Val Glu Ala Val Met Lys Arg Glu Glu Ile Val
420 425 430
Lys Ala Val Asp Ile Leu Met Gly Ser Gly Gln Glu Ser Ile Glu Met
435 440 445
Arg Met Arg Ala Lys Lys Leu Gly Asp Ala Ala Lys Arg Thr Ile Glu
450 455 460
Glu Gly Gly His Ser Tyr Asn Thr Leu Ile Gln Leu Ile Asp Glu Leu
465 470 475 480
Lys Ser Leu Lys Lys Ser Lys Ala Leu Asp Val Lys Val Asn
485 490
<210> 4
<211> 1485
<212> DNA
<213> Artificial Sequence
<220>
<223> genes of enzymes for highly efficient synthesis of Jiang Zhuang sanchinoside R1 and/or ginsenoside Ro
<400> 4
atgatggaat ctcaacaatt gcatgttgtt tttgttccat ttccaactcc tggtcatatg 60
ttgcctatga ttgatactgc tagattgttt gctaaacatg gtgttaatgt cactattatc 120
actacacatt caaacgcatc tacttttcag aaatcaatcg attctgattt taattctggt 180
tactctatca aaactcaatt gattcaattt ccatctgctc aagttggttt gcctgatggt 240
gttgaaaata ttaaagatgg tacttctaga gaaattttgg gtaaaatttc tagaggtatc 300
tggatgttgc aaaacccaat tgaaattttg tttaaagatt tgcaacctga ttgtattgtt 360
actgatatga tgcatgcttg gactgttgaa gctgcagcta aattgggtat tcctaggatt 420
cattactact catcttcata tttttctaat tgtgcatacc attttattat gaaatataga 480
ccacatgatg gtttggtttc tgatactcaa aaatttacta ttcctggttt tccacattca 540
attgaaatga caagattgca aattcctgat tggttgagag aaaaatctgc tgcaactgca 600
tattttgagc ctatctatca atctgaaaaa agatcttatg gtactttgta taattctttt 660
cacgagttgg agtcagatta tgaaaaattg aatagatcta ctttgggtat taaatcttgg 720
tctgttggtc ctgtttcttc ttgggttaat aaagatgatg aaaaaaaagc taatagaggt 780
catatggaag aacatggtaa agaagctgag tggttgaatt ggttgaattc taaagagaat 840
gaatctgttt tgtatgtttc ttttggttct ttgactagat tggctcatgc tcaattggtt 900
gaaattgctc atggtttgga aaattctggt agaaatttta tttgggttgt taggaagaat 960
gatagagatg aaggtgagaa ttcattcttg caagattttg aacaaagaat gaaagaatct 1020
aaaaaaggtt atattatttg gaattgggct ccacaattgt tgattttgga tcatcctgct 1080
actggtggta ttgttactca ttgtggttgg aattctattt tggaatcttt gaatgctggt 1140
ttgccaatga ttgcttggcc aatgtttgct gatcaatttt ataatgaaaa attgttggtt 1200
gatgttttga aaattggtgt tcctgttggt gctaaagaaa ataaattgtg gaattctgtt 1260
tcagttgaag ctgtcatgaa aagagaagaa attgttaaag ctgttgatat tttgatgggt 1320
tctggtcaag aatctattga aatgaggatg agagctaaaa aattgggtga tgctgctaaa 1380
agaactatcg aggaaggtgg tcactcttat aatactttaa ttcaattgat cgacgaattg 1440
aaatcattaa agaagtctaa agctttggat gttaaagtta attaa 1485
<210> 5
<211> 497
<212> PRT
<213> Artificial Sequence
<220>
<223> enzyme for highly efficient Synthesis of Panax japonicus saponin IVa and/or ginsenoside Ro
<400> 5
Met Glu Gly Val Glu Val Glu Gln Pro Leu Lys Val Tyr Phe Ile Pro
1 5 10 15
Phe Leu Ala Ser Gly His Met Ile Pro Leu Phe Asp Ile Ala Thr Met
20 25 30
Phe Ala Ser Arg Gly Gln Gln Val Thr Val Ile Thr Thr Pro Ala Asn
35 40 45
Ala Lys Ser Leu Thr Lys Ser Leu Ser Ser Asp Ala Pro Ser Phe Leu
50 55 60
Arg Leu His Thr Val Asp Phe Pro Ser Gln Gln Val Gly Leu Pro Glu
65 70 75 80
Gly Ile Glu Ser Met Ser Ser Thr Thr Asp Pro Thr Thr Thr Trp Lys
85 90 95
Ile His Thr Gly Ala Met Leu Leu Lys Glu Pro Ile Gly Asp Phe Ile
100 105 110
Glu Asn Asp Pro Pro Asp Cys Ile Ile Ser Asp Ser Thr Tyr Pro Trp
115 120 125
Val Asn Asp Leu Ala Asp Lys Phe Gln Ile Pro Asn Ile Thr Phe Asn
130 135 140
Gly Leu Cys Leu Phe Ala Val Ser Leu Val Glu Thr Leu Lys Thr Asn
145 150 155 160
Asn Leu Leu Lys Ser Gln Thr Asp Ser Asp Ser Asp Ser Ser Ser Phe
165 170 175
Val Val Pro Asn Phe Pro His His Ile Thr Leu Cys Gly Lys Pro Pro
180 185 190
Lys Val Ile Gly Ile Phe Met Gly Met Met Leu Glu Thr Val Leu Lys
195 200 205
Ser Lys Ala Leu Ile Ile Asn Asn Phe Ser Glu Leu Asp Gly Glu Glu
210 215 220
Cys Ile Gln His Tyr Glu Lys Ala Thr Gly His Lys Val Trp His Leu
225 230 235 240
Gly Pro Thr Ser Leu Ile Arg Lys Thr Ala Gln Glu Lys Ser Glu Arg
245 250 255
Gly Asn Glu Gly Ala Val Asn Val His Glu Ser Leu Ser Trp Leu Asp
260 265 270
Ser Glu Arg Val Asn Ser Val Leu Tyr Ile Cys Phe Gly Ser Ile Asn
275 280 285
Tyr Phe Ser Asp Lys Gln Leu Tyr Glu Met Ala Cys Ala Ile Glu Ala
290 295 300
Ser Gly His Pro Phe Ile Trp Val Val Pro Glu Lys Lys Gly Lys Glu
305 310 315 320
Asp Glu Ser Glu Glu Glu Lys Glu Lys Trp Leu Pro Lys Gly Phe Glu
325 330 335
Glu Arg Asn Ile Gly Lys Lys Gly Leu Ile Ile Arg Gly Trp Ala Pro
340 345 350
Gln Val Lys Ile Leu Ser His Pro Ala Val Gly Gly Phe Met Thr His
355 360 365
Cys Gly Gly Asn Ser Thr Val Glu Ala Val Ser Ala Gly Val Pro Met
370 375 380
Ile Thr Trp Pro Val His Gly Asp Gln Phe Tyr Asn Glu Lys Leu Ile
385 390 395 400
Thr Gln Phe Arg Gly Ile Gly Val Glu Val Gly Ala Thr Glu Trp Cys
405 410 415
Thr Ser Gly Val Ala Glu Arg Lys Lys Leu Val Ser Arg Asp Ser Ile
420 425 430
Glu Lys Ala Val Arg Arg Leu Met Asp Gly Gly Asp Glu Ala Glu Asn
435 440 445
Ile Arg Leu Arg Ala Arg Glu Phe Gly Glu Lys Ala Ile Gln Ala Ile
450 455 460
Gln Glu Gly Gly Ser Ser Tyr Asn Asn Leu Leu Ala Leu Ile Asp Glu
465 470 475 480
Leu Lys Arg Ser Arg Asp Leu Lys Arg Leu Arg Asp Leu Lys Leu Asp
485 490 495
Asp
<210> 6
<211> 1494
<212> DNA
<213> Artificial Sequence
<220>
<223> Gene of enzyme for highly efficient Synthesis of Panax japonicus saponin IVa and/or ginsenoside Ro
<400> 6
atggaagggg ttgaagttga acaaccattg aaagtttatt ttattccatt tttggcctct 60
gggcatatga ttccattgtt tgatattgct actatgtttg cttctagagg tcaacaagtt 120
actgttatta ctactcctgc taatgctaaa tctttgacta agtctttgtc tagtgatgct 180
ccatcttttt tgagattgca tactgttgat tttccatctc aacaagttgg tttgcctgaa 240
ggtattgaat ctatgtcttc tactactgat ccaactacta cttggaaaat tcatactggt 300
gctatgttgt tgaaagaacc aattggtgat tttattgaaa atgatccacc tgattgtatt 360
atttctgatt ctacttatcc atgggtgaat gatttggctg ataaatttca aattccaaac 420
atcactttta atggtttgtg tctttttgct gtctctttgg ttgaaactct gaaaaccaac 480
aatttgctaa agtctcaaac tgattctgat tctgattctt cttcttttgt tgttccaaat 540
tttccacatc atattacttt gtgtggtaaa ccaccaaaag ttattggtat ttttatgggt 600
atgatgttgg agactgtttt gaagtctaag gctttgatta ttaataattt ttcagaattg 660
gatggtgaag aatgtattca acattatgaa aaagctactg gtcataaagt ttggcatttg 720
ggtccaactt ctttgattag aaaaactgct caagaaaaat ctgaaagagg taatgaaggt 780
gctgttaatg ttcatgagtc attgtcttgg ttggactctg aaagagtaaa ttctgtctta 840
tatatatgtt ttggatcaat aaactatttc tctgataaac aattgtatga aatggcttgt 900
gctattgaag catctggtca tccattcatt tgggttgttc ctgaaaaaaa aggtaaagaa 960
gatgaatctg aggaagaaaa ggaaaaatgg ttgccaaaag ggtttgaaga aagaaacatt 1020
ggtaaaaaag gtttgattat tagaggttgg gctccacaag ttaaaatttt gtctcatcct 1080
gctgttggtg gttttatgac tcattgtggt ggtaattcta ctgttgaagc tgtttctgct 1140
ggtgttccaa tgattacttg gcctgttcat ggtgatcaat tttataatga aaaattgatt 1200
actcaattta gaggtattgg agttgaagtt ggtgctactg aatggtgtac ttctggtgtt 1260
gctgaaagaa aaaaattggt ttctagagat tctattgaaa aagctgttag aagattgatg 1320
gatggtggtg atgaagctga aaatattaga ttgagagcta gagaatttgg tgaaaaagct 1380
attcaagcta ttcaagaggg tggttcttct tataataatc tcttggcatt gattgatgaa 1440
ttgaaaagat ctagagattt aaagagattg agagacctca aattggatga ctaa 1494
Claims (6)
1. Engineering saccharomycete capable of efficiently synthesizing calendula glycoside ESaccharomyces cerevisiaeCE-P1, characterized in that the preservation number is CGMCC No.23731.
2. A method for efficiently synthesizing calendula extract E is characterized in that engineering saccharomycetes CE-P1 with the preservation number of CGMCC No.23731 are cultured, wherein the culturing refers to inoculating the engineering saccharomycetes CE-P1 into YPD culture medium to be cultured for 3-7 days; the inoculation means that the OD 600 2-5 percent of seed solution of engineering saccharomycete CE-P1 is inoculated into YPD culture medium according to the volume ratio of 5-15 percent; the culture conditions were 28-30 ℃.
3. The method for efficiently synthesizing calendula E according to claim 2, wherein the culturing means culturing by inoculating the engineering yeast CE-P1 into YPD medium for 5 days.
4. A method for the efficient synthesis of calendered glycoside E according to claim 2 or 3, wherein the inoculation is directed at the OD 600 2-5 of engineering yeast CE-P1 is inoculated into YPD culture medium according to the volume ratio of 10%.
The use of the enzyme shown in SEQ ID No.1 in the efficient synthesis of calendula glycoside E is characterized in that the enzyme is expressed by engineering saccharomycete CE-P1 with a preservation number of CGMCC No.23731.
6. The gene of the enzyme for efficiently synthesizing the calendula glycoside E is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2; the enzyme is expressed by engineering saccharomycete CE-P1 with a preservation number of CGMCC No.23731.
Priority Applications (2)
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| CN202111498550.7A CN114134060B (en) | 2021-12-09 | 2021-12-09 | Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method |
| PCT/CN2022/104358 WO2023103365A1 (en) | 2021-12-09 | 2022-07-07 | Four engineering yeasts capable of efficiently synthesizing intermediate product or end product in synthetic route of ginsenoside ro and method |
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| CN202111498550.7A CN114134060B (en) | 2021-12-09 | 2021-12-09 | Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method |
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| CN114134060B (en) * | 2021-12-09 | 2023-09-19 | 北京理工大学 | Engineering saccharomycete capable of efficiently synthesizing intermediate product or final product of ginsenoside Ro synthesis path and method |
| CN115725620B (en) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | Method for synthesizing panax japonicus saponins in pseudo-ginseng cells |
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| Publication number | Publication date |
|---|---|
| WO2023103365A1 (en) | 2023-06-15 |
| CN114134060A (en) | 2022-03-04 |
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