CN112852653A - Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof - Google Patents
Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof Download PDFInfo
- Publication number
- CN112852653A CN112852653A CN202110106054.6A CN202110106054A CN112852653A CN 112852653 A CN112852653 A CN 112852653A CN 202110106054 A CN202110106054 A CN 202110106054A CN 112852653 A CN112852653 A CN 112852653A
- Authority
- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- gene
- udp
- glucosyltransferase
- ncbi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13078—Ent-kaurene oxidase (1.14.13.78)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13079—Ent-kaurenoic acid oxidase (1.14.13.79)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03019—Ent-kaurene synthase (4.2.3.19)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a saccharomyces cerevisiae engineering bacterium for synthesizing rebaudioside M from head and application thereof, belonging to the technical field of metabolic engineering. The saccharomyces cerevisiae engineering bacteria of the invention integrally express kaurene synthase fpps, kaurene synthase ks, kaurene oxidase ko, steviol synthase kah, UDP glucosyltransferase ugt74g1, UDP glucosyltransferase ugt85C2, UDP glucosyltransferase eugt11 and UDP glucosyltransferase ugt76g 1. The recombinant saccharomyces cerevisiae provided by the invention can realize fermentation production of rebaudioside M by taking glucose as a substrate, and can be accumulated in cells, thereby laying a foundation for de novo synthesis of rebaudioside M by saccharomyces cerevisiae modified by metabolic engineering. The recombinant saccharomyces cerevisiae provided by the invention is simple in construction method, convenient to use and has good application prospect.
Description
Technical Field
The invention relates to a saccharomyces cerevisiae engineering bacterium for synthesizing rebaudioside M from head and application thereof, belonging to the technical field of metabolic engineering.
Background
Rebaudioside M, is a sweet component of stevia sugar that tastes good and has no aftertaste. Stevioside, also known as stevioside, has the characteristics of high sweetness (300-450 times of sucrose), low calorific value (1/300 of sucrose) and the like, and natural sucrose substitutes are known as the third world's original sugar. Stevia sugar is a purified product obtained by extracting stevioside from stevia rebaudiana Bertoni. The steviol glycosides extracted from stevia rebaudiana by manufacturers are mostly of the Reb a type. This type of steviol glycoside is the easiest to extract because of its high content, but is a source of bitterness. The research finds new steviol glycosides, Reb D and Reb M. These two steviol glycosides are not bitter, but are very low in stevia rebaudiana, Reb a accounts for 1/3, while Reb D and Reb M account for less than 1% in the dry leaves of stevia rebaudiana. To solve this problem, new stevia varieties rich in Reb D and Reb M need to be grown, but the advanced technology and equipment required by them are very capital intensive. Even in the largest export country of stevia, most manufacturers are not affordable. At present, the genetic engineering method is adopted to construct a genetic engineering strain to synthesize a natural product from the beginning, and the natural product has the advantages of low cost, unlimited raw materials, simple extraction process, no seasonality, short production time, small environmental pollution and the like, so that the natural product is favored by broad scholars.
The microbial fermentation method has sustainable, green and environment-friendly social and economic benefits, and the method for producing rebaudioside-M by adopting the microbial fermentation method is an effective way for solving the problems. Since the microorganisms do not have a Reb M synthetic pathway, the research uses Reb M as a final target product to carry out metabolic modification. At present, no report is related to the synthesis of rebaudioside M from head to head by using glucose as a substrate in a microbial fermentation method.
Saccharomyces cerevisiae is also known as baker's yeast or budding yeast. Saccharomyces cerevisiae is the yeast most widely related to human beings, and has been used as a food safety strain in the food and wine industry for making bread, steamed bread and the like. In recent years, researchers have begun to study the production of natural products, such as artemisinic acid, notoginsenoside, etc., using saccharomyces cerevisiae. Compared with Escherichia coli, the Saccharomyces cerevisiae has the advantages of high safety, low pathogenicity, high stress resistance, low probability of being polluted by phage and the like, so that the Saccharomyces cerevisiae also plays an important role in the field of genetic engineering. However, there is no metabolic pathway for Reb M synthesis in saccharomyces cerevisiae.
Disclosure of Invention
In order to solve the technical problems, the CRISPR Cas9 technology is adopted to integrate the metabolic pathway of Reb M into saccharomyces cerevisiae, and since accumulation of precursor pyrophosphoric acid isoprene and dimethyl propenyl diphosphate synthesized by Reb M is toxic to cell growth, a glucose repression type promoter P is selectedgal1/10The cell growth and the production of the rebaudioside M are separated, so that the effect of efficiently synthesizing the rebaudioside M is achieved.
The first purpose of the invention is to provide a saccharomyces cerevisiae engineering bacterium for synthesizing rebaudioside M from the beginning, and the saccharomyces cerevisiae engineering bacterium integrates and expresses kaurene synthase fpps, kaurene synthase ks, kaurene oxidase ko, steviol synthase kah, UDP glucosyltransferase ugt74g1, UDP glucosyltransferase ugt85C2, UDP glucosyltransferase eugt11 and UDP glucosyltransferase ugt76g 1.
Further, it is characterized byThe saccharomyces cerevisiae engineering bacteria are promoter Pgal1/10Controlling gene expression.
Further, the promoter Pgal1/10Is a bidirectional promoter consisting of promoters Gal1 and Gal10 induced by beta-galactose and glucose together.
Further, the promoter Pgal1/10The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
Further, the NCBI of kaurene synthase fpps is numbered P08836.2, kaurene synthase ks is numbered Q9UVY5, kaurene oxidase ko is numbered AAQ63464.1, steviol synthase kah is numbered NP _197872.1, UDP glucosyltransferase ugt74g1 is numbered Q6VAA6.1, UDP glucosyltransferase ugt85C2 is numbered Q6VAB0.1, UDP glucosyltransferase eugt11 is numbered AAS07253.1, and UDP glucosyltransferase ugt76g1 is numbered AGL 95113.1.
Further, the kaurene synthase gene and the kaurene synthase gene are inserted into a 308a site of a saccharomyces cerevisiae genome; the kaurene oxidase gene is inserted into 1014a locus of a saccharomyces cerevisiae genome; inserting a steviol synthase gene into a 1309a locus of a saccharomyces cerevisiae genome; inserting 1622a sites of UDP glucosyltransferase gene and UDP glucosyltransferase gene; the UDP-glucosyltransferase gene and the UDP-glucosyltransferase gene were inserted into gal80 site.
Further, the saccharomyces cerevisiae engineering bacteria also comprise Pgal1/10The isopentenyl diphosphate-isomerase gene idi in the mevalonate pathway controlled by the bidirectional promoter and the hydroxymethylglutaryl-CoA reductase gene thmg1 from which the N-terminal endoplasmic reticulum localization signal peptide has been excised are integrated into the 208a locus of the genome.
The second purpose of the invention is to provide the application of the saccharomyces cerevisiae engineering bacteria in fermentation production of rebaudioside M.
Further, the application is that the saccharomyces cerevisiae engineering bacteria are inoculated into a fermentation culture medium according to the inoculation amount of 1-5%, and cultured for 90-100h under the conditions of 28-32 ℃ and 200-250 rpm.
Further, the fermentation medium comprises (g/L): 30-50 parts of glucose, 10-30 parts of peptone and 5-15 parts of yeast powder.
The invention has the beneficial effects that:
the recombinant saccharomyces cerevisiae provided by the invention can realize fermentation production of rebaudioside M by taking glucose as a substrate, and can be accumulated in cells, thereby laying a foundation for de novo synthesis of rebaudioside M by saccharomyces cerevisiae modified by metabolic engineering. The recombinant saccharomyces cerevisiae provided by the invention is simple in construction method, convenient to use and has good application prospect.
Drawings
FIG. 1 is a graph showing the results of qualitative mass spectrometry;
FIG. 2 is a graph showing the results of chromatography.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Detection method of rebaudioside M: high performance liquid chromatography-mass spectrometry (LC-MS) detection method: waters (micromass QUATTRO MICRO), BEH-C18 column, mobile phase acetonitrile and 0.1% aqueous formic acid, column temperature 45 ℃, sample size 1. mu.L.
Seed medium (g/L): yeast Nitrigon Base 6.7, glucose 20, histidine, tryptophan, leucine and uracil as required, to a final concentration of 50g/mL in the medium, at natural pH.
Fermentation medium (g/L): glucose 40, peptone 20, yeast powder 10, natural pH.
Example 1: construction of rebaudioside M de novo synthesis recombinant Saccharomyces cerevisiae strains
According to the rebaudioside M key enzyme synthesis fpps (NCBI ID: P08836.2), kaurene synthase ks (NCBI ID: Q9UVY5), kaurene oxidase ko (NCBI ID: AAQ63464.1), steviol synthase kah (NCBI ID: NP-197872.1), UDP glucosyltransferase UGT74g1(NCBI ID: Q6VAA6.1), UDP glucosyltransferase UGT85C2(NCBI ID: Q6VAB0.1), UDP glucosyltransferase eugt11(NCBI ID: AAS07253.1) and UDP glucosyltransferase UGT76g1(NCBI ID: AGL95113.1) published on NCBI, codon optimization and whole gene synthesis were performed according to the codon preference of Saccharomyces cerevisiae. A20 bp PAM sequence of CRISPR Cas9 is selected according to the integration sites of saccharomyces cerevisiae genomes 308a, 416d, 1309a, 1622b and gal80, upstream and downstream homologous arm amplification primers of a gene integration frame are designed on two sides of the PAM sequence, and a left arm and a right arm are amplified by PCR respectively by taking saccharomyces cerevisiae genome DNA as templates.
According to the design method of the overlapping derivative PCR primer, the key enzyme and the promoter P in the rebaudioside-M synthetic pathway are designed and amplified by the primergal1/10And a terminator. And using gfks, fpps, ko, kah, ugt76g1, ugt85c2, ugt74g1 and eugt11 plasmids synthesized by whole genes as templates to amplify and obtain corresponding gene fragments. Taking saccharomyces cerevisiae genome DNA as a template, and amplifying to obtain a promoter Pgal1/10Terminator CYC1t and ADH1 t. The gene integration cassette was obtained by overlap extension PCR.
Constructing sgRNA plasmid of CRISPR Cas9, using plasmid pML104 as a template, using 20bp PAM sequence design primers shown in SEQ ID NO. 2-6 to carry out circular PCR, and obtaining the plasmid with corresponding PAM sequence through sequencing.
Transforming the constructed pML104-PAM and the gene integration frame into a saccharomyces cerevisiae competent cell, specifically inserting fpps and gfks gene integration frames into a 308a locus of a saccharomyces cerevisiae genome to obtain a kaurene synthetic strain RebM-1; inserting a ko gene integration frame into 416d locus in a genome by taking Reb M-1 as an initial strain to obtain a kaurenoic acid synthetic strain Reb M-2; taking Reb M-2 as an original strain, inserting an kah gene integration frame into a 1309a site to obtain a steviol production strain Reb M-3; inserting UGT74G1 and UGT85C integration frames into 1622b site by taking Reb M-3 as an original strain to obtain Reb M-4; and (3) taking RebM-4 as an original strain, inserting an EUGT11 and UGT76G1 integration frame into a gal80 site to obtain the rebaudioside M producing recombinant saccharomyces cerevisiae strain Reb M-5.
Example 2: enhancement of key rate-limiting isopentenyl diphosphate-isomerase gene idi and hydroxymethylglutaryl-CoA reductase gene hmg1 for synthesizing rebaudioside M precursor
Rebaudioside M synthesis belongs to the group of secondary metabolism and prodromal productLess tiredness, and the copy number of the key rate-limiting enzyme gene idi in the mevalonate pathway is increased for improving the synthesis of the precursor; cutting off endoplasmic reticulum localization signal peptide at N terminal of hydroxymethyl glutaryl-CoA reductase gene hmg1 to obtain thmg1, increasing copy number of thmg1, specifically, Pgal1/10The isopentenyl diphosphate-isomerase gene idi in the mevalonate pathway controlled by the bidirectional promoter and the hydroxymethyl glutaryl-CoA reductase gene thmg1 from which the N-terminal endoplasmic reticulum positioning signal peptide is cut are integrated into the 208a site of the genome to obtain the rebaudioside M recombinant saccharomyces cerevisiae strain Reb M-6.
Example 3: rebaudioside M produced by fermenting recombinant saccharomyces cerevisiae
The saccharomyces cerevisiae recombinant strain Reb M-6 with rebaudioside M synthesis pathway is streaked and subjected to SD plate (uracil is added to the plate at 50mg/L), and the plate is cultured at 30 ℃ until a large number of colonies are grown.
Inoculating a ring of single colony to a seed culture medium, and culturing at 30 ℃ and 220rpm for 18-20 h until the early logarithmic phase of cell growth.
The seed culture was inoculated into the fermentation medium at an initial 3% inoculum size and incubated at 30 ℃ for 96h at 220 rpm. Taking the bacterial liquid every 12h, and measuring OD600And the remaining amount of glucose. And stopping culturing for 96h, transferring the bacterial liquid to a 50mL centrifuge tube, and centrifuging at the normal temperature of 6000rpm for 10 min. The medium was discarded, and the cells were resuspended in 1mL of water, and homogenized by adding 0.1mm glass beads. The ground sample was centrifuged at 8000rpm for 10min, and the supernatant was filtered through a 0.22 μm aqueous membrane and analyzed by LC-MS. Finally, a de novo synthetic strain with a RebM yield of 1.5mg/L was obtained. The qualitative mass spectrometry results of RebM are shown in FIG. 1, and the chromatographic analysis results are shown in FIG. 2.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of south of the Yangtze river
<120> saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 668
<212> DNA
<213> (Artificial sequence)
<400> 1
ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60
atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120
tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180
taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240
acagccctcc gacggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg 300
aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360
tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaattaac 420
gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg 480
taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat ggaaaagctg 540
cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct tattcaaatg 600
tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660
aaactata 668
<210> 2
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 2
cacttgtcaa acagaatata 20
<210> 3
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 3
tagtgcactt accccacgtt 20
<210> 4
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 4
cctgtggtga ctacgtatcc 20
<210> 5
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 5
taaagccacc acatcgcaaa 20
<210> 6
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 6
ccatactgca actatcgtca 20
Claims (10)
1. The saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from the beginning are characterized by integrally expressing kaurene synthase fpps, kaurene synthase ks, kaurene oxidase ko, steviol synthase kah, UDP glucosyltransferase ugt74g1, UDP glucosyltransferase ugt85C2, UDP glucosyltransferase eugt11 and UDP glucosyltransferase ugt76g 1.
2. The engineered saccharomyces cerevisiae strain as claimed in claim 1, wherein the engineered saccharomyces cerevisiae strain is promoter Pgal1/10Controlling gene expression.
3. The engineered saccharomyces cerevisiae strain as claimed in claim 2, wherein the promoter P is selected from the group consisting ofgal1/10Is a bidirectional promoter consisting of promoters Gal1 and Gal10 induced by beta-galactose and glucose together.
4. The engineered saccharomyces cerevisiae strain as claimed in claim 3, wherein said promoter P is selected from the group consisting ofgal1/10The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
5. The engineered saccharomyces cerevisiae as claimed in claim 1, wherein NCBI of kaurene synthase fpps is numbered P08836.2, NCBI of kaurene synthase ks is numbered Q9UVY5, NCBI of kaurene oxidase ko is numbered AAQ63464.1, NCBI of steviol synthase kah is numbered NP _197872.1, NCBI of UDP glucosyltransferase ugt74g1 is numbered q6vaa6.1, NCBI of UDP glucosyltransferase ugt85C2 is numbered qvab0.1, NCBI of UDP glucosyltransferase eugt11 is numbered AAS07253.1, and NCBI of UDP glucosyltransferase ugt76g1 is numbered AGL 95113.1.
6. The engineered saccharomyces cerevisiae strain of claim 1, wherein the kaurene synthase gene and the kaurene synthase gene are inserted into a 308a locus of a saccharomyces cerevisiae genome; the kaurene oxidase gene is inserted into 1014a locus of a saccharomyces cerevisiae genome; inserting a steviol synthase gene into a 1309a locus of a saccharomyces cerevisiae genome; inserting 1622a sites of UDP glucosyltransferase gene and UDP glucosyltransferase gene; the UDP-glucosyltransferase gene and the UDP-glucosyltransferase gene were inserted into gal80 site.
7. The engineered saccharomyces cerevisiae strain as claimed in claim 1, further comprising Pgal1/10The isopentenyl diphosphate-isomerase gene idi in the mevalonate pathway controlled by the bidirectional promoter and the hydroxymethylglutaryl-CoA reductase gene thmg1 from which the N-terminal endoplasmic reticulum localization signal peptide has been excised are integrated into the 208a locus of the genome.
8. The application of the saccharomyces cerevisiae engineering bacteria of any one of claims 1-7 in fermentation production of rebaudioside M.
9. The application of the strain as claimed in claim 8, wherein the strain is inoculated into the fermentation medium at an inoculum size of 1-5%, and cultured at the temperature of 28-32 ℃ and at the speed of 200-250rpm for 90-100 h.
10. The use according to claim 9, wherein the fermentation medium comprises (g/L): 30-50 parts of glucose, 10-30 parts of peptone and 5-15 parts of yeast powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110106054.6A CN112852653A (en) | 2021-01-26 | 2021-01-26 | Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110106054.6A CN112852653A (en) | 2021-01-26 | 2021-01-26 | Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112852653A true CN112852653A (en) | 2021-05-28 |
Family
ID=76009367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110106054.6A Pending CN112852653A (en) | 2021-01-26 | 2021-01-26 | Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112852653A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214378A (en) * | 2021-11-23 | 2022-03-22 | 安徽金禾实业股份有限公司 | Method for preparing rebaudioside M by utilizing fermentation catalysis of bacillus subtilis |
| CN114561310A (en) * | 2022-03-17 | 2022-05-31 | 江南大学 | Saccharomyces cerevisiae for producing rubusoside and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2899276A1 (en) * | 2013-02-06 | 2014-08-14 | Evolva Sa | Methods for improved production of rebaudioside d and rebaudioside m |
| CN104726523A (en) * | 2015-03-28 | 2015-06-24 | 南京工业大学 | Method for preparing rebaudioside-M by enzymic method |
| CN105200098A (en) * | 2015-06-30 | 2015-12-30 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside M according to saccharomyces cerevisiae enzymatic method |
| CN106471128A (en) * | 2014-01-28 | 2017-03-01 | 百事可乐公司 | A kind of method that enzyme process prepares rebaudioside M |
| CA3071029A1 (en) * | 2017-08-11 | 2019-02-14 | Amyris, Inc. | Pisum sativum kaurene oxidase for high efficiency production of rebaudiosides |
| US20190078105A1 (en) * | 2015-10-05 | 2019-03-14 | Dsm Ip Assets B.V. | Kaurenoic acid hydroxylases |
| CN110734944A (en) * | 2019-11-11 | 2020-01-31 | 中化健康产业发展有限公司 | method for synthesizing rebaudioside M |
-
2021
- 2021-01-26 CN CN202110106054.6A patent/CN112852653A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2899276A1 (en) * | 2013-02-06 | 2014-08-14 | Evolva Sa | Methods for improved production of rebaudioside d and rebaudioside m |
| CN105051195A (en) * | 2013-02-06 | 2015-11-11 | 埃沃尔瓦公司 | Methods for improved production of rebaudioside d and rebaudioside m |
| CN106471128A (en) * | 2014-01-28 | 2017-03-01 | 百事可乐公司 | A kind of method that enzyme process prepares rebaudioside M |
| CN104726523A (en) * | 2015-03-28 | 2015-06-24 | 南京工业大学 | Method for preparing rebaudioside-M by enzymic method |
| CN105200098A (en) * | 2015-06-30 | 2015-12-30 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside M according to saccharomyces cerevisiae enzymatic method |
| WO2017000366A1 (en) * | 2015-06-30 | 2017-01-05 | 苏州汉酶生物技术有限公司 | Method for preparing rebaudioside m by using saccharomyces cerevisiae enzymatic process |
| US20190078105A1 (en) * | 2015-10-05 | 2019-03-14 | Dsm Ip Assets B.V. | Kaurenoic acid hydroxylases |
| CA3071029A1 (en) * | 2017-08-11 | 2019-02-14 | Amyris, Inc. | Pisum sativum kaurene oxidase for high efficiency production of rebaudiosides |
| WO2019033064A1 (en) * | 2017-08-11 | 2019-02-14 | Amyris, Inc. | Pisum sativum kaurene oxidase for high efficiency production of rebaudiosides |
| CN111263815A (en) * | 2017-08-11 | 2020-06-09 | 阿迈瑞斯公司 | Pea kaurene oxidase for efficiently generating rebaudioside |
| CN110734944A (en) * | 2019-11-11 | 2020-01-31 | 中化健康产业发展有限公司 | method for synthesizing rebaudioside M |
Non-Patent Citations (1)
| Title |
|---|
| 刘欢等: "重组酿酒酵母全细胞催化合成莱鲍迪苷A", 《食品与发酵工业》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214378A (en) * | 2021-11-23 | 2022-03-22 | 安徽金禾实业股份有限公司 | Method for preparing rebaudioside M by utilizing fermentation catalysis of bacillus subtilis |
| CN114561310A (en) * | 2022-03-17 | 2022-05-31 | 江南大学 | Saccharomyces cerevisiae for producing rubusoside and application thereof |
| CN114561310B (en) * | 2022-03-17 | 2022-12-02 | 江南大学 | Saccharomyces cerevisiae for producing rubusoside and application thereof |
| WO2023173566A1 (en) * | 2022-03-17 | 2023-09-21 | 江南大学 | Saccharomyces cerevisiae for producing rubusoside and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111712570B (en) | Engineering strain for producing psicose and derivatives thereof, construction method and application thereof | |
| CN103710318A (en) | Method for producing stevioside compounds by using microorganisms | |
| CN112852653A (en) | Saccharomyces cerevisiae engineering bacteria for synthesizing rebaudioside M from head and application thereof | |
| CN113755354B (en) | Recombinant saccharomyces cerevisiae for producing gastrodin by utilizing glucose and application thereof | |
| CN113755422B (en) | Recombinant amycolatopsis capable of highly producing vanillin, construction method and application thereof | |
| CN112094797B (en) | Genetically engineered bacterium and application thereof in preparation of 9 alpha, 22-dihydroxy-23, 24-bis-cholesta-4-en-3-one | |
| CN106947705B (en) | Gene recombination monascus purpureus M-piy strain with low-yield citrinin and high-yield monascus yellow pigment as well as preparation method and application thereof | |
| CN105002204A (en) | High yield 5-KGA oxidation gluconobacter cerinus genetically engineered bacteria and preparing method and application thereof | |
| CN114480465A (en) | Bacillus subtilis for producing 2' -fucosyllactose and application thereof | |
| CN112029701B (en) | Genetically engineered bacterium and application thereof in preparation of 22-hydroxy-23, 24-bis-cholesta-4-en-3-one | |
| CN112080452B (en) | High-yield phenyllactic acid bacillus licheniformis genetically engineered bacterium, method for producing phenyllactic acid and application | |
| CN111197020B (en) | Recombinant bacterium for producing milbemycins as well as construction method and application thereof | |
| CN113789292B (en) | Gene-deficient amycolatopsis capable of producing vanillin at high yield, and construction method and application thereof | |
| CN116042425A (en) | Yeast engineering bacteria for producing patchouli alcohol and application thereof | |
| CN110892068A (en) | UDP-glycosyltransferase | |
| CN110343650B (en) | Recombinant streptomyces tuberculatus for producing amphotericin B and application thereof | |
| CN109722455B (en) | Method for producing glutacoside by microbial fermentation, engineering bacteria and application | |
| CN110072995B (en) | Kaurenoic acid hydroxylase | |
| CN107541481B (en) | Genetic engineering bacterium for producing epirubicin and application thereof | |
| CN116590161B (en) | Recombinant amycolatopsis for producing vanillin, construction method and application thereof | |
| CN117965500B (en) | Alpha-L rhamnosidase AfRhase and product, application and production process thereof | |
| CN114561310B (en) | Saccharomyces cerevisiae for producing rubusoside and application thereof | |
| CN113930349B (en) | Engineering bacterium for biosynthesis of resveratrol by taking L-tyrosine as substrate, construction and application | |
| CN117887599A (en) | Construction method of engineering yeast for synthesizing mogroside from head | |
| CN114717281B (en) | Method for improving fermentation yield of heterologous spinosad expression strain by optimizing carbon source |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |