CN116411013A - Method for synthesizing delta-tocotrienol from saccharomyces cerevisiae through metabolic engineering modification - Google Patents

Method for synthesizing delta-tocotrienol from saccharomyces cerevisiae through metabolic engineering modification Download PDF

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CN116411013A
CN116411013A CN202310158802.4A CN202310158802A CN116411013A CN 116411013 A CN116411013 A CN 116411013A CN 202310158802 A CN202310158802 A CN 202310158802A CN 116411013 A CN116411013 A CN 116411013A
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saccharomyces cerevisiae
ttc
tocotrienol
hggt
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
韩璐瑶
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Jiangnan University
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Abstract

The invention discloses a method for synthesizing delta-tocotrienol from the head of saccharomyces cerevisiae by metabolic engineering modification. The construction method of the substrate transmission channel provided by the invention is simple and effective, and can solve the problem of low catalytic efficiency caused by improper assembly of various exogenous enzymes.

Description

Method for synthesizing delta-tocotrienol from saccharomyces cerevisiae through metabolic engineering modification
Technical Field
The invention relates to a method for synthesizing delta-tocotrienol from saccharomyces cerevisiae head by metabolic engineering, belonging to the technical field of metabolic engineering.
Background
Delta-tocotrienol, an isomer in vitamin E, has been attracting attention as having a richer biological activity against human health, particularly anticancer activity, in addition to an antioxidant effect. Vitamin E is a generic term for tocopherols and tocotrienols, and comprises 8 compounds of alpha, beta, gamma, delta-tocopherols and alpha, beta, gamma, delta-tocotrienols, and is one of the most main antioxidants, is essential vitamin for maintaining the metabolism of the organism, has wide efficacy and has increasingly high market demand. Although the chemical full synthesis of vitamin E has been achieved since 1938, the synthesis of two intermediates, mainly using 2,3, 5-trimethylhydroquinone (main ring) and isophytol (branched chain), in a "one-step condensation process" is relatively mature in the current technology, but it is widely used as an animal feed additive because it is unable to synthesize vitamin monomers of specific steric configuration, thereby affecting the activity of vitamin E. The combination of biological and chemical methods to synthesize vitamin E was realized in 2018 at home, but isophytol (C) could be synthesized only by microbial fermentation 20 H 40 ) Farnesene (C) 15 H 24 ) The synthesis process is still complex and relies mainly on chemical synthesis. At present, the health care and medical treatment are carried outIn the aspects of therapy, cosmetology and the like, people tend to select natural and synthetic vitamin E. The existing natural vitamin E still mainly depends on plant extraction, and is limited by the limitation of plant resources and the excessive cost. Thus, the use of genetic engineering to artificially construct vitamin E biosynthetic pathways in microorganisms to achieve de novo synthesis of a particular configuration of natural vitamin E is a more convenient and economical way.
As GRAS (Generally Regard as Safe) strain, saccharomyces cerevisiae has been widely used for biosynthesis of natural products such as terpenes and aromatic compounds. Compared with the escherichia coli, the saccharomyces cerevisiae has the advantages of high safety, low pathogenicity, high stress resistance, low probability of phage pollution and the like, and in addition, the saccharomyces cerevisiae has higher tolerance to several isoprene compounds, such as farnesyl diphosphate (FPP) and geranyl diphosphate (GGPP), than the escherichia coli, so that the saccharomyces cerevisiae is more suitable for chassis cells for producing isoprene compounds at high level. Since GGPP is one of the key precursors in the delta-tocotrienol biosynthetic pathway, saccharomyces cerevisiae is a host strain that is more conducive to delta-tocotrienol production. The Saccharomyces cerevisiae does not have a delta-tocotrienol synthetic pathway, so that the delta-tocotrienol synthetic pathway needs to be introduced into the Saccharomyces cerevisiae by utilizing metabolic engineering to construct a delta-tocotrienol production engineering strain. In recent years, the introduction of the delta-tocotrienol synthesis pathway of photosynthetic organisms into Saccharomyces cerevisiae has been by scholars, achieving de novo synthesis of delta-tocotrienol in Saccharomyces cerevisiae.
At present, although de novo synthesis of delta-tocotrienol in s.cerevisiae has been achieved, there are uncertainties in expression, localization and assembly patterns after introduction of genes of photosynthetic organism origin into s.cerevisiae, and a series of problems are caused: the mislocalization of heterologous enzymes results in separation of the enzyme from the substrate, consumption of precursors required for synthesis of the product by endogenous competing pathways, imbalance in expression between upstream and downstream enzymes, difficulty in achieving synergistic catalysis due to improper assembly of multiple exogenous enzymes, and the like. These problems greatly limit the functionality of the exogenous gene in the cell factory, resulting in low delta-tocotrienol biosynthesis efficiency.
Disclosure of Invention
In order to solve the problem of low biosynthesis efficiency of delta-tocotrienol at present, the invention provides a saccharomyces cerevisiae for efficiently synthesizing delta-tocotrienol, which can be used for efficiently secreting and producing delta-tocotrienol extracellular by integrating a delta-tocotrienol synthesis path from a photosynthetic organism source, strengthening a precursor path, mutating key enzymes in a delta-tocotrienol synthesis module, constructing a substrate transmission channel to assemble the key enzymes in the delta-tocotrienol synthesis module and overexpressing a transporter PDR11.
A first object of the present invention is to provide a method for the de novo synthesis of delta-tocotrienol by metabolically engineered Saccharomyces cerevisiae by integration of the p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC on the Saccharomyces cerevisiae genome and overexpression of GGPP synthase CrtE and FPP synthase mutant FPS F112A 。
Further, NCBI number NP-172144.3 of the p-hydroxyphenylpyruvate dioxygenase HPPD; the NCBI number of geranylgeranyl transferase HGGT is BAA17774; the amino acid sequence of the truncated tocopherol cyclase tTC is shown in SEQ ID NO. 1; NCBI accession number of GGPP synthetase CrtE is AAS49033.1; FPP synthase mutant FPS F112A The amino acid sequence of (2) is shown in SEQ ID NO. 2.
Further, the gene encoding p-hydroxyphenylpyruvate dioxygenase HPPD is inserted into the HO locus of the Saccharomyces cerevisiae genome; the coding genes of geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC are inserted into the DPP1 locus of the Saccharomyces cerevisiae genome; GGPP synthase CrtE and FPP synthase mutant FPS F112A The coding gene of the gene is inserted into GAL 1-7 loci of saccharomyces cerevisiae genome.
Further, the method comprises mutating the truncated tocopherol cyclase tTC to mutate the asparagine (N) at the 331 st position of the parent sequence with the amino acid sequence shown in SEQ ID NO.1 into proline (P).
Further, the method further comprises the step of over-expressing the protein scaffoldSH 3 Assembled HPPD and HGGT-tTC N331P A complex.
Further, the complex is inserted into the 416d position of the Saccharomyces cerevisiae genome.
Further, HGGT and tTC N331P Short protein connector (GGGGS) 3 Fusion expression.
Further, the protein scaffold SH 3 The nucleotide sequence of (2) is shown as SEQ ID NO. 4.
Further, the method also includes obtaining Saccharomyces cerevisiae that secretes delta-tocotrienol using overexpression of the endogenous transporter PDR11.
Further, the method takes Saccharomyces cerevisiae CEN PK2-1C as a host.
A second object of the present invention is to provide a Saccharomyces cerevisiae for the de novo synthesis of delta-tocotrienol by the integrated expression of p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC on the genome of the Saccharomyces cerevisiae host, and over-expression of GGPP synthase CrtE and FPP synthase mutant FPS F112A 。
Further, NCBI number NP-172144.3 of the p-hydroxyphenylpyruvate dioxygenase HPPD; the NCBI number of geranylgeranyl transferase HGGT is BAA17774; the amino acid sequence of the truncated tocopherol cyclase tTC is shown in SEQ ID NO. 1; NCBI accession number of GGPP synthetase CrtE is AAS49033.1; FPP synthase mutant FPS F112A The amino acid sequence of (2) is shown in SEQ ID NO. 2.
Further, by promoter P ADH1 And P PGK1 Control of Gene expression in the delta-tocotrienol synthetic pathway, P ADH1 The nucleotide sequence of (B) is shown as SEQ ID NO.5, P PGK1 The nucleotide sequence of (2) is shown as SEQ ID NO. 6.
Further, the gene encoding p-hydroxyphenylpyruvate dioxygenase HPPD is inserted into the HO locus of the Saccharomyces cerevisiae genome; insertion of genes encoding geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC into Saccharomyces cerevisiae genesA group DPP1 site; GGPP synthase CrtE and FPP synthase mutant FPS F112A The coding gene of the gene is inserted into GAL 1-7 loci of saccharomyces cerevisiae genome.
Further, in the saccharomyces cerevisiae, the truncated tocopherol cyclase tTC is a truncated tocopherol cyclase tTC mutant with the amino acid sequence shown as SEQ ID NO.1 and with asparagine (N) at the 331 st site of a parent sequence mutated into proline (P).
Further, in the Saccharomyces cerevisiae, protein scaffold SH is used for overexpression 3 Assembled HPPD and HGGT-tTC N331P A complex.
Further, HGGT and tTC N331P Short protein connector (GGGGS) 3 Fusion expression.
Further, the protein scaffold SH 3 The nucleotide sequence of (2) is shown as SEQ ID NO. 4.
Further, in the Saccharomyces cerevisiae, the endogenous transporter PDR11 is overexpressed.
Further, the Saccharomyces cerevisiae host is Saccharomyces cerevisiae CEN PK2-1C.
A third object of the present invention is to provide the use of said Saccharomyces cerevisiae for the fermentative production of delta-tocotrienol.
The beneficial effects of the invention are as follows:
the invention constructs and optimizes the synthesis path of delta-tocotrienol in saccharomyces cerevisiae, and then obtains the saccharomyces cerevisiae strain for efficiently producing delta-tocotrienol by extracellular secretion by constructing a substrate transmission channel and over-expressing a transporter PDR11, thereby laying a foundation for the de novo synthesis of natural vitamin E of a specific configuration by metabolic engineering modified saccharomyces cerevisiae. The construction method of the substrate transmission channel provided by the invention is simple and effective, and can solve the problem of low catalytic efficiency caused by improper assembly of various exogenous enzymes.
Description of the drawings:
FIG. 1 is delta-tocotrienol production by VE-2 and VE-3 strains;
FIG. 2 shows delta-tocotrienol production by VE-3 and VE-4 strains;
FIG. 3 shows delta-tocotrienol production by VE-4 and VE-5 strains;
FIG. 4 shows delta-tocotrienol production by VE-5 and VE-6 strains.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The detection method comprises the following steps: triple four-stage rod composite linear ion trap liquid chromatography-mass spectrometry (QTRAP 5500,ChromCore C8) column, mobile phase A phase of water (containing 0.1% FA), mobile phase B phase of acetonitrile, column temperature of 50 ℃, sample injection amount of 2 mu L, flow rate of 0.35mL/min, gradient elution program of 80% mobile phase B phase (0-1 min); 99% mobile phase B phase (1-8 min); 80% mobile phase B phase (8-10.5 min), cation mode, ion pairs 137/397 and 177/397.
Example 1: construction of recombinant Saccharomyces cerevisiae Strain from de novo Synthesis of delta-tocotrienol
According to the published NCBI-derived p-hydroxyphenylpyruvate dioxygenase HPPD (NCBI ID: NP-172144.3), coptis-derived geranylgeranyl transferase HGGT (NCBIID: BAA 17774), arabidopsis-derived truncated tocopherol cyclase tTC (truncated by 47 amino acids based on NCBIID: NP-567906.1, the amino acid sequence of which is shown in SEQ ID NO. 1), taxus-derived GGPP synthase CrtE (NCBIID: AAS 49033.1) and chickens-derived FPP synthase mutant FPS F112A (NCBI ID of unmutated FPP synthase: P08836.2, on the basis of which phenylalanine at position 112 is mutated to alanine, the amino acid sequence after mutation is shown as SEQ ID NO. 2), codon optimization is performed according to the codon preference of Saccharomyces cerevisiae and total gene synthesis is performed.
Since Saccharomyces cerevisiae has homologous recombination capability, primers are designed according to the overlapping part of 45-50bp of each adjacent fragment of the gene integration box. And designing upstream and downstream homology arm amplification primers of a gene integration frame at two sides of the sequences of the integration sites of the HO, DPP1 and GAL 1-7 of the saccharomyces cerevisiae genome, and amplifying the upstream and downstream homology arms by using the saccharomyces cerevisiae genome DNA as a template through PCR. With plasmid pML104-HISAnd pML104-TRP as a template to design a primer, and amplifying to obtain a corresponding amino acid screening tag. And designing primers to amplify the delta-tocotrienol synthetic pathway enzyme, the promoter and the terminator by combining with an overlapping extension PCR primer design method. HPPD, HGGT, tTC, crtE and FPS synthesized by total genes F112A The plasmid is used as a template, and the corresponding gene fragment is obtained through amplification. The saccharomyces cerevisiae genome DNA is used as a template to obtain a promoter P by amplification ADH1 And P PGK1 Terminator T CYC1 And T ADH1 . The corresponding gene expression cassette was obtained by overlap extension PCR.
According to the Cre-loxp system, the amplified upstream and downstream homology arms, amino acid screening tags and gene integration frame are transformed into saccharomyces cerevisiae competent cells, correct transformants of colony PCR are selected for sequencing verification, and the recombinant saccharomyces cerevisiae strain VE-2 with delta-tocotrienol de novo synthesis pathway is obtained.
To enhance the supply of precursor GGPP, taxus chinensis-derived GGPP synthase CrtE and chicken-derived FPP synthase mutant FPS were overexpressed on the basis of VE-2 strain F112A . And designing upstream and downstream homology arm amplification primers of a gene integration frame at two sides of a GAL 1-7 integration site sequence of a saccharomyces cerevisiae genome, and amplifying the upstream and downstream homology arms by using a saccharomyces cerevisiae genome DNA as a template through PCR. The plasmid pML104-LEU is used as a template to design a primer, and leucine screening tag is obtained through amplification. CrtE and FPS synthesized by total genes F112A The plasmid is used as a template, and the corresponding gene fragment is obtained through amplification. The saccharomyces cerevisiae genome DNA is used as a template to obtain a promoter P by amplification ADH1 And P PGK1 Terminator T CYC1 And T ADH1 . The corresponding gene expression cassette was obtained by overlap extension PCR. According to the Cre-loxp system gene editing method, the gene engineering strain VE-3 for synthesizing delta-tocotrienol after optimizing the precursor path is obtained.
Construction tTC N331P The mutant plasmid takes the tTC plasmid synthesized by the whole gene as a template, uses the primers N331-F and N331-R to carry out circular PCR, and obtains tTC by sequencing N331P Mutant plasmid.
According to the above gene integration method, VE-3 is used as starting strain inDPP1 integration expression tTC N331P The mutant replaces the unmutated tTC, and finally the genetically engineered strain VE-4 for synthesizing delta-tocotrienol from the head is obtained.
Primer sequence:
N331-F：CTGAAAACGAACCACATGTTGTTGAATTAGAAGCTAGAACCAAC
N331-R：TCAACAACATGTGGTTCGTTTTCAGCAGTGATGTACCA。
example 2: construction of substrate transport channels in the delta-tocotrienol synthetic pathway
Construction of HGGT and tTC N331P Fusion expression plasmid pY16-HGGT- (GGGGS) 3 -tTC N331P The HGGT plasmid and the constructed tTC are synthesized by total genes respectively N331P The mutant plasmid is used as a template, and the HGGT and the primer tTC are amplified through the primer HGGT-F and the primer HGGT-R N331P F and primer tTC N331P R amplification tTC N331P The vector frame was amplified by primers pY16-F1 and pY16-R1 using the pY16-URA plasmid as a template. The obtained DNA fragment was purified, inserted into pY16-URA vector by Gibson assembly method, and sequenced to obtain plasmid pY16-HGGT- (GGGGS) 3 -tTC N331P 。
Construction of HPPD and HGGT-tTC N331P Complex assembly expression plasmid pY16-HPPD-HGGT-tTC N331P -SH 3 The whole gene synthesized HPPD plasmid is used as a template, the HPPD is amplified by a primer HPPD-F and a primer HPPD-R, and the plasmid pY16-HGGT- (GGGGS) is used 3 -tTC N331P By containing SH as template 3 Ligand sequence primer HGGT-tTC N331P -F and primer HGGT-tTC N331P -R amplification HGGT-tTC N331P Complex (SH) 3 The nucleotide sequence of-ligands is shown in SEQ ID NO. 3) to synthesize the sequence SH 3 Domain as template (SH 3 The nucleotide sequence of-domain is shown as SEQ ID NO. 4) through primer SH 3 -F and SH 3 R amplification of SH 3 Domain, using Saccharomyces cerevisiae genome DNA as template, obtaining promoter P by amplification of primers P-F and P-R ADH1 And P PGK1 The vector frame was amplified by primers pY16-F2 and pY16-R2 using the pY16-URA plasmid as a template. The obtained DNA fragment was purified, and then inserted into pY16-URA vector by Gibson assembly method, followed bySequencing to obtain plasmid pY16-HPPD-HGGT-tTC N331P -SH 3 。
SH was used for integration at position 416d using VE-4 as starting strain according to the gene integration method described in example 1 3 Assembled HPPD and HGGT-tTC N331P And constructing a substrate transmission channel by the complex to obtain the genetically engineered strain VE-5 for efficiently synthesizing delta-tocotrienol.
Primer sequence:
HGGT-F：CAAATATAAAACAATGGCTACTATTCAAGCTTTTTGGAG
HGGT-R：GCCACCGCCGCTTCCACCGCCACCAAAAATAGTATTAGAAAAATTTGGC AACCACAAAG
tTC N331P -F：GCGGTGGAAGCGGCGGTGGCGGAAGCATGGCTTCTATTAGTACTCCAA ACTCTG
tTC N331P -R：CATAAGAAATTCGCTCATAATCCAGGTGGCTTGAAGAATG
pY16-F1：CTGGATTATGAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAG
pY16-R1：TAGTAGCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTG A
HPPD-F：GTACGGTGGAGGAGGAAGCGGCGGTGGCGGATCCGGTCACCAAAATGC TGCCG
HPPD-R：TAAGAAATTCGCTTAACCGACCAATTGCTTGGC
HGGT-tTC N331P -F：GAGGTAAGGCTGGTGGGGGGCTTCCGCCACCGCCGCTTCCACC GCCACCTAATCCAGGTGGCTTGAAGAATG
HGGT-tTC N331P -R：CATACAATCAACTATGGCTACTATTCAAGCTTTTTGGAG
SH3-F：ATATAAAACAATGGCAGAGTATGTGCGTGCT
SH3-R：ACCGGATCCGCCACCGCCGCTTCCTCCTCCACCGTACTTCTCCACATAAGG AACG
P-F：GAATAGTAGCCATAGTTGATTGTATGCTTGGTATAGCTTG
P-R：TACTCTGCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTC
pY16-F2：TGGTCGGTTAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGpY16-R2：AAGCCCCCCACCAGCCTTACCTCCTAAGCGTAGACGTTAATCATGTAATT AGTTATGTCACGCTTACATTC。
example 3: extracellular secretion production of delta-tocotrienol
Constructing a pY16-PDR11 plasmid, amplifying PDR11 by using a yeast genome as a template through a primer PDR11-F and a primer PDR11-R, and amplifying a vector frame by using a pY16-URA plasmid as a template through primers pY16-F3 and pY 16-R3. After purifying the obtained DNA fragment, the plasmid pY16-PDR11 is obtained by inserting the DNA fragment into a pY16-URA vector by a Gibson assembly method and sequencing the DNA fragment.
The plasmid pY16-PDR11 is transformed into VE-5 strain to obtain the delta-tocotrienol-producing strain VE-6 by extracellular secretion.
The shake flask fermentation method comprises the following steps:
(1) Recombinant strains VE-2, VE-3, VE-4, VE-5 and VE-6 were streaked onto YPD plates and incubated at 30℃until a large number of colonies were developed.
(2) A loop of single colony was inoculated to the seed medium and cultured at 220rpm at 30℃for 20 hours.
(3) Seed culture broth was inoculated into the fermentation medium at an initial inoculum size of 2%, and after 48 hours of cultivation at 30℃and 220rpm, 50% (v/v) dodecane was added, and cultivation was continued at 30℃and 220rpm for 144 hours. Measuring OD 600 After that, the bacterial liquid was transferred to a 50mL centrifuge tube and centrifuged at 6000rpm at room temperature for 10min. Taking 1mL of organic phase, spin-evaporating in a vacuum rotary evaporator, re-dissolving with 1mL of acetonitrile, filtering with an organic system filter membrane with the thickness of 0.22 mu m, and quantitatively analyzing by using a triple quaternary rod composite linear ion trap liquid chromatography-mass spectrometer.
Primer sequence:
PDR11-F：TAGAACTAGTGGATCCCCCGGCGGATGTCTCTTTCCAAATAT
PDR11-R：GACGGTATCGATAAGCTTGATTATACGCTTTGTTCGTTTGGAT
pY16-F3：CGGGGGATCCACTAGTTCTA
pY16-R3：TCAAGCTTATCGATACCGTCG。
as shown in FIG. 1, the shake flask fermentation results of the strains VE-2 and VE-3 show that the delta-tocotrienol yields of the strains VE-2 and VE-3 are 59.2 mug/L and 384.5 mug/L, respectively, and the delta-tocotrienol yield of the strain VE-3 is improved by 5.5 times compared with the strain VE-2 to verify that the taxus-derived GGPP synthetase CrtE and the chicken-derived FPP synthetase mutant FPS are overexpressed F112A Enhanced GGPP delivery followed by enhanced delta-tocotrienol synthesisEffects.
The results of shake flask fermentation of the strains VE-3 and VE-4 show in FIG. 2, the delta-tocotrienol production of the strain VE-4 was 1.8 times that of the strain VE-3, reaching 703.7. Mu.g/L, to verify the effect of site-directed mutagenesis of tocopherol cyclase tTC on the increase of delta-tocotrienol production.
The results of shake flask fermentation of the strains VE-4 and VE-5 show in FIG. 3, the delta-tocotrienol yield of the strain VE-5 was 1801.5. Mu.g/L, and the delta-tocotrienol yield of the strain VE-5 was increased by 1.6 times as compared to the strain VE-4, to verify the effect of constructing a substrate transfer channel to increase delta-tocotrienol yield.
The results of shake flask fermentation of the strains VE-5 and VE-6 are shown in FIG. 4, and the delta-tocotrienol yield of the strain VE-6 is 1.7 times that of the strain VE-5 and reaches 3062.6 mug/L, so that the effect of extracellular secretion in efficiently producing delta-tocotrienol is verified.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A method for synthesizing delta-tocotrienol from saccharomyces cerevisiae through metabolic engineering is characterized in that p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC are integrated on the genome of the saccharomyces cerevisiae, and GGPP synthetase CrtE and FPP synthetase mutant FPS are overexpressed F112A 。
2. The method of claim 1, further comprising mutating the truncated tocopherol cyclase tTC to mutate asparagine at position 331 of the parent sequence having the amino acid sequence shown in SEQ ID No.1 to proline.
3. The method according to claim 1 or 2, characterized in that theThe method further comprises the step of over-expressing the protein scaffold SH 3 Assembled HPPD and HGGT-tTC N331P A complex, wherein the protein scaffold SH 3 The nucleotide sequence of (2) is shown as SEQ ID NO. 4.
4. A method according to claim 3, wherein HGGT and tTC N331P Short protein connector (GGGGS) 3 Fusion expression.
5. The method of claim 1, further comprising obtaining s.cerevisiae that secretes delta-tocotrienol using overexpression of the endogenous transporter PDR11.
6. A Saccharomyces cerevisiae for synthesizing delta-tocotrienol from head to head, which is characterized in that the Saccharomyces cerevisiae is obtained by integrating and expressing p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC on the genome of a Saccharomyces cerevisiae host, and overexpressing GGPP synthase CrtE and FPP synthase mutant FPS F112A 。
7. The Saccharomyces cerevisiae according to claim 6, wherein NCBI number of the p-hydroxyphenylpyruvate dioxygenase HPPD is NP-172144.3; the NCBI number of geranylgeranyl transferase HGGT is BAA17774; the amino acid sequence of the truncated tocopherol cyclase tTC is shown as SEQ ID NO.1, or asparagine at the 331 st site of a parent sequence with the amino acid sequence shown as SEQ ID NO.1 is mutated into proline; NCBI accession number of GGPP synthetase CrtE is AAS49033.1; FPP synthase mutant FPS F112A The amino acid sequence of (2) is shown in SEQ ID NO. 2.
8. The Saccharomyces cerevisiae according to claim 6, wherein the protein scaffold SH is overexpressed in the Saccharomyces cerevisiae 3 Assembled HPPD and HGGT-tTC N331P A complex; wherein the protein scaffold SH 3 The nucleotide sequence of the polypeptide is shown as SEQ ID NO.4Shown are HGGT and tTC N331P Short protein connector (GGGGS) 3 Fusion expression.
9. The saccharomyces cerevisiae according to claim 6 wherein the endogenous transporter PDR11 is overexpressed.
10. Use of a saccharomyces cerevisiae according to any of claims 6-9 for the fermentative production of delta-tocotrienol.
CN202310158802.4A 2023-02-24 2023-02-24 Method for synthesizing delta-tocotrienol from saccharomyces cerevisiae through metabolic engineering modification Pending CN116411013A (en)

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