CN116411013B - A method for metabolically engineering Saccharomyces cerevisiae to synthesize δ-tocotrienol from scratch - Google Patents
A method for metabolically engineering Saccharomyces cerevisiae to synthesize δ-tocotrienol from scratch Download PDFInfo
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- CN116411013B CN116411013B CN202310158802.4A CN202310158802A CN116411013B CN 116411013 B CN116411013 B CN 116411013B CN 202310158802 A CN202310158802 A CN 202310158802A CN 116411013 B CN116411013 B CN 116411013B
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Abstract
本发明公开了一种代谢工程改造酿酒酵母从头合成δ‑生育三烯醇的方法，本发明在酿酒酵母中构建并优化了δ‑生育三烯酚合成途径，再通过构建底物传输通道并过表达转运体PDR11，获得了胞外分泌高效生产δ‑生育三烯酚的基因工程菌株，为代谢工程改造酿酒酵母从头合成特定构型的天然维生素E奠定了基础。本发明提供的底物传输通道的构建方法简单有效，可解决多种外源酶组装不当导致的催化效率低问题。
The present invention discloses a method for metabolically engineering Saccharomyces cerevisiae to synthesize δ-tocotrienol from scratch. The present invention constructs and optimizes the δ-tocotrienol synthesis pathway in Saccharomyces cerevisiae, and then obtains a genetically engineered strain that efficiently produces δ-tocotrienol through extracellular secretion by constructing a substrate transport channel and overexpressing the transporter PDR11, laying a foundation for metabolically engineering Saccharomyces cerevisiae to synthesize natural vitamin E of a specific configuration from scratch. The method for constructing the substrate transport channel provided by the present invention is simple and effective, and can solve the problem of low catalytic efficiency caused by improper assembly of multiple exogenous enzymes.
Description
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 in China, but the synthesis of the precursor substance farnesene (C 15H24) of isophytol (C 20H40) can only be realized by utilizing a microbial fermentation method, and the synthesis process is still relatively complex and mainly depends on chemical synthesis. At present, people tend to select natural and synthetic vitamin E in health care, medical treatment, beauty treatment and other aspects. 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 PDR 11.
It is a first object of the present invention to provide a method for metabolically engineering the de novo delta-tocotrienol synthesis by Saccharomyces cerevisiae by integrating the p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC on the Saccharomyces cerevisiae genome and overexpressing 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 as SEQ ID NO. 1; NCBI accession number AAS49033.1 for GGPP synthetase CrtE; the amino acid sequence of FPP synthase mutant FPS F112A is shown as SEQ ID NO. 2.
Further, the gene encoding p-hydroxyphenylpyruvate dioxygenase HPPD is inserted into the HO locus of the Saccharomyces cerevisiae genome; the genes encoding geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC are inserted into the DPP1 site of the saccharomyces cerevisiae genome; the coding genes of GGPP synthetase CrtE and FPP synthetase mutant FPS F112A are inserted into GAL 1-7 sites of Saccharomyces cerevisiae genome.
Further, the method comprises mutating the truncated tocopherol cyclase tTC to mutate asparagine (N) at position 331 of the parent sequence with the amino acid sequence shown in SEQ ID NO.1 to proline (P).
Further, the method further comprises overexpressing the HPPD and HGGT-tTC N331P complex assembled using the protein scaffold SH 3.
Further, the complex is inserted into the 416d position of the Saccharomyces cerevisiae genome.
Further, a short protein linker (GGGGS) 3 fusion expression was used between HGGT and tTC N331P.
Further, the nucleotide sequence of the protein scaffold SH 3 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 PDR 11.
Further, the method takes Saccharomyces cerevisiae CEN PK2-1C as a host.
It is a second object of the present invention to provide a Saccharomyces cerevisiae for the de novo synthesis of delta-tocotrienol which integrates expression of p-hydroxyphenylpyruvate dioxygenase HPPD, geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC on the genome of the Saccharomyces cerevisiae host and over-expresses 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 as SEQ ID NO. 1; NCBI accession number AAS49033.1 for GGPP synthetase CrtE; the amino acid sequence of FPP synthase mutant FPS F112A is shown as SEQ ID NO. 2.
Further, promoters P ADH1 and P PGK1 are used for controlling gene expression in the delta-tocotrienol synthesis pathway, the nucleotide sequence of P ADH1 is shown as SEQ ID NO.5, and the nucleotide sequence of P PGK1 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; the genes encoding geranylgeranyl transferase HGGT and truncated tocopherol cyclase tTC are inserted into the DPP1 site of the saccharomyces cerevisiae genome; the coding genes of GGPP synthetase CrtE and FPP synthetase mutant FPS F112A are inserted into GAL 1-7 sites 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 the parent sequence mutated into proline (P).
Further, in the Saccharomyces cerevisiae, HPPD and HGGT-tTC N331P complex assembled using the protein scaffold SH 3 are overexpressed.
Further, a short protein linker (GGGGS) 3 fusion expression was used between HGGT and tTC N331P.
Further, the nucleotide sequence of the protein scaffold SH 3 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.
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-level rod composite linear ion trap liquid chromatography-mass spectrometry (QTRAP 5500,ChromCore C8) column, mobile phase A phase is water (containing 0.1% FA), mobile phase B phase is acetonitrile, column temperature is 50 ℃, sample injection amount is 2 mu L, flow rate is 0.35mL/min, and gradient elution program is 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 of Arabidopsis thaliana-derived P-hydroxyphenylpyruvate dioxygenase HPPD (NCBI ID: NP-172144.3), coptis-derived geranylgeranyl transferase HGGT (NCBIID: BAA 17774), arabidopsis thaliana-derived truncated tocopherol cyclase tTC (47 amino acids were truncated on the basis of NCBIID: NP-567906.1, the amino acid sequence was shown as SEQ ID NO. 1), taxus chinensis-derived GGPP synthase CrtE (NCBIID: AAS 49033.1) and the chicken-derived FPP synthase mutant FPS F112A (non-mutated FPP synthase NCBIID: P08836.2, on the basis of which phenylalanine at position 112 was mutated to alanine, the amino acid sequence after mutation was shown as SEQ ID NO. 2), a codon optimization was carried out in accordance with the yeast codon preference and a total gene synthesis was carried out.
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. The plasmids pML104-HIS and pML104-TRP are used as templates to design primers, and corresponding amino acid screening labels are obtained through amplification. 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. The HPPD, HGGT, tTC, crtE and FPS F112A plasmids synthesized by the whole genes are used as templates, and the corresponding gene fragments are obtained through amplification. The promoters P ADH1 and P PGK1 and the terminators T CYC1 and T ADH1 are obtained by amplification with Saccharomyces cerevisiae genome DNA as a template. 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-derived GGPP synthase CrtE and chicken-derived FPP synthase mutant FPS F112A were overexpressed on the basis of VE-2 strain. 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. The CrtE and FPS F112A plasmids synthesized by the whole genes are used as templates, and the corresponding gene fragments are obtained through amplification. The promoters P ADH1 and P PGK1 and the terminators T CYC1 and T ADH1 are obtained by amplification with Saccharomyces cerevisiae genome DNA as a template. 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 of mutant plasmid, using tTC plasmid synthesized by whole gene as template, using primers N331-F and N331-R to make circular PCR, and making sequencing so as to obtain tTC N331P mutant plasmid.
According to the gene integration method, VE-3 is taken as an original strain, a tTC N331P mutant is integrated and expressed in DPP1 to replace 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 plasmids pY16-HGGT- (GGGGS) 3-tTCN331P, full-gene synthesis HGGT plasmid and constructed tTC N331P mutant plasmid are respectively used as templates, amplification HGGT is carried out by a primer HGGT-F and a primer HGGT-R, amplification tTC N331P is carried out by a primer tTC N331P -F and a primer tTC N331P -R, pY16-URA plasmid is used as a template, and a vector frame is amplified by a primer pY16-F1 and a primer pY 16-R1. After purifying the obtained DNA fragment, the plasmid pY16-HGGT- (GGGGS) 3-tTCN331P was obtained by inserting the DNA fragment into a pY16-URA vector by Gibson assembly method and sequencing the DNA fragment.
Constructing HPPD and HGGT-tTC N331P complex assembly expression plasmid pY16-HPPD-HGGT-tTC N331P-SH3, using whole-gene synthesis HPPD plasmid as template, amplifying HPPD by using primer HPPD-F and primer HPPD-R, using plasmid pY16-HGGT- (GGGGS) 3-tTCN331P as template, The HGGT-tTC N331P complex was amplified by primers HGGT-tTC N331P -F and HGGT-tTC N331P -R containing the SH 3 -ligands sequence (the nucleotide sequence of SH 3 -ligands is shown in SEQ ID NO. 3), SH 3 -domain is amplified by primers SH 3 -F and SH 3 -R by taking a synthetic sequence SH 3 -domain as a template (the nucleotide sequence of SH 3 -domain is shown as SEQ ID NO. 4), Promoters P ADH1 and P PGK1 are obtained by amplification of primers P-F and P-R by using Saccharomyces cerevisiae genome DNA as a template, and a vector frame is amplified by primers pY16-F2 and pY16-R2 by using pY16-URA plasmid as a template. After purifying the obtained DNA fragment, the plasmid pY16-HPPD-HGGT-tTC N331P-SH3 was obtained by inserting the DNA fragment into a pY16-URA vector by Gibson assembly method and sequencing the DNA fragment.
According to the gene integration method described in example 1, VE-4 was used as an initial strain, and a substrate transfer channel was constructed by integrating the HPPD assembled by SH 3 and HGGT-tTC N331P complex at the 416d site, thereby obtaining a genetically engineered strain VE-5 for efficiently synthesizing delta-tocotrienol.
Primer sequence:
HGGT-F：CAAATATAAAACAATGGCTACTATTCAAGCTTTTTGGAG
HGGT-R：GCCACCGCCGCTTCCACCGCCACCAAAAATAGTATTAGAAAAATTTGGC AACCACAAAG
tTCN331P-F：GCGGTGGAAGCGGCGGTGGCGGAAGCATGGCTTCTATTAGTACTCCAA ACTCTG
tTCN331P-R：CATAAGAAATTCGCTCATAATCCAGGTGGCTTGAAGAATG
pY16-F1：CTGGATTATGAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAG
pY16-R1：TAGTAGCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTG A
HPPD-F：GTACGGTGGAGGAGGAAGCGGCGGTGGCGGATCCGGTCACCAAAATGC TGCCG
HPPD-R：TAAGAAATTCGCTTAACCGACCAATTGCTTGGC
HGGT-tTCN331P-F：GAGGTAAGGCTGGTGGGGGGCTTCCGCCACCGCCGCTTCCACC GCCACCTAATCCAGGTGGCTTGAAGAATG
HGGT-tTCN331P-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. After OD 600 was measured, the bacterial solution was transferred to a 50mL centrifuge tube and centrifuged at 6000rpm for 10min at room temperature. 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, so as to verify that the over-expression of the taxus chinensis-derived GGPP synthetase CrtE and the chicken-derived FPP synthetase mutant FPS F112A are improved, and the effect of the GGPP after supply on the improvement of delta-tocotrienol synthesis is enhanced.
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.
As shown in FIG. 3, the shake flask fermentation results of the strains VE-4 and VE-5 show that the delta-tocotrienol yield of the strain VE-5 is 1801.5 mu g/L, and compared with the strain VE-4, the delta-tocotrienol yield of the strain VE-5 is improved by 1.6 times, so that the effect of building a substrate transmission channel on improving the delta-tocotrienol yield is verified.
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.
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| CN111235044A (en) * | 2019-12-31 | 2020-06-05 | 天津大学 | Synthesis of delta-tocotrienol recombinant Saccharomyces cerevisiae strain and construction method and use |
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