CN103849643A - Synthesis method for increasing squalene in saccharomyces cerevisiae by using protein scaffolds - Google Patents
Synthesis method for increasing squalene in saccharomyces cerevisiae by using protein scaffolds Download PDFInfo
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- CN103849643A CN103849643A CN201410088307.1A CN201410088307A CN103849643A CN 103849643 A CN103849643 A CN 103849643A CN 201410088307 A CN201410088307 A CN 201410088307A CN 103849643 A CN103849643 A CN 103849643A
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Abstract
The invention relates to a synthesis method for increasing squalene in saccharomyces cerevisiae by using protein scaffolds, namely, in saccharomyces cerevisiae, isoprene pyrophosphate isomerase derived from escherichia coli and farnesyl pyrophosphate synthase and squalene synthase derived from the saccharomyces cerevisiae are built into a three-enzyme artificial multi-enzyme complex by using three pairs of interacted proteins GBD, WH1 and PDZ as well as ligands thereof and a multi-enzyme connector, and a natural substrate tunnel effect is simulated, thereby reducing the mass transfer resistance and improving the squalene producing capacity of saccharomyces cerevisiae. Compared with chemical extraction methods, the squalene production method provided by the invention is low in cost, easy to operate, and high in safety of produced squalene.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of synthetic method of utilizing albumen support to improve MF59 in yeast saccharomyces cerevisiae.
Background technology
MF59 (squalene) has another name called squalene, squalene, Squalene, chemistry by name 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa carbon six alkene, are a kind of height unsaturated hydrocarbons compounds, are the synthetic important precursor of terpene and steroid compound.In black shark liver oil, found in 1906 by Japanization scholar Tsujimoto at first.The aspects such as MF59 has extremely strong oxygen delivery capacity, the quantity of leucocyte minimizing causing because of chemotherapy in anticancer generation, diffusion and alleviation have significant curative effect, to cancer of the stomach, esophagus cancer, lung cancer, and ovarian cancer, tool has significant therapeutic effect.MF59 also has good anti-infective, and anti-fatigue effect, is mainly used in healthcare products and medical and health care industry.
The method of industrial abstract squalene is to extract from shark liver oil at present.Due to the impact of the mankind on marine ecology in recent years, marine ecology situation is increasingly severe, and marine fishery resources is on the verge of exhaustion, thereby the traditional technology that uses animal grease especially to extract squalene in shark liver oil must be eliminated gradually.And the squalene extracting from marine animal grease, with the peculiar smell that is difficult to remove, causes it to be unfavorable for joining in makeup and protective foods as additive.Therefore traditional extraction production technology must be substituted by other modes of production.For a long time, it is found that MF59 all has a small amount of distribution in sweet oil, soybean oil, Rice pollard oil, from plant, extract MF59 production cost too high, be difficult to by realizing large-scale industrial production to meet the growing market requirement.Synthesize not only complex steps of MF59 by the method for chemosynthesis, and the product obtaining is difficult to separation and purification, can not be used for the high medicine of purity requirement and health products trade.Therefore, seek a kind of production cost low, the simple MF59 large scale production method efficiently of separation and Extraction is very necessary.
Produce MF59 with microorganism cells and caused scientist's concern.Yeast saccharomyces cerevisiae is as a kind of mode trickle biology, and not only growth and breeding is fast, and genetic background is clear, and food safety.Yeast saccharomyces cerevisiae itself also can synthesize MF59, but there is rate-limiting step in MF59 route of synthesis, metabolism shunting, the problem such as toxic action and multienzyme catalysis resistance to mass transfer of intermediate product to cell, cause the synthesis capability of MF59 low, cannot realize and produce in a large number MF59 with microorganism.
Summary of the invention
The object of the invention is, for solving the low limitation of yeast saccharomyces cerevisiae production MF59 ability, provides a kind of and utilizes by albumen support colocalization MF59 route of synthesis key enzyme, thereby improves the method for yeast saccharomyces cerevisiae production MF59 ability.
For achieving the above object, the GBD that the artificial chemosynthesis codon of the present invention is optimized, WH1, tri-protein structure domains of PDZ, make a fusion rotein by two sections of polypeptide junctors in order by three structural domains.Start and stop transcribing of this fusion rotein with deriving from the promotor FBA1 of yeast saccharomyces cerevisiae and corresponding terminator FBA1.
Clone is from the Isopentenyl diphosphate isomerase (IDI) of E.coli BL21 (DE3), from farnesyl pyrophosphate synthase (FPPS) and the squalene synthase (SQS) of Saccharomyces cerevisiae INVSC1, by GBD, the aglucon of WH1 and PDZ is respectively by polypeptide junctor and IDI, N end or the C end of FPPS and SQS merge, and make three fusion roteins.With three promotor Nam2 that derive from yeast saccharomyces cerevisiae, Ala1, Gpm1 and corresponding terminator Nam2, Ala1, Gpm1 starts respectively and stops transcribing of three fusion roteins.
Four fusion roteins that build are above proceeded in yeast saccharomyces cerevisiae INVSc1 jointly with the single copy plasmid pRS41H after Sal I and BamH I double digestion, realize the connection of four fusion roteins and plasmid by the homologous recombination of yeast saccharomyces cerevisiae self, the multienzyme complex of expressing enzyme and the common composition of albumen support with plasmid form, the engineering strain of structure is pRS41H-scaf.
Yeast saccharomyces cerevisiae engineering pRS41H-scaf of the present invention, for therein by the IDI of overexpression, FPPS, SQS builds artificial multienzyme complex by albumen support colocalization, improves the throughput of yeast saccharomyces cerevisiae MF59.
The inventive method has the following advantages:
1, the present invention, by the form of albumen support by the key enzyme colocalization of MF59 route of synthesis, simulates natural substrate tunnel effect, effectively reduces resistance to mass transfer.
2, the present invention, by FPP and SQS colocalization can be reduced to the peripherad diffusion of poisonous intermediate metabolites FPP on the one hand, on the other hand, also can accelerate the rapid conversion of FPP to MF59, reduces its toxic action to cell of FPP.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.
Embodiment 1: with connector combination 1 construction of fusion protein engineering bacteria
1, clone is through codon optimized GBD, WH1, tri-genes of PDZ, use the junctor of two sections of 9 glycine and Serine alternative arrangement composition by GBD by OE-PCR, WH1, PDZ forms a fusion gene, connects into a large fusion gene by OE-PCR then equally with promotor FBA1 and terminator FBA1.
2, clone IDI from E.coli BL21 (DE3), from Saccharomyces cerevisiae INVSC1, clone FPPS and SQS, by GBD, the aglucon of WH1 and PDZ is respectively by junctor and the IDI of 9 glycine and Serine alternative arrangement composition, N end or the C end of FPPS and SQS merge, and make three fusion genes.With three promotor Nam2 that derive from yeast saccharomyces cerevisiae, Ala1, Gpm1 and corresponding terminator Nam2, Ala1, Gpm1 starts respectively and stops transcribing of three fusion genes.
3, single copy plasmid pRS41H is used after restriction enzyme Sal I and BamH I double digestion, mix with above-mentioned fusion gene, after dehydrated alcohol precipitation, dissolve with 5 μ L sterilized waters.The competent cell Saccharomyces cerevisiae INVSC1 of electricity transformed saccharomyces cerevisiae, transforms successful engineering strain and grows on Hygromycin B resistant flat board, and bacterium colony PCR screens positive saccharomyces cerevisiae engineered yeast, further fermentation culture checking.
Embodiment 2: with connector combination 2 construction of fusion protein engineering bacterias
1, clone is through codon optimized GBD, WH1, tri-genes of PDZ, use the junctor of two sections of 12 glycine and Serine alternative arrangement composition by GBD by OE-PCR, WH1, PDZ forms a fusion gene, connects into a large fusion gene by OE-PCR then equally with promotor Tys and terminator Tys.
2, clone IDI from E.coli BL21 (DE3), from Saccharomyces cerevisiae INVSC1, clone FPPS and SQS, by GBD, the aglucon of WH1 and PDZ is respectively by junctor and the IDI of 12 glycine and Serine alternative arrangement composition, N end or the C end of FPPS and SQS merge, and make three fusion genes.With same promotor Tys and the terminator Nam2 that derives from yeast saccharomyces cerevisiae, Ala1, Gpm1 starts respectively and stops transcribing of three fusion genes.
3, single copy plasmid pRS41H is used after restriction enzyme Sal I and BamH I double digestion, mix with above-mentioned fusion gene, after dehydrated alcohol precipitation, dissolve with 5 μ L sterilized waters.The competent cell Saccharomyces cerevisiae INVSC1 of electricity transformed saccharomyces cerevisiae, transforms successful engineering strain and grows on Hygromycin B resistant flat board, and bacterium colony PCR screens positive saccharomyces cerevisiae engineered yeast, further fermentation culture checking.
Embodiment 3: saccharomyces cerevisiae engineered yeast fermentative production MF59
Select the saccharomyces cerevisiae engineered yeast that embodiment 1 filters out, 30 ℃ of shake-flask culture in the substratum that contains 2% glucose, 2% peptone and 1% yeast powder, sampling time point is 8h, 12h, 16h, 20h, 24h, 36h, 48h, 60h, 72h, 64h, 96h, 108h, 120h.Each sampling amount is 40mL, sample preparation process is as follows: the centrifugal 5min of 5000rpm, precipitate resuspended with 20% potassium hydroxide solution (containing 50% ethanol) of 10mL, then in boiling water, boil 10min, cooling rear n-hexane extraction 2 times of using of room temperature, extraction liquid is concentrated into 1mL after merging, and then utilizes Agilent chromatographic column DB-1(30m*0.25mm*0.25um is installed) Shimadzu gas-chromatography-the carry out analysis of saccharomyces cerevisiae engineered yeast fermentative production MF59.Found that, saccharomyces cerevisiae engineered yeast is MF59 output maximum in the time of fermentation 16h.
Claims (5)
1. improve the synthetic method of MF59 in yeast saccharomyces cerevisiae, it is characterized in that, utilize albumen support by the Isopentenyl diphosphate isomerase of heterogenous expression MF59 route of synthesis in yeast saccharomyces cerevisiae, farnesyl pyrophosphate synthase and squalene synthase colocalization.
2. the synthetic method of MF59 in raising yeast saccharomyces cerevisiae as claimed in claim 1, is characterized in that, three enzymes of described MF59 route of synthesis, from E.coli, have the enzyme of same or similar function in Saccharomyces cerevisiae or other biological.
3. the synthetic method of MF59 in raising yeast saccharomyces cerevisiae as claimed in claim 1, it is characterized in that, described albumen support is to play the albumen that can make the effect of enzyme colocalization, comprise the albumen support building by paired interacting protein and polypeptide junctor, the albumen support building by self-assembled protein and by the albumen support building by chemically modified protein.
4. protein scaffolds as claimed in claim 3, derives from one or more in animal, plant or microorganism.
5. polypeptide junctor as claimed in claim 3, is constituted by more than one amino acid, and junctor length is between 5 to 20 amino acid.
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Cited By (1)
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| CN104497107A (en) * | 2014-12-08 | 2015-04-08 | 重庆医科大学 | Self-assembling oligopeptide and application thereof in three-dimensional cell culture |
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| CN102257149A (en) * | 2008-08-28 | 2011-11-23 | 诺华有限公司 | Production of squalene from hyper-producing yeasts |
| CN103387944A (en) * | 2012-05-09 | 2013-11-13 | 中国科学院大连化学物理研究所 | Construction method for high-yield sclareol strain |
| CN103492558A (en) * | 2011-02-28 | 2014-01-01 | 器官平衡有限责任公司 | Yeast cell for the production of terpenes and uses thereof |
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| CN102257149A (en) * | 2008-08-28 | 2011-11-23 | 诺华有限公司 | Production of squalene from hyper-producing yeasts |
| WO2011053079A2 (en) * | 2009-10-30 | 2011-05-05 | 한국생명공학연구원 | Mutant yeast and method for producing squalene using same |
| CN103492558A (en) * | 2011-02-28 | 2014-01-01 | 器官平衡有限责任公司 | Yeast cell for the production of terpenes and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104497107A (en) * | 2014-12-08 | 2015-04-08 | 重庆医科大学 | Self-assembling oligopeptide and application thereof in three-dimensional cell culture |
| CN104497107B (en) * | 2014-12-08 | 2017-09-19 | 重庆医科大学 | A kind of self-assembled short peptide and its application in three-dimensional cell cultivation |
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Application publication date: 20140611 |