CN114015587B - Recombinant strain for producing squalene, construction method and application thereof - Google Patents

Recombinant strain for producing squalene, construction method and application thereof Download PDF

Info

Publication number
CN114015587B
CN114015587B CN202111145425.8A CN202111145425A CN114015587B CN 114015587 B CN114015587 B CN 114015587B CN 202111145425 A CN202111145425 A CN 202111145425A CN 114015587 B CN114015587 B CN 114015587B
Authority
CN
China
Prior art keywords
squalene
strain
synthesis
mitochondrial
engineering
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.)
Active
Application number
CN202111145425.8A
Other languages
Chinese (zh)
Other versions
CN114015587A (en
Inventor
魏东芝
王风清
朱占涛
杜蒙蒙
张格格
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baikaisheng Shanghai Biotechnology Co ltd
Original Assignee
East China University of Science and Technology
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by East China University of Science and Technology filed Critical East China University of Science and Technology
Priority to CN202111145425.8A priority Critical patent/CN114015587B/en
Publication of CN114015587A publication Critical patent/CN114015587A/en
Application granted granted Critical
Publication of CN114015587B publication Critical patent/CN114015587B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
    • C12Y203/0301—Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01036—Mevalonate kinase (2.7.1.36)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04002—Phosphomevalonate kinase (2.7.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
    • C12Y503/03002—Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01001—Acetate-CoA ligase (6.2.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (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 recombinant strain for producing squalene, a construction method and application thereof. The method comprises the following steps: the preparation method comprises the steps of taking saccharomyces cerevisiae as an original strain, firstly reconstructing a mevalonate pathway in an online granulocyte to construct a mitochondrial engineering strain, then strengthening mevalonate synthesis in cytoplasm of the mitochondrial engineering strain to obviously improve strain growth, finally carrying out cytoplasm engineering policy optimization on the basis, and obtaining a recombinant strain of high-yield squalene through joint optimization of mitochondria and cytoplasm double-procedure. The invention utilizes the combined regulation and control strategy of the cytoplasm and mitochondria of Saccharomyces cerevisiae to ensure that the yield of squalene has a superposition effect, solves the problem of poor growth of mitochondrial engineering strains, and greatly improves the capability of producing squalene by the yeast, even more than 20g/L. The recombinant strain obtained by the invention is suitable for industrialized production of squalene and other terpenoid, and provides a powerful research foundation for biosynthesis of squalene.

Description

Recombinant strain for producing squalene, construction method and application thereof
Technical Field
The invention relates to the field of biosynthesis application, in particular to a recombinant strain for producing squalene, a construction method and application thereof.
Background
Squalene (Squalene), also known as Squalene, is an all-trans linear triterpene compound with a chemical formula of C30H50, CAS registry number 111-02-4, and a relative molecular mass of 410.70 and a density of 0.8584mg/mL. From the chemical structure, squalene molecules contain 6 double bonds, belonging to polyunsaturated olefins and are chemically named 2,6,10,15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracosahexaene. Squalene is colorless or yellow, is transparent oily liquid, can give out pleasant smell, becomes tacky after oxygen inhalation, is in a linseed oil state, is easily dissolved in diethyl ether, acetone and the like, is insoluble in water, is slightly dissolved in glacial acetic acid and ethanol, and is decomposed at 330 ℃ under normal pressure. Squalene is widely used in the food, cosmetic, pharmaceutical and other industries for its antioxidant, antistatic and anticancer properties. Importantly, squalene can stimulate and enhance immune responses against antigens and has been used as an additive to a variety of vaccines to improve the efficacy of vaccines, including vaccines for pandemic influenza, malaria, and covd-19.
At present, squalene is mainly obtained by methods of chemical synthesis, animal and plant extraction, microbial fermentation method and the like. Chemical synthesis of squalene mainly includes a "complex synthetic route" and a "farnesyl derivative route". These two methods are complex, low in yield and limited to laboratory studies. Shark liver has been the major source of squalene for decades. However, the production mode is not environment-friendly and sustainable, and is strictly forbidden by the international trade convention of endangered wild animal and plant species. Meanwhile, the plant extraction method is difficult to meet the requirements of industrial application due to long production period and complex production conditions. The microbial fermentation method is favored because of the advantages of simple production method, short period, low price and abundant sources of production raw materials, small environmental influence, good economic benefit and the like. Therefore, the genetic engineering strain for developing high-yield squalene by utilizing genetic modification is a technical direction with good application prospect. Squalene synthesized in Saccharomyces cerevisiae shows equally effective and safe vaccine adjuvant effect in mouse model experiments, so that production of squalene in engineering Saccharomyces cerevisiae is advantageous.
At present, a saccharomyces cerevisiae engineering strain for producing squalene is mainly constructed by adopting a simple cytoplasmic engineering technology, has insufficient production capacity and cannot meet industrial application requirements. In addition to cytoplasmic engineering, the organelle engineering is considered another potentially effective method for synthesizing target products in yeast, given the unique physiological and physical advantages of highly functional specialized subcellular organelles, such as specific biochemical environments, rich precursor and cofactor supply, compartmentalization of metabolic pathways, both isolation of competing pathways and efficient increase of concentration of reactive precursors and enzymes, improving efficiency of cascade enzyme catalysis, and furthermore, the organelle favors product storage, reducing product toxicity. Mitochondria are the main sites of eukaryotic cell central metabolism and energy production, and engineering mitochondria (mitochondrial engineering) to synthesize terpenoids has the following advantages: the precursor species acetyl-coa content for terpene synthesis is about 20-30 times that in the cytosol (Weinert et al, 2014), high redox potential, abundant ATP and sufficient redox enzyme cofactors (Hu et al, 2008), and compact space can concentrate substrates, enzymes, intermediates and cofactors to accelerate enzymatic reactions (Avalos et al, 2013). The feasibility of using mitochondria to efficiently synthesize terpenes has been widely studied so far. Yuan et al (Yuan and Ching, 2016) have first demonstrated that mitochondria can be used as compartments that promote the production of sesquiterpene amolpha-4, 11-diene by rebuilding the farnesyl pyrophosphate (FPP) biosynthetic pathway in yeast mitochondria. Recently, yee et al (Yee et al, 2019) indicated that mitochondrial engineering is an efficient method for producing monoterpenes, and can increase the yield of geraniol in yeast by a factor of 6 compared to cytoplasmic engineering. Lv et al increased isoprene production in yeast by 2.5g/L through double metabolic engineering of the MVA pathway in mitochondria and cytoplasm (Lv et al, 2016) by 2.1 times the isoprene production obtained by mitochondrial engineering and 1.6 times the isoprene production obtained by cytoplasmic engineering, respectively. These cases fully demonstrate that mitochondria play a great role in yeast terpene production. However, mitochondria act as the core organelle of eukaryotic cells, and mitochondrial engineering strategies often lead to severe metabolic burden, manifested by poor growth of the engineered strain and low product titers. This severely affects the exploitation of the advantages of mitochondrial engineering strategies and needs to be addressed.
Disclosure of Invention
The invention aims to provide a recombinant strain for producing squalene, a construction method and application thereof, thereby solving the problem of lack of high-yield squalene genetic engineering bacteria in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to a first aspect of the present invention there is provided a method of constructing a recombinant strain for the production of squalene comprising the steps of: the method comprises the steps of taking saccharomyces cerevisiae as an original strain, firstly reconstructing a mevalonate pathway in a line granule body to construct a mitochondrial engineering strain, then strengthening mevalonate synthesis in cytoplasm of the mitochondrial engineering strain, finally carrying out cytoplasm engineering strategy optimization on the basis, and obtaining a recombinant strain of high-yield squalene through joint optimization of mitochondria and cytoplasm duplex operation.
As an implementation mode of the invention, the invention randomly utilizes a strain of Saccharomyces cerevisiae CEN.PK2-1C to carry out metabolic engineering transformation, and obtains genetically engineered bacteria with obviously enhanced squalene production capacity. It should be understood that the invention is not limited to this strain of Saccharomyces cerevisiae.
According to the construction method provided by the invention, firstly, a mitochondrial engineering strain is constructed. As one embodiment of the present invention, the present invention utilizes mitochondrial localization signal peptides (e.g., MLS) to localize 7 key enzymes (ERG 10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI 1) of the MVA pathway from acetyl-CoA to dimethylallyl pyrophosphate (DMAPP) within the mitochondria of the above strain, and to effect synthesis from acetyl-CoA to isopentenyl pyrophosphate (IPP) and DMAPP within the wire granules. The invention utilizes GAL1-GAL10, GAL2, GAL3, GAL7 and other inducible promoters to express the gene connected with the mitochondrial localization signal peptide.
The 7 key enzymes located within the mitochondria of saccharomyces cerevisiae are: ERG10 (Genbank: nm_ 001183842.1), ERG13 (Genbank: NM_ 001182489.1), tHMG1 (Genbank: NM_ 001182434.1), ERG12 (Genbank: NM_ 001182715.1), ERG8 (Genbank: NM_ 001182727.1), ERG19 (Genbank: NM_ 001183220.1) and IDI1 (Genbank: NM_ 001183931.1).
Secondly, in order to solve the growth defect of the mitochondrial engineering strain, the invention further strengthens the synthesis of mevalonic acid in cytoplasm of the mitochondrial engineering strain so as to improve the growth state of the mitochondrial engineering strain. As one embodiment of the present invention, tHMG1 (Genbank: NM-001182434.1) is overexpressed in the cytoplasm of the mitochondrial engineered strain, which significantly improves the growth of the mitochondrial engineered strain and squalene production. Wherein mitochondrial engineering localizes 7 key enzymes in the MVA pathway into mitochondria, IPP/DMAPP synthesized in-line with the mitochondrial membrane can penetrate into the cytoplasm for squalene synthesis. The strain realizes that the squalene synthesis product IPP/DMAPP is provided by utilizing the combination of mitochondria and cytoplasm, so that the yield of squalene is greatly improved, and the yield is about the sum of the yield of squalene of pure mitochondrial engineering and the yield of squalene of pure cytoplasmic engineering, so that the strain has the superposition effect of the yields of mitochondrial engineering and cytoplasmic engineering.
Finally, on the basis of the cytoplasmic and mitochondrial double engineering combined strain, the invention optimizes the cytoplasmic engineering strategy, and further improves the yield of squalene. As one embodiment of the invention, the invention over-expresses rate-limiting enzymes in squalene synthesis pathways, including but not limited to enhancing the supply of acetyl-CoA, weakening the expression level or enzyme activity of key enzymes in pathways consuming the target product, in the cytoplasm of the combined cytoplasmic and mitochondrial duplex strain. All promoters of the cytoplasmic engineering which are required to enhance the expressed genes are inducible promoters such as GAL1-GAL10, GAL2, GAL7 and the like.
Wherein according to a preferred embodiment of the invention the expression enhanced enzyme comprises ERG10 (Genbank: NM-001183842.1) and/or ACS1 (Genbank: NM-001178197.1) and/or ACS2 (Genbank: NM-001182040.1), the expression enhanced enzyme is ERG1 (Genbank: NM-001181304.1).
In order to evaluate the production capacity of squalene engineering strains, fermentation amplification tests are carried out. As one embodiment of the invention, the invention complements the auxotroph gene of the squalene engineering strain. The invention realizes a high-density fermentation method of double-stage regulation and control in a growth stage and a product synthesis stage by utilizing a high-yield squalene engineering strain induced expression system in the fermentation amplification process, and realizes high yield of squalene.
According to a second aspect of the present invention there is provided a recombinant strain producing squalene obtainable according to the above construction method.
According to a particularly preferred embodiment of the invention, the nucleotide sequence of the recombinant strain of high-yield squalene obtained is shown in SEQ ID No. 8.
According to a third aspect of the present invention there is also provided the use of a recombinant strain as described above for the production of squalene, during fermentation culture, adding to the fermentation system a feed medium comprising: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
The fermentation culture process is divided into two stages: the first stage makes the thallus grow fast, and after the thallus biomass reaches certain concentration, the second stage fermentation is started; the second stage is the production stage of squalene, and a large amount of squalene synthesis is started by adding an inducer, so that high yield of squalene is finally obtained. According to the method provided by the invention, the yield of squalene exceeds 20g/L through a two-stage fermentation process finally, and the method is the highest international production level at present, and has room for further improvement.
In the invention, a mevalonic acid pathway (MVA pathway) is introduced into online granules by a mitochondrial engineering technology in saccharomyces cerevisiae, so that a mitochondrial engineering strain with obviously improved squalene yield is constructed, however, the biomass of the strain is obviously reduced, and the yield of squalene per unit volume is reduced. Further studies have shown that this is due to the fact that metabolites produced by the MVA pathway in the in-line granule have a severe toxic effect on saccharomyces cerevisiae. In order to solve the toxic and side effects of mitochondrial engineering on mitochondria, the invention proves that increasing the synthesis of mevalonic acid in the cytoplasm of the mitochondrial engineering strain can obviously improve the growth of thalli and the yield of squalene, and therefore, a combined optimization strategy of cytoplasm and mitochondrial engineering is further provided to relieve the metabolic burden caused by introducing MVA pathway in mitochondria and improve the growth of cells. Furthermore, the present invention demonstrates that this strategy also has a additive effect on the production of squalene for cytoplasmic engineering and mitochondrial engineering squalene production. By means of the combined optimization of cytoplasm and mitochondrial double-process, negative influence of mitochondrial engineering on squalene products can be relieved, and the yield of squalene is greatly improved.
In conclusion, the invention obtains the mitochondrial engineering strain by reconstructing the MVA pathway in yeast mitochondria, enhances the synthesis of mevalonic acid in cytoplasm of the mitochondrial engineering strain to improve the growth of the mitochondrial engineering strain to obtain the cytoplasmic and mitochondrial double-process combined strain, and finally optimizes the cytoplasm engineering in cytoplasm of the cytoplasmic and mitochondrial double-process combined strain, thereby realizing the development of the high-yield squalene of the yeast mitochondrial engineering combined cytoplasm engineering. Through fermentation amplification test, the yield of squalene of the genetic engineering strain developed by the invention can reach 20g/L, is the highest yield at present, and is nearly twice as high as that of squalene of the peroxisome engineering strain with the second highest yield developed in the laboratory in advance. The invention proves the advantages of utilizing mitochondrial engineering and cytoplasmic engineering to combine and optimize and develop the high-yield squalene gene engineering bacteria, and provides a new idea for realizing the high-efficiency production of squalene and other terpenes in yeast.
Drawings
FIG. 1 is a schematic diagram of engineering strain construction;
FIG. 2 is a map of plasmids PCZT3-MLS-GFP and PCZT3-GFP in example 1;
FIG. 3 is a graph showing specificity and stability verification of the mitochondrial localization signal peptide MLS in example 1;
FIG. 4 is a map of the mitochondrial localized gene expression cassette construction tool plasmid PZT-MLS in example 2;
FIG. 5 is a map of the plasmid PZT20 for the construction of the cytoplasmic gene expression cassette in example 2;
FIG. 6 is a schematic representation of the knock-in of BMLS-ERG13-ERG12 into ADH3 sites by the CRISPR-Cas9 gene editing tool of example 2;
FIG. 7 is a gas phase diagram of squalene standards and engineering strain fermentation assays in example 2.
Detailed Description
The present invention will be further described with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are those commercially available.
The solution preparation methods referred to herein are as follows:
YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose (YPD solid medium, 20g/L agar powder was added).
And (3) feeding a fermentation basic culture medium: adding 8g/L KH into YPD 2 P0 4 ，3g/L MgSO 4 ，0.72g/L ZnSO 4 ·7H 2 O,10mL/L trace element liquid and 12mL/L vitamin liquid.
Feeding a feed medium: 500g/L glucose, 9g/L KH 2 P0 4 ，2.5g/L MgSO 4 ，3.5g/L K2SO 4 ，0.28g/L Na 2 SO 4 10mL/L of trace element liquid, 12mL/L of vitamin liquid, 10g/L of yeast extract and 20g/L of peptone.
The squalene extraction method comprises the following steps: to extract squalene, 600. Mu.L of cultured cells and 600. Mu.L of ethyl acetate were mixed with 1.5g of zirconia beads (diameter 0.5 mm) in a 2mL centrifuge tube and crushed with a cryomill (Shanghai Jingxin China) at-4℃for 30min at 55 Hz. The shaken mixture was centrifuged at 13,000rpm for 10 minutes and the upper organic phase was dehydrated over anhydrous sodium sulfate for GC detection.
The detection method of squalene comprises the following steps:
squalene standards were purchased from Shanghai Ala Biochemical technologies Co.
Preparing squalene standard substance solution: 50mg squalene standard is weighed, dissolved in ethyl acetate, and fixed in a 5mL volumetric flask, and the concentration of the standard mother solution is 10mg/mL.
The invention adopts gas chromatography to carry out qualitative and quantitative analysis on squalene. The GC system (Agilent 7820A, USA) was equipped with an HP-5 column (30 m. Times.0.25 mm,0.25 μm film thickness) and a Flame Ionization Detector (FID). The oven temperature was held at 90 ℃ for 0.5 minutes, then gradually increased to 170 ℃ at 20 ℃/min for 0.5 minutes, then increased to 190 ℃ at 10 ℃/min for 0.5 minutes, and finally increased to 280 ℃ at 20 ℃/min for 7 minutes. Squalene was quantified by external standard method.
Original strain of Saccharomyces cerevisiae CEN.PK2-1C was purchased from European Saccharomyces cerevisiae functional analysis Collection (European Saccharomyces cerevisiae Archive for Functional Analysis, EUROSCRF), accession number: 30000A.
FIG. 1 is a schematic diagram of a combined cytoplasmic and mitochondrial duplex strain regulation strategy employed in the present invention. The following describes the same in detail by way of examples.
Example 1: method for stably positioning target protein in mitochondria
In order to ensure stable localization of the target protein in the s.cerevisiae mitochondria, the present invention selects the N-terminal mitochondrial localization signal MLS from mitochondrial cytochrome oxidase subunit IV to direct accurate localization of the target gene protein in the mitochondrial matrix. The invention verifies the ability of MLS to pinpoint proteins in mitochondria by using green fluorescent protein GFP as a reporter gene. The mitochondrial localization signal peptide MLS is introduced into the front end of GFP, the promoter is Saccharomyces cerevisiae constitutive strong promoter PTDH3, and plasmid PCZT3-MLS-GFP is constructed, and the map is shown as b in FIG. 2. Meanwhile, as a control, the plasmid PCZT3-GFP without the signal peptide added to the front end of GFP is constructed, and the map is shown as a in figure 2. The invention amplifies GFP through primers BamHI-GFP-F and SalI-GFP-R (specific sequences of the primers are shown in Table 1), and constructs plasmid PCZT3-GFP by connecting the PCZT3 after double enzyme cutting by BamHI and SalI through a seamless cloning method. The primers BamHI-MLS-GFP-F and MLS-GFP-R1 (specific sequences of the primers are shown in Table 1) were used to amplify the MLS, and the plasmid PCZT3-MLS-GFP was obtained by seamless cloning and ligation after single cleavage of the plasmid PCZT3-GFP from BamHI. The invention integrates PTDH3-MLS-GFP and PTDH3-GFP into GPD1 gene loci of yeast CEN.PK2-1C genome respectively through CRISPR-Cas9 gene editing tools. The primers GPD1-UF and GPD1-TPGK1-UR are used for PCR amplification of upstream homology arm GPD1-UP by using the saccharomyces cerevisiae genome as a template. The primer GPD1-TTPS1-DF and GPD1-DR are used to PCR amplify the downstream homologous arm GPD1-DN by using the Saccharomyces cerevisiae genome as template. The specific primers of the expression cassettes PTDH3-GFP and PTDH3-MLS-GFP obtained by PCR amplification with the primers GPD1-TPGK1-F, GPD1-TTPS1-R and the plasmids PCZT3-GFP and PCZT3-MLS-GFP as templates are shown in Table 1. GPD1-UP, GPD1-DN, PTDH3-GFP or PTDH3-MLS-GFP and gRNA plasmids were transformed into yeasts by chemical transformation, screened and cultured on SD-Leu-URA double-defect medium plates, and the localization of fluorescent proteins was identified by fluorescence confocal microscopy. The localization results are shown in FIG. 3, in which the fluorescent protein fused with the MLS sequence is concentrated only in a localized region of mitochondria, whereas the fluorescent protein without the fusion sequence fills the whole yeast cell, and the results indicate that the target gene can be stably and specifically localized in mitochondria by fusing the MLS sequence. This example demonstrates the accuracy and stability of the present invention to the localization of enzymes in mitochondria.
TABLE 1
Example 2: construction of high-yield squalene mitochondrial engineering strain
In order to utilize the advantage of mitochondria in squalene biosynthesis, the invention marks MLS to the N end of a gene, and positions 7 key enzymes of the MVA pathway from acetyl coenzyme A to IPP/DMAPP to mitochondria, thereby obtaining a mitochondrial engineering strain. Firstly, the invention constructs successful PZT20 by cyclizing the plasmid skeleton derived from yeast promoters GAL1-GAL10, terminator TCYC1 and TADH1 and from PUC19, the map is shown in figure 5, and the nucleotide sequence is shown in SEQ ID No. 1. The invention inserts mitochondrial localization signal peptide MLS into two promoters of PZT20 to obtain PZT-MLS, the spectrum is shown in figure 4, and the nucleotide sequence is shown in SEQ ID No. 2. The invention constructs 7 enzymes of MVA pathway to the multiple cloning site of inducible plasmid PZT-MLS containing MLS sequence. And the present invention constructs squalene synthesis-related ERG9 and ERG20 to the multiple cloning site of PZT 20. The invention respectively constructs five recombinant plasmids of PZT-MLS-ERG10-tHMG1, PZT-MLS-ERG13-ERG12, PZT-MLS-ERG8-ERG19, PZT-MLS-tHMG1-IDI1 and PZT-20-ERG 9-ERG20 for constructing mitochondrial engineering strains with high squalene yield. The above gene sequences are derived from Saccharomyces cerevisiae CEN.PK2-1C (yeast genome database (SGD): CEN.PK2-1Ca_SGD_2015_JRIV01000000). Taking PZT-MLS-ERG13-ERG12 as an example, primers MLS-ERG12-F, MLS-ERG12-R and MLS-ERG13-F, MLS-ERG13-R are used for ERG12 and ERG13 respectively, the saccharomyces cerevisiae genome is used as a template for PCR amplification, and fragments are inserted into corresponding enzyme cutting sites of PZT-MLS to obtain plasmids PZT-MLS-ERG13-ERG12. The MLS-ERG13-ERG12 is transferred into the ADH3 gene locus through a CRISPR-Cas9 gene editing tool. The primers ADH3-UF and ADH3-TADH1-UR are used for PCR amplification of the upstream homology arm ADH3-UP by using the Saccharomyces cerevisiae genome as a template. The primers ADH3-TCYC1-DF and ADH3-DR are used, and the saccharomyces cerevisiae genome is used as a template for PCR amplification of the downstream homology arm ADH3-DN. The primer ADH3-TADH1-F, ADH3-TCYC1-R and the plasmid PZT-MLS-ERG13-ERG12 are used as templates for PCR amplification to obtain the expression cassette BMLS-ERG13-ERG12. According to the invention, ADH3-UP, ADH3-DN, BMLS-ERG13-ERG12 and gRNA plasmids are transformed into yeast cells by a chemical transformation method, a CRISPR-Cas9 gene editing tool is utilized to knock BMLS-ERG13-ERG12 into an ADH3 site, a schematic diagram is shown in figure 6, and a nucleotide sequence is shown in SEQ ID No. 3. Screening was performed with SD-Leu-URA double-defect medium plates. Similarly, the genome integration methods of the remaining genes were all constructed as described above, and specific primers can be seen in Table 2. The mitochondrial engineering strain with high squalene yield is obtained after metabolic engineering optimization, and the squalene yield is detected through gas phase by oxygen consumption fermentation culture for 7 days, and a squalene standard and engineering strain fermentation detection gas phase diagram is shown in figure 7. The squalene yield of the engineering strain reaches 401.97mg/L, which is 63.1 times of that of the original strain CEN.PK2-1C.
TABLE 2
Example 3: intracellular overexpression of tHMG1 significantly improves the growth state and squalene yield of mitochondrial engineering strains and construction of mitochondrial engineering and cytoplasmic engineering combined genetic engineering strains
Although 7 key genes of the MVA pathway are positioned in mitochondria through mitochondrial engineering, the construction of the MVA pathway in mitochondria is realized, and the squalene yield of engineering bacteria is remarkably improved, the mitochondrial engineering generates great burden on yeast growth and metabolism, so that the squalene yield is inferior to that of corresponding cytoplasmic engineering. Therefore, in order to better utilize mitochondria to synthesize squalene, new strategies should be explored to reduce the metabolic burden of mitochondrial engineering on mitochondria.
When 7 key enzymes of MVA pathway are sequentially introduced into mitochondria, after ERG10, ERG13 and tHMG1 are introduced, namely, after local pathway from acetyl coenzyme A to mevalonate is successfully introduced into the online granules, the growth of the thalli is not reduced, but is greatly improved, which is contrary to the result of all 7 key enzymes introduced into MVA pathway, so that the synthesis of mevalonate in the online granules is not toxic to the thalli, and the growth of the thalli is facilitated. However, when mevalonate starts to extend down to IPP and DMAPP again, the growth state of the cells is suddenly deteriorated, indicating that the metabolic products after mevalonate have significant toxicity. In view of the remarkable promoting effect of mevalonic acid on cell growth, the present invention proposes: the problem of poor cell growth of the mitochondrial engineering bacteria is improved by the enhanced expression of mevalonate synthase tHMG1 in the cytoplasm of the mitochondrial engineering strain. Experimental results show that the method is a very effective strategy, not only can remarkably improve the growth state of mitochondrial engineering bacteria, but also can greatly improve the yield of squalene. Specifically, primers PZT-tHMGR1-F and MLS-tHMGR1-R are used for PCR amplification of tHMG1 by taking the saccharomyces cerevisiae genome as a template, and the primers are inserted into corresponding loci of PZT20 to obtain the PZT20-tHMG1, and the nucleotide sequence is shown as SEQ ID No. 4. In the invention, tHMG1 is transferred into an ADH4 gene locus through a CRISPR-Cas9 gene editing tool. The primers ADH4-UF and ADH4-TADH1-UR are used for PCR amplification of the upstream homology arm ADH4-UP by using the Saccharomyces cerevisiae genome as a template. The primers ADH4-TCYC1-DF and ADH4-DR are used, and the saccharomyces cerevisiae genome is used as a template for PCR amplification of the downstream homology arm ADH4-DN. The expression cassette BGAL1-tHMG1 is obtained by PCR amplification using the primer ADH4-TADH1-F, ADH4-TCYC1-R and the plasmid PZT20-tHMG1 as a template. ADH4-UP, ADH4-DN, BGAL1-tHMG1 and gRNA plasmids were transformed into the above mitochondrial engineering strains by chemical transformation, and the mitochondrial and cytoplasmic combination strains were obtained by plate screening with SD-Leu-URA double defect medium, and specific primers can be seen in Table 3. Meanwhile, ADH4-UP, ADH4-DN, BGAL1-tHMG1 and gRNA plasmids are converted into non-mitochondrial engineering strains through a chemical conversion method, SD-LEU-URA double-defect culture medium plates are used for screening, cytoplasmic engineering strains are obtained, and the yield of squalene reaches 1003.87mg/L through gas phase detection after 7 days of oxygen consumption fermentation culture. The combined engineering strain of mitochondria and cytoplasm is cultured for 7 days by oxygen consumption fermentation, and the biomass is improved by 32.0 percent compared with the pure mitochondrial engineering strain. This indicates that overexpression of tmg 1 in the cytoplasm of the mitochondrial metabolic engineering strain significantly improved mitochondrial engineering strain growth. Meanwhile, the yield of squalene of the mitochondrial and cytoplasmic combined strain reaches 1382.88mg/L through gas phase detection, and the yield of squalene is almost the sum of the yields of squalene of the pure cytoplasmic engineering strain and the pure mitochondrial engineering strain. This shows that the combination of mitochondria and cytoplasm strains realize the use of the combination of mitochondria and cytoplasm to provide the terpene synthesis precursor IPP/DMAPP, so that the yield of squalene has a superposition effect.
TABLE 3 Table 3
Example 4: the combined engineering of mitochondria and cytoplasm optimizes the cytoplasm engineering of the strain, namely, enhances the supply of acetyl coenzyme A in cytoplasm and weakens the consumption path of target products.
Enhanced acetyl-CoA supply
Acetyl-coa is a direct precursor to terpenes synthesis, whereas acetyl-coa synthesis in the cytoplasm is the rate-limiting step. In order to further improve the squalene yield, the invention strengthens the first gene ERG10 of acetyl coenzyme A synthesis pathway and MVA pathway in the cytoplasm of the strain on the basis of the mitochondrial and cytoplasmic combined engineering strain. The invention uses the plasmid pZT110 with GAL1-GAL10 as promoter and TPGK1 and TTPS1 as terminator to construct the plasmids pZT110-ACS2-ERG10 and pZT110-ACS1-ERG10 for strengthening the genes. The primers BamHI-ACS2-F and XhoI-ACS2-R, GAL110-ACS1-F and XhoI-ACS1-R, MLS-ERG10-F and NotI-ERG10-R are used for PCR amplification by taking the saccharomyces cerevisiae genome as a template to respectively obtain ACS2, ACS1 and ERG10, and the ACS2, ACS1 and NotI-ERG10 are respectively inserted into corresponding sites of PZT110 to obtain plasmids pZT110-ACS2-ERG10 (the nucleotide sequence is shown as SEQ ID No: 5) and pZT110-ACS1-ERG10 (the nucleotide sequence is shown as SEQ ID No: 6), and specific primers can be seen in Table 4. The invention integrates BACS2-ERG10 and BACS1-ERG10 into GPD1 and ARS911 sites of the genome of the mitochondrial and cytoplasmic combined strain respectively through CRISPR-Cas9 gene editing tools.
The invention uses primers GPD1-UF and GPD1-TPGK1-UR, uses the saccharomyces cerevisiae genome as a template to PCR amplify the upstream homologous arm GPD1-UP. The primer GPD1-TTPS1-DF and GPD1-DR are used to PCR amplify the downstream homologous arm GPD1-DN by using the Saccharomyces cerevisiae genome as template. PCR amplification was performed using the primers GPD1-TPGK1-F, GPD1-TTPS1-R and the plasmid pZT110-ACS2-ERG10 as a template to obtain the expression cassette BACS2-ERG10 (specific primers can be seen in Table 1). The invention uses primers ARS911-UF and ARS911-TPGK-UR, uses the Saccharomyces cerevisiae genome as a template to PCR amplify the upstream homologous arm ARS911-UP. The primer ARS911-TTPS1-DF and ARS911-DR are used to amplify the downstream homologous arm ARS911-DN by PCR with the Saccharomyces cerevisiae genome as template. The expression cassette BACS1-ERG10 is obtained by PCR amplification using the primer ARS911-TPGK1-F, ARS911-TTPS1-R and the plasmid pZT110-ACS1-ERG10 as a template, and specific primers are shown in Table 4. The invention firstly converts GPD1-UP, GPD1-DN, BACS2-ERG10 and gRNA plasmid into GPD1 locus of the genome of the combined mitochondrial and cytoplasmic strains by a chemical conversion method, and screens correct clone by using SD-Leu-URA double-defect culture medium plates. Then ARS911-UP, ARS911-DN, BACS1-ERG10 and gRNA plasmid are transformed into ARS911 locus of the genome of the strain by a chemical transformation method, SD-Leu-URA double defect culture medium plates are used for screening correct clones, the supplied mitochondria and cytoplasm combined strain for enhancing acetyl coenzyme A is obtained, and the yield of squalene reaches 2489.25mg/L through gas phase detection after 7 days of oxygen-consuming fermentation culture.
Attenuation of squalene consumption
Squalene is an intermediate product of ergosterol synthesis in yeast, and is an essential component for cell growth. The present invention dynamically regulates the transcription of ERG1 by replacing the ERG1 promoter pERG1 with pHXT1 in order to reduce squalene excess consumption for ergosterol synthesis. The method comprises the following steps:
the invention uses the primers BproERG1-F and ERG1-UF, uses the saccharomyces cerevisiae genome as a template to PCR amplify the upstream homologous arm ERG1-UP. The primers ATF2-DR and BproERG1-R were used to PCR amplify the downstream homology arm ERG1-DN using the Saccharomyces cerevisiae genome as template. Primers pHXT1-ERG1-F and pHXT1-TCYC1-R, TCYC1-R and TCYC1-ATF2-F are used, and the Saccharomyces cerevisiae genome is used as a template for PCR amplification to obtain pHXT1 and TCYC1 respectively. The fragments pHXT1 and TCYC1 were fused into fragments pHXT1-TCYC1 with primers pHXT1-ERG1-F and TCYC1-ATF 2-F. According to the invention, ERG1-UP, ERG1-DN, pHXT1-TCYC1 and gRNA plasmids are converted into pERG1 sites of the genome of the mitochondrial and cytoplasmic combined strain by a chemical conversion method, so that the pHXT1 is used for replacing the promoter pERG1 of the ERG1 of the engineering strain genome, the nucleotide sequence is shown as SEQ ID No. 7, and SD-Leu-URA double-defect culture medium plates are used for screening correct clones, and specific primers can be seen in Table 4. The combined mitochondrial and cytoplasmic strain with weakened squalene consumption was obtained, and the squalene yield was further increased to 3000.63mg/L by gas phase detection through 7 days of oxygen-consuming fermentation culture.
TABLE 4 Table 4
Example 5: evaluation of squalene production ability of engineering Strain
Auxotroph amino acid gene complementation
To assess the productivity of the cytoplasmic-mitochondrial double engineering strain, fed-batch fermentation was performed in a 5L tank fermentor. Since these strains are auxotrophs, their growth is unsatisfactory resulting in limited squalene production. Thus, the present invention complements the deleted four amino acid genes of HIS3, LEU2, URA3 and TRP 1. The method comprises the following steps:
the invention obtains promoters and genes of four auxotroph genes of URA3, TRP1, HIS3 and LEU2 by PCR amplification by taking a Saccharomyces cerevisiae S288C genome as a template through four pairs of primers ZZT-URA3-F and ZZT-URA3-R, ZZT-TRP1-F and ZZT-TRP1-R, ZZT-His3-F and ZZT-His3-R, ZZT-Leu2-F and ZZT-Leu2-R, and obtains plasmids PURA3-PTRP1 and PHIS-PLeuu 2 by seamless cloning by taking plasmids PZT20 and PZT110 as skeletons respectively, and specific primers can be seen in Table 5. The invention integrates the expression cassettes BPURA3-PTRP1 and BPHIS-PLeu2 of four auxotrophic genes into the his3 locus of the genome of the mitochondrial and cytoplasmic combined strain weakening squalene consumption through a CRISPR-Cas9 gene editing tool.
The invention uses primers CHiS3-UF and His3-TADH1-UR, uses the saccharomyces cerevisiae genome as a template for PCR amplification of an upstream homology arm CHiS3-UP. The primers TTPS1-His3-DF and CHiS3-DR are used for PCR amplification of downstream homology arm CHiS3-DN by taking the Saccharomyces cerevisiae genome as a template. The expression cassettes BPURA3-PTRP1 and BPHIS-PLeu2 of four auxotrophic genes are obtained by PCR amplification using the primers His3-TADH1-F and CYC1-TPGK1-R, CYC1-TPGK1-F and TTPS1-His3-R and the plasmids PURA3-PTRP1 and PHIS-PLeu2 as templates, and specific primers can be seen in Table 5. The invention firstly converts CHiS3-UP, CHiS3-DN, BPURA3-PTRP1, BPHIS-PLeu2 and gRNA plasmid into the HIS3 locus of the genome of the mitochondrial and cytoplasmic combined strain consumed by weakening squalene through a chemical conversion method, screens correct clone by using an SD-Leu-URA double-defect culture medium plate, and finally realizes the retrieval and the supplement of four amino acid genes of high-yield squalene mitochondrial and cytoplasmic combined engineering strains HIS3, LEU2, URA3 and TRP1, and the nucleotide sequence is shown as SEQ ID No. 8.
TABLE 5
2) Fermentation in upper tank
In view of the significant growth burden caused by the accumulation of squalene in the early stages, a two-stage fermentation strategy consisting of a cell growth stage and a squalene accumulation stage was realized by modifying the GAL regulation system and controlling the supply of glucose. The first stage was set to 48 hours, in which the biomass of the engineering strain was increased to OD 600 Without significant squalene accumulation. In the second stage, squalene is mainly produced by the induction of GAL1-GAL10 promoter by galactose, and the squalene titer reaches 21.1g/L, which is the highest yield of squalene known to us. The method comprises the following steps:
the basic culture medium for fed-batch fermentation of the invention consists of YPD, 8g/L KH 2 PO 4 、3g/L MgSO 4 、0.72g/L ZnSO 4 ·7H 2 O,10mL/L trace element solution and 12mL/L vitamin solution.
The fermentation seed culture process in the upper tank of the invention comprises the following steps: first, a seed culture was prepared by inoculating a monoclonal into a 5mL YPD tube, and then kept at 30℃for about 18 hours in a rotary shaker at 220 rpm. Next, the seed culture was subcultured by transferring 0.15mL of the first-stage culture into a 250mL Erlenmeyer flask containing 15mL of YPD, and then kept at 30℃for about 14 hours in a rotary shaker at 220 rpm. Third, seed cultures were subcultured by transferring 3mL of the secondary culture into a 500mL flask containing 100mL of YPD. These cultures were kept at 30℃for about 24 hours in a rotating shaker at 230rpm, and then 10% (vol/vol) of the third stage seed culture was inoculated into a 5L bioreactor (Bai Lun, china) containing 2.7L of the above fermentation basal medium. The batch fermentation in the upper tank of the invention is carried out at 30 ℃, and the pH in the fermentation system is maintained at 5.0 by automatically adding 5M ammonium hydroxide. The air flow ranges from 1vvm to 2vvm (air volume/working volume/minute).
The present invention employs a two-stage fed-batch strategy. In the first stage, the composition contains 500g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution. When the glucose concentration in the batch culture was reduced to 1g/L, a feed solution was supplied to adjust the residual glucose concentration between 1g/L and 2 g/L. When the cells start to slowly increase (to stationary phase), the first phase ends and the inducer galactose is added. In the second stage, 800g/L glucose was added in addition to the same mineral salts and trace elements as contained in the feed solution. The concentrated medium is used to induce the accumulation of squalene.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of the present application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
SEQUENCE LISTING
<110> university of Industy of Huadong
<120> recombinant strain for producing squalene, construction method and application thereof
<160> 114
<170> PatentIn version 3.5
<210> 1
<211> 3157
<212> DNA
<213> Synthesis
<400> 1
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 780
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 840
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 900
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 960
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1020
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1080
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1140
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1200
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1260
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1320
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1380
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1440
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1500
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1560
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 1620
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 1680
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 1740
agtgcaaagc tggagctggc cttgtctgaa ttggagcgac ctcatgctat acctgagaaa 1800
gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt aaaacctaag 1860
agtcacttta aaatttgtat acacttattt tttttataac ttatttaata ataaaaatca 1920
taaatcataa gaaattcgct tatttagaag tgtcaacaac gtatctacca acgatttgac 1980
ccttttccat cttttcgtaa atttctggca aggtagacaa gccgacaacc ttgattggag 2040
acttgaccaa acctctggcg aagaattgtt aattaagagc tcagatctta tcgtcgtcat 2100
ccttgtaatc catcgatact agtgcggccg ccctttagtg agggttgaat tcgaattttc 2160
aacatttctt acttttttca tggatggacg caaagaagtt taataatcat attacatggc 2220
attaccacca tatacatatc catatacata tccatatcta atcttactta tatgttgtgg 2280
aaatgtaaag agccccatta tcttagccta aaacatcctt ctctttggaa ctttcagtaa 2340
tacgcttaac tgctcattgc tatattgaag tacggattag aagccgccga gcgggtgaca 2400
gccctccgaa ggaagactct cctccgtgcg tcctcgtctt caccggtcgc gttcctgaaa 2460
cgcagcattg cctcgcgccg cactgctccg aacaataaag attctacaat actagcatgt 2520
atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca aatgaacgaa 2580
tcaaattaac aaccatagga tgataatgcg attagatgtt tagccttatt tctggggtaa 2640
ttaatcagcg aagcgatgaa tgttgatcta ttaacagata tataaatgca aaaactgcat 2700
aaccacttta actaatactt tcaacatttt cggtttgtat tacttcttat tcaaatgtaa 2760
taaaagtatc aacatgaaat tgttaatata cctctatact ttaacgtcaa ggagatgaaa 2820
ccccggatcc gtaatacgac tcactatagg gcccgggcgt cgacatggaa cagaagttga 2880
tttccgaaga agacctcgag taagcttggt accgcggcta gctaagatcc gctctaaccg 2940
aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 3000
tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 3060
acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggcgc 3120
tcgacaaccc ttaatataag gccgcgttgc tggcgtt 3157
<210> 2
<211> 3285
<212> DNA
<213> Synthesis
<400> 2
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 780
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 840
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 900
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 960
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1020
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1080
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1140
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1200
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1260
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1320
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1380
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1440
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1500
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1560
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 1620
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 1680
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 1740
agtgcaaagc tggagctggc cttgtctgaa ttggagcgac ctcatgctat acctgagaaa 1800
gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt aaaacctaag 1860
agtcacttta aaatttgtat acacttattt tttttataac ttatttaata ataaaaatca 1920
taaatcataa gaaattcgct tatttagaag tgtcaacaac gtatctacca acgatttgac 1980
ccttttccat cttttcgtaa atttctggca aggtagacaa gccgacaacc ttgattggag 2040
acttgaccaa acctctggcg aagaattgtt aattaagagc tcagatctta tcgtcgtcat 2100
ccttgtaatc catcgatact agtctgaagc agatatctag agctacacaa agttcttgtg 2160
gctggcttga aaaatcttat agattgacgt agtgaaagca tgcggccgcc ccatagtgag 2220
gcatgaattc gaattttcaa catttcttac ttttttcatg gatggacgca aagaagttta 2280
ataatcatat tacatggcat taccaccata tacatatcca tatacatatc catatctaat 2340
cttacttata tgttgtggaa atgtaaagag ccccattatc ttagcctaaa acatccttct 2400
ctttggaact ttcagtaata cgcttaactg ctcattgcta tattgaagta cggattagaa 2460
gccgccgagc gggtgacagc cctccgaagg aagactctcc tccgtgcgtc ctcgtcttca 2520
ccggtcgcgt tcctgaaacg cagcattgcc tcgcgccgca ctgctccgaa caataaagat 2580
tctacaatac tagcatgtat ggttatgaag aggaaaaatt ggcagtaacc tggccccaca 2640
aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat tagatgttta 2700
gccttatttc tggggtaatt aatcagcgaa gcgatgaatg ttgatctatt aacagatata 2760
taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg gtttgtatta 2820
cttcttattc aaatgtaata aaagtatcaa catgaaattg ttaatatacc tctatacttt 2880
aacgtcaagg agatgaaacc ccggatccat gctttcacta cgtcaatcta taagattttt 2940
caagccagcc acaagaactt tgtgtagctc tagatatctg cttcaggtcg acatggaaca 3000
gaagttgatt tccgaagaag acctcgagta agcttggtac cgcggctagc taagatccgc 3060
tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 3120
agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 3180
acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 3240
gaaggcgctc gacaaccctt aatataaggc cgcgttgctg gcgtt 3285
<210> 3
<211> 6125
<212> DNA
<213> Synthesis
<400> 3
atagtggctg ctcatacaca acgaaaaaaa ttgttaacaa aatagaagga ttcgtctatg 60
cccttttttc agtgcggaaa ttatatacca cacaagccgt cgaagcccat agggtccttt 120
aacaataaag actctcctaa aaaaattttt cttgtgacac gtcagtgcag gatataaatg 180
tagcaagcgt atataacaaa tgacaaaggg ttcttgcgca aggttacttt agcgttgctg 240
gataatgatc ggattgtttt tagatcacga tagcacattt gaggaatcgt atcgaaacgg 300
acagggcaag agtacgaggg cgaaatgaac ttataacacc attgatattt tctcctcaat 360
cttatcattc cacgcacatt attttcctac tatgttatgc gggaacagtt cctaaggaat 420
ttttctaatg gcatttcggc agcgtcatca aatgtacgca cccacttcaa gtgatcattc 480
gctcgttact acctatcacg tgtcgcatca ctcgcgacgt ccatgcgcat gctgtgccgc 540
aggagaattc gagatgcgaa tgagcagcag ccattttgat gttgtgagca tcggaacgtt 600
tctgcgtccg tacactgtcc ttttgttact tagataatgg ctaaggcaag cagtccgggc 660
cacaggagtc aaaggctttt cgccagctcc taaacgctgg aagtgtaatt ttttttcgtc 720
ttataaaatt aaacaaaccc ttttagaaag gaacactcgc tttatctctt cgaccgaatt 780
tactatacat ggatatatat atattatctt ctgttcacag ttaaaactag gaatagtata 840
gtcataagtt aacaccatcc tgaattggag cgacctcatg ctatacctga gaaagcaacc 900
tgacctacag gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac 960
tttaaaattt gtatacactt atttttttta taacttattt aataataaaa atcataaatc 1020
ataagaaatt cgcttattta gaagtgtcaa caacgtatct accaacgatt tgaccctttt 1080
ccatcttttc gtaaatttct ggcaaggtag acaagccgac aaccttgatt ggagacttga 1140
ccaaacctct ggcgaagaat tgttaattaa gagctcagat cttatcgtcg tcatccttgt 1200
aatccatcga tactagttta tgaagtccat ggtaaattcg tgtttcctgg caataataga 1260
tcgtcaattt gttgctttgt ggtagtttta ttttcaaata attggaatac tagggatttg 1320
attttaagat ctttattcaa attttttgcg cttaacaaac agcagccagt cccacccaag 1380
tctgtttcaa atgtctcgta actaaaatca tcttgcaatt tctttttgaa actgtcaatt 1440
tgctcttgag taatgtctct tcgtaacaaa gtcaaagagc aaccgccgcc accagcaccg 1500
gtaagttttg tggagccaat tctcaaatca tcgctcagat ttttaataag ttctaatcca 1560
ggatgagaaa caccgattga gacaagcagt ccatgattta ttcttatcaa ttccaatagt 1620
tgttcataca gttcattatt agtttctaca gcctcgtcat cggtgccttt acatttactt 1680
aacttagtca tgatctctaa gccttgtagg gcacattcac ccatggcatc tagaattggc 1740
ttcataactt caggaaattt ctcggtgacc aacacacgaa cgcgagcaac aagatctttt 1800
gtagaccttg gaattctagt ataggttagg atcattggaa tggctgggaa atcatctaag 1860
aacttactgt tgtttgtgtt tattgttcca ttatgtgagt ctttttcaaa tagcagggca 1920
ttaccataag tggccacagc gttatctatt cctgaagggg taccgtgaat acacttttca 1980
cctatgaagg cccattgatt cactatatgc ttatcgtttt ctgacagctt ttccaagtca 2040
ttagatccta ttaacccccc caagtaggcc atagctaagg ccagtgatac agaaatagag 2100
gcgcttgagc ccaacccagc accgatgggt aaagtagact ttaaagaaaa cttaatattc 2160
ttggcatggg ggcataggca aacaaacata tacaggaaac aaaacgctgc atggtagtgg 2220
aaggattcgg atagttgagc taacaacgga tccaaaagac taacgagttc ctgagacaag 2280
ccatcggtgg cttgttgagc cttggccaat ttttgggagt ttacttgatc ctcggtgatg 2340
gcattgaaat cattgatgga ccacttatga ttaaagctaa tgtccgggaa gtccaattca 2400
atagtatctg gtgcagatga ctcgcttatt agcaggtagg ttctcaacgc agacacacta 2460
gcagcgacgg caggcttgtt gtacacagca gagtgttcac caaaaataat aacctttccc 2520
ggtgcagaag ttaagaacgg taatgatttc tgaagcagat atctagagct acacaaagtt 2580
cttgtggctg gcttgaaaaa tcttatagat tgacgtagtg aaagcatgcg gccgccccat 2640
agtgaggcat gaattcgaat tttcaacatt tcttactttt ttcatggatg gacgcaaaga 2700
agtttaataa tcatattaca tggcattacc accatataca tatccatata catatccata 2760
tctaatctta cttatatgtt gtggaaatgt aaagagcccc attatcttag cctaaaacat 2820
ccttctcttt ggaactttca gtaatacgct taactgctca ttgctatatt gaagtacgga 2880
ttagaagccg ccgagcgggt gacagccctc cgaaggaaga ctctcctccg tgcgtcctcg 2940
tcttcaccgg tcgcgttcct gaaacgcagc attgcctcgc gccgcactgc tccgaacaat 3000
aaagattcta caatactagc atgtatggtt atgaagagga aaaattggca gtaacctggc 3060
cccacaaacc ttcaaatgaa cgaatcaaat taacaaccat aggatgataa tgcgattaga 3120
tgtttagcct tatttctggg gtaattaatc agcgaagcga tgaatgttga tctattaaca 3180
gatatataaa tgcaaaaact gcataaccac tttaactaat actttcaaca ttttcggttt 3240
gtattacttc ttattcaaat gtaataaaag tatcaacatg aaattgttaa tatacctcta 3300
tactttaacg tcaaggagat gaaaccccgg atccatgctt tcactacgtc aatctataag 3360
atttttcaag ccagccacaa gaactttgtg tagctctaga tatctgcttc agtttaaact 3420
ctcaactaaa ctttgttggt gtggtattaa aggaagactt aggccgcaaa agcaacaaca 3480
attacacaat acaaacttgc aaatgactga actaaaaaaa caaaagaccg ctgaacaaaa 3540
aaccagacct caaaatgtcg gtattaaagg tatccaaatt tacatcccaa ctcaatgtgt 3600
caaccaatct gagctagaga aatttgatgg cgtttctcaa ggtaaataca caattggtct 3660
gggccaaacc aacatgtctt ttgtcaatga cagagaagat atctactcga tgtccctaac 3720
tgttttgtct aagttgatca agagttacaa catcgacacc aacaaaattg gtagattaga 3780
agtcggtact gaaactctga ttgacaagtc caagtctgtc aagtctgtct tgatgcaatt 3840
gtttggtgaa aacactgacg tcgaaggtat tgacacgctt aatgcctgtt acggtggtac 3900
caacgcgttg ttcaactctt tgaactggat tgaatctaac gcatgggatg gtagagacgc 3960
cattgtagtt tgcggtgata ttgccatcta cgataagggt gccgcaagac caaccggtgg 4020
tgccggtact gttgctatgt ggatcggtcc tgatgctcca attgtatttg actctgtaag 4080
agcttcttac atggaacacg cctacgaatg ttacaagcca gatttcacca gcgaatatcc 4140
ttacgtcgat ggtcattttt cattaacttg ttacgtcaag gctcttgatc aagtttacaa 4200
gagttattcc aagaaggcta tttctaaagg gttggttagc gatcccgctg gttcggatgc 4260
tttgaacgtt ttgaaatatt tcgactacaa cgttttccat gttccaacct gtaaattggt 4320
cacaaaatca tacggtagat tactatataa cgatttcaga gccaatcctc aattgttccc 4380
agaagttgac gccgaattag ctactcgcga ttatgacgaa tctttaaccg ataagaacat 4440
tgatgaaact tttgttaatg ttgctaagcc attccacaaa gagagagttg cccaatcttt 4500
gattgttcca acaaacacag gtaacatgta caccgcatct gtttatgccg cctttgcatc 4560
tctattaaac tatgttggat ctgacgactt acaaggcaag cgtgttggtt tattttctta 4620
cggttccggt ttagctgcat ctctatattc ttgcaaaatt gttggtgacg tccaacatat 4680
tatcaaggaa ttagatatta ctaacaaatt agccaagaga atcaccgaaa ctccaaagga 4740
ttacgaagct gccatcgaat tgagagaaaa tgcccatttg aagaagaact tcaaacctca 4800
aggttccatt gagcatttgc aaagtggtgt ttactacttg accaacatcg atgacaaatt 4860
tagaagatct tacgatgtta aaaaataagt cgacatggaa cagaagttga tttccgaaga 4920
agacctcgag taagcttggt accgcggcta gctaagatcc gctctaaccg aaaaggaagg 4980
agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 5040
aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 5100
cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagtagc gtgttacgca 5160
cccaaacttt ttatgaaagt ctttgtttat aatgatgagg tttataaata tatagtggag 5220
caaagattaa tcactaaatc aagaagcagt accagtattt tttctatatc aagtagtgat 5280
aatggaaata gcccaaattt ggcttccgtc gacacataga acgtttgaga gacattatca 5340
ccatcaagca tcgagccgcc caaacctaac cgtataagtt ttttcacgtt tttgattttt 5400
ccttgcacac ttcgatatta ctctcacgat aaaagggccg aagagaatat ttttcttgaa 5460
catccagaat tttaattcgg agaaatttca caagccgcca atttaagggt cctgtgttct 5520
taataatcag cctctctcaa agcaggtaag aggcagtctt tcttttaaca atgagagaca 5580
ttcgaactaa aacatcagcc ccaaaaatgc gcttgaaggt cattagaatt tggatttctt 5640
cctcattttt aaaggcgccc cttttcgttt tagtcatgct gggctacact agcacgtagt 5700
gtatggaatc aaactgatgc cgtcccctga attatattta caattcagaa acgtctttga 5760
acatttataa ttaaattata aaacaaaagg aatttgtaaa aaagactttc aaaagatgat 5820
ccaatcaact agattaaaaa cccaatataa gaagaagaat gtgtggcatt tttggctatt 5880
gcaatttttt aattgggaag acaagaggag aaatcatcga cactttaatc gaagggttac 5940
aggcattgga gtacaaggaa tatgactcca gtggaatttc gatccaaggt gatgagctga 6000
agtctctcaa tatttataag caaaccggta aaataagcag tttgaaggaa gaaatcgacc 6060
tttacaatct taataagaac ctgcccttta tttcgcattg tggcattgcc catactaggc 6120
gggca 6125
<210> 4
<211> 4707
<212> DNA
<213> Synthesis
<400> 4
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 780
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 840
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 900
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 960
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1020
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1080
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1140
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1200
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1260
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1320
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1380
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1440
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1500
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1560
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 1620
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 1680
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 1740
agtgcaaagc tggagctggc cttgtctgaa ttggagcgac ctcatgctat acctgagaaa 1800
gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt aaaacctaag 1860
agtcacttta aaatttgtat acacttattt tttttataac ttatttaata ataaaaatca 1920
taaatcataa gaaattcgct tatttagaag tgtcaacaac gtatctacca acgatttgac 1980
ccttttccat cttttcgtaa atttctggca aggtagacaa gccgacaacc ttgattggag 2040
acttgaccaa acctctggcg aagaattgtt aattaagagc tcagatctta tcgtcgtcat 2100
ccttgtaatc catcgatact agtgcggccg ccctttagtg agggttgaat tcgaattttc 2160
aacatttctt acttttttca tggatggacg caaagaagtt taataatcat attacatggc 2220
attaccacca tatacatatc catatacata tccatatcta atcttactta tatgttgtgg 2280
aaatgtaaag agccccatta tcttagccta aaacatcctt ctctttggaa ctttcagtaa 2340
tacgcttaac tgctcattgc tatattgaag tacggattag aagccgccga gcgggtgaca 2400
gccctccgaa ggaagactct cctccgtgcg tcctcgtctt caccggtcgc gttcctgaaa 2460
cgcagcattg cctcgcgccg cactgctccg aacaataaag attctacaat actagcatgt 2520
atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca aatgaacgaa 2580
tcaaattaac aaccatagga tgataatgcg attagatgtt tagccttatt tctggggtaa 2640
ttaatcagcg aagcgatgaa tgttgatcta ttaacagata tataaatgca aaaactgcat 2700
aaccacttta actaatactt tcaacatttt cggtttgtat tacttcttat tcaaatgtaa 2760
taaaagtatc aacatgaaat tgttaatata cctctatact ttaacgtcaa ggagatgaaa 2820
ccccggatcc atggaccaat tggtgaagac tgaagtcacc aagaagtcat gtactgctcc 2880
tgtacaaaag gcttctacac cagttttaac caataaaaca gtcatttctg gatcgaaagt 2940
caaaagttta tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga 3000
tgattcccgc gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc 3060
attattaagt agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat 3120
tcacggtaag ttacctttgt acgctttgga gaaaatgtta ggtgatacta cgagagcggt 3180
tgcggtacgt aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg 3240
tttaccatat aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat 3300
aggttacatg cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta 3360
tcatatacca atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa 3420
ggcaatcaat gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg 3480
cccagtagtc cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc 3540
agaagaggga caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct 3600
gcaacatatt caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac 3660
tggtgacgca atgggtatga atatgatttc taagggtgtc gaatactcat taaagcaaat 3720
ggtagaagag tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac 3780
cgacaaaaaa ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga 3840
agctactatt cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt 3900
tgagttgaac attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt 3960
taacgcacat gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc 4020
acatgatgtc gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag 4080
aatttccgta tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga 4140
accacaaggt gccatgttgg acttattagg tgtaagaggc ccacatgcta ccgctcctgg 4200
taccaacgca cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc 4260
cttatgtgct gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa 4320
acctgctgaa ccaaccatac ctaacaattt ggacgccact gatataaatc gtttgaaaga 4380
tgggtccgtc acctgcatta aatcctaagt cgacatggaa cagaagttga tttccgaaga 4440
agacctcgag taagcttggt accgcggcta gctaagatcc gctctaaccg aaaaggaagg 4500
agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 4560
aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 4620
cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggcgc tcgacaaccc 4680
ttaatataag gccgcgttgc tggcgtt 4707
<210> 5
<211> 6459
<212> DNA
<213> Synthesis
<400> 5
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 780
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 840
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 900
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 960
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1020
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1080
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1140
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1200
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1260
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1320
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1380
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1440
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1500
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1560
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 1620
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 1680
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 1740
agtgcaaagc tggagctggc cttgttaacg aacgcagaat tttcgagtta ttaaacttaa 1800
aatacgctga acccgaacat agaaatatcg aatgggaaaa aaaaactgca taaaggcatt 1860
aaaagaggag cgaatttttt tttaataaaa atcttaataa tcattaaaag ataaataata 1920
gtctatatat acgtatataa ataaaaaata ttcaaaaaat aaaataaact attattttag 1980
cgtaaaggat ggggaaagag aaaagaaaaa aattgatcta tcgatttcaa ttcaattcaa 2040
tgttaattaa gagctcagat cttatcgtcg tcatccttgt aatccatcga tactagttca 2100
tatcttttca atgacaatag aggaagcacc accaccacca ttacaaatgg cggcaacacc 2160
gatcttacct ccttcttgct gtaagatgga tagcagtgta acaaccactc tagcaccaga 2220
acaacccaat gggtgaccta gagcaacagc accaccatat acattaacct tagatgggtc 2280
tagcttcaaa atcttagtgt tcaccaaacc gacaaccgaa aaggcttcat tgaattcaaa 2340
gtaatcaaca gaattgatgt cttcgatgcc agcatgtttc aaagcctttg gaactgcaag 2400
agatggagcc catgtaaaat cagctggttg atgagcggcc tcaccccaac ctttgataat 2460
agccaaaggc ttcaaattcc attccttcaa aactttttcg gaaaccaaga tgacggctgc 2520
agcaccatcg ttgattggag aagcgttagc ggcagtaaca gtaccgtttt ctttttggaa 2580
aacagtcctt gcagatctca acatttcaac gtgtaatcta gcaggttcct cgtccttcgt 2640
gacttgagta tcaggcttac ctctaaatcc cttaatggta acaggtacaa tttcattgtc 2700
gaatttacct tccttttgag atttttgaga catttggtag gattcgatgg caaaattgtc 2760
ttgttgttct ctagtaatat cccaatcacg ggcacacttt tctgcgtgta cacccatggc 2820
tagaccatcg tacgcatcgt tcaacccatc tctttcgaca ccatcaacaa gaacagtttg 2880
gccaaatttg gcacccgcac gggctgctgg catgtagtat ggtgcgttag tcatagattc 2940
acaaccacca gctacgacaa catcagcatt accacatttg atggattgag cacccaaaat 3000
gattgccttc atagcggatg cacagacctt gttaactgtg cttgcaacga tatgattact 3060
caaaccggca gccaaagcaa cttgtctggc cggagcttgg cccaaattgg cagaaagaac 3120
gttacccata ataatttcgt caaaatcctt ggatgcatcc aattctggaa ccttagccaa 3180
ggcgcccatt aaagcaacag cacccaattc cactgctgtc ttggaggata gagaaccctg 3240
gaatgaacca attggggttc tggcagtcga tacaatgtaa acgttctgag acatgcggcc 3300
gccctttagt gagggttgaa ttcgaatttt caacatttct tacttttttc atggatggac 3360
gcaaagaagt ttaataatca tattacatgg cattaccacc atatacatat ccatatacat 3420
atccatatct aatcttactt atatgttgtg gaaatgtaaa gagccccatt atcttagcct 3480
aaaacatcct tctctttgga actttcagta atacgcttaa ctgctcattg ctatattgaa 3540
gtacggatta gaagccgccg agcgggtgac agccctccga aggaagactc tcctccgtgc 3600
gtcctcgtct tcaccggtcg cgttcctgaa acgcagcatt gcctcgcgcc gcactgctcc 3660
gaacaataaa gattctacaa tactagcatg tatggttatg aagaggaaaa attggcagta 3720
acctggcccc acaaaccttc aaatgaacga atcaaattaa caaccatagg atgataatgc 3780
gattagatgt ttagccttat ttctggggta attaatcagc gaagcgatga atgttgatct 3840
attaacagat atataaatgc aaaaactgca taaccacttt aactaatact ttcaacattt 3900
tcggtttgta ttacttctta ttcaaatgta ataaaagtat caacatgaaa ttgttaatat 3960
acctctatac tttaacgtca aggagatgaa accccggatc catgacaatc aaggaacata 4020
aagtagttta tgaagctcac aacgtaaagg ctcttaaggc tcctcaacaa tgttacaaca 4080
gccaacccgg caagggttac gttactgata tgcaacatta tcaagaaatg tatcaacaat 4140
ctatcaatga gccagaaaaa ttctttgata agatggctaa ggaatacttg cattgggatg 4200
ctccatacac caaagttcaa tctggttcat tgaacaatgg tgatgttgca tggtttttga 4260
acggtaaatt gaatgcatca tacaattgtg ttgacagaca tgcctttgct aatcccgaca 4320
agccagcttt gatctatgaa gctgatgacg aatccgacaa caaaatcatc acatttggtg 4380
aattactcag aaaagtttcc caaatcgctg gtgtcttcat aagctggggc gttaagaaag 4440
gtgacacagt ggctatctat ttgccaatga ttccagaagc ggtcattgct atgttggctg 4500
tggctcgtat tggtgctatt cactctgttg tctttgctgg gttctccgct ggttcgttga 4560
aagatcgtgt cgttgacgct aattctaaag tggtcatcac ttgtgatgaa ggtaaaagag 4620
gtggtaagac catcaacact atgaaaattg ttgacgaagg tttgaacgga gtcgatttgg 4680
tttcccgtat cttggttttc caaagaactg gtactgaagg tattccaatg aaggccggta 4740
gagattactg gtggcatgag gaggccgcta agcagagaac ttacctacct cctgtttcat 4800
gtgacgctga agatcctcta tttttattat acacttccgg ttccactggt tctccaaagg 4860
gtgtcgttca cactacaggt ggttatttat taggtgccgc tttaacaact agatacgttt 4920
ttgatattca cccagaagat gttctcttca ctgccggtga cgtcggctgg atcacgggtc 4980
acacctatgc tctatatggt ccattaacct tgggtaccgc ctcaataatt ttcgaatcca 5040
ctcctgccta cccagattat ggtagatatt ggagaattat ccaacgtcac aaggctaccc 5100
atttctatgt ggctccaact gctttaagat taatcaaacg tgtaggtgaa gccgaaattg 5160
ccaaatatga cacttcctca ttacgtgtct tgggttccgt cggtgaacca atctctccag 5220
acttatggga atggtatcat gatgaagtgg gtaacaaaaa ctgtgtcatt tgtgacacta 5280
tgtggcaaac agagtctggt tctcatttaa ttgctccttt ggcaggtgct gtcccaacaa 5340
aacctggttc tgctaccgtg ccattctttg gtattaacgc ttgtatcatt gaccctgtta 5400
caggtgtgga attagaaggt aatgatgtcg aaggtgtcct tgccgttaaa tcaccatggc 5460
catcaatggc tagatctgtt tggaaccacc acgaccgtta catggatact tacttgaaac 5520
cttatcctgg tcactatttc acaggtgatg gtgctggtag agatcatgat ggttactact 5580
ggatcagggg tagagttgac gacgttgtaa atgtttccgg tcatagatta tccacatcag 5640
aaattgaagc atctatctca aatcacgaaa acgtctcgga agctgctgtt gtcggtattc 5700
cagatgaatt gaccggtcaa accgtcgttg catatgtttc cctaaaagat ggttatctac 5760
aaaacaacgc tactgaaggt gatgcagaac acatcacacc agataattta cgtagagaat 5820
tgatcttaca agttaggggt gagattggtc ctttcgcctc acccataacc attattctag 5880
ttagagatct accaagaaca aggtcaggaa agattatgag aagagttcta agaaaggttg 5940
cttctaacga agccgaacag ctaggtgacc taactacttt ggccaaccca gaagttgtac 6000
ctgccatcat ttctgctgta gagaaccaat ttttctctca aaaaaagaaa taactcgagt 6060
aagcttggta ccgcggctag ctaagtgaac ccgatgcaaa tgagacgatc gtctattcct 6120
ggtccggttt tctctgccct ctcttctatt cacttttttt atactttata taaaattata 6180
taaatgacat aactgaaacg ccacacgtcc tctcctattc gttaacgcct gtctgtagcg 6240
ctgttactga agctgcgcaa gtagtttttt caccgtatag gccctctttt tctctctctt 6300
tctttctctc ccgcgctgat ctcttcttcg aaacatcatg aataaaaaga aaaaggaaat 6360
caagaaaaaa aagccataat ttatcccaca ttttttttta ttgtcgctgt tcacaccggc 6420
gctcgacaac ccttaatata aggccgcgtt gctggcgtt 6459
<210> 6
<211> 6549
<212> DNA
<213> Synthesis
<400> 6
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 600
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 660
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 720
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 780
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 840
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 900
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 960
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1020
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1080
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1140
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1200
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1260
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1320
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1380
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1440
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1500
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1560
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 1620
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 1680
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 1740
agtgcaaagc tggagctggc cttgttaacg aacgcagaat tttcgagtta ttaaacttaa 1800
aatacgctga acccgaacat agaaatatcg aatgggaaaa aaaaactgca taaaggcatt 1860
aaaagaggag cgaatttttt tttaataaaa atcttaataa tcattaaaag ataaataata 1920
gtctatatat acgtatataa ataaaaaata ttcaaaaaat aaaataaact attattttag 1980
cgtaaaggat ggggaaagag aaaagaaaaa aattgatcta tcgatttcaa ttcaattcaa 2040
tgttaattaa gagctcagat cttatcgtcg tcatccttgt aatccatcga tactagttca 2100
tatcttttca atgacaatag aggaagcacc accaccacca ttacaaatgg cggcaacacc 2160
gatcttacct ccttcttgct gtaagatgga tagcagtgta acaaccactc tagcaccaga 2220
acaacccaat gggtgaccta gagcaacagc accaccatat acattaacct tagatgggtc 2280
tagcttcaaa atcttagtgt tcaccaaacc gacaaccgaa aaggcttcat tgaattcaaa 2340
gtaatcaaca gaattgatgt cttcgatgcc agcatgtttc aaagcctttg gaactgcaag 2400
agatggagcc catgtaaaat cagctggttg atgagcggcc tcaccccaac ctttgataat 2460
agccaaaggc ttcaaattcc attccttcaa aactttttcg gaaaccaaga tgacggctgc 2520
agcaccatcg ttgattggag aagcgttagc ggcagtaaca gtaccgtttt ctttttggaa 2580
aacagtcctt gcagatctca acatttcaac gtgtaatcta gcaggttcct cgtccttcgt 2640
gacttgagta tcaggcttac ctctaaatcc cttaatggta acaggtacaa tttcattgtc 2700
gaatttacct tccttttgag atttttgaga catttggtag gattcgatgg caaaattgtc 2760
ttgttgttct ctagtaatat cccaatcacg ggcacacttt tctgcgtgta cacccatggc 2820
tagaccatcg tacgcatcgt tcaacccatc tctttcgaca ccatcaacaa gaacagtttg 2880
gccaaatttg gcacccgcac gggctgctgg catgtagtat ggtgcgttag tcatagattc 2940
acaaccacca gctacgacaa catcagcatt accacatttg atggattgag cacccaaaat 3000
gattgccttc atagcggatg cacagacctt gttaactgtg cttgcaacga tatgattact 3060
caaaccggca gccaaagcaa cttgtctggc cggagcttgg cccaaattgg cagaaagaac 3120
gttacccata ataatttcgt caaaatcctt ggatgcatcc aattctggaa ccttagccaa 3180
ggcgcccatt aaagcaacag cacccaattc cactgctgtc ttggaggata gagaaccctg 3240
gaatgaacca attggggttc tggcagtcga tacaatgtaa acgttctgag acatgcggcc 3300
gccctttagt gagggttgaa ttcgaatttt caacatttct tacttttttc atggatggac 3360
gcaaagaagt ttaataatca tattacatgg cattaccacc atatacatat ccatatacat 3420
atccatatct aatcttactt atatgttgtg gaaatgtaaa gagccccatt atcttagcct 3480
aaaacatcct tctctttgga actttcagta atacgcttaa ctgctcattg ctatattgaa 3540
gtacggatta gaagccgccg agcgggtgac agccctccga aggaagactc tcctccgtgc 3600
gtcctcgtct tcaccggtcg cgttcctgaa acgcagcatt gcctcgcgcc gcactgctcc 3660
gaacaataaa gattctacaa tactagcatg tatggttatg aagaggaaaa attggcagta 3720
acctggcccc acaaaccttc aaatgaacga atcaaattaa caaccatagg atgataatgc 3780
gattagatgt ttagccttat ttctggggta attaatcagc gaagcgatga atgttgatct 3840
attaacagat atataaatgc aaaaactgca taaccacttt aactaatact ttcaacattt 3900
tcggtttgta ttacttctta ttcaaatgta ataaaagtat caacatgaaa ttgttaatat 3960
acctctatac tttaacgtca aggagatgaa accccggatc catgtcgccc tctgccgtac 4020
aatcatcaaa actagaagaa cagtcaagtg aaattgacaa gttgaaagcc ataatgtccc 4080
agtctgcctc cactgcgcag cagaagaagg aacatgagta tgaacatttg acctcggtca 4140
agatcgtgcc acaacggccc atctcagata gactgcagcc cgcaattgct acccactatt 4200
ctccacactt ggacgggttg caggactatc agcgcttgca caaggagtct attgaagacc 4260
ctgctaagtt cttcggttct aaagctaccc aatttttaaa ctggtctaag ccattcgata 4320
aggtgttcat cccagactct aaaacgggta ggccctcctt ccagaacaat gcatggttcc 4380
tcaacggcca attaaacgcc tgttacaact gtgttgacag acatgccttg aagaccccta 4440
acaagaaagc cattattttc gaaggtgacg agcctggcca aggctattcc attacctaca 4500
aggaactact tgaagaagtt tgtcaagtgg cacaagtgct gacttactct atgggcgttc 4560
gcaagggcga tactgttgcc gtgtacatgc ctatggtccc agaagcaatc ataaccttgt 4620
tggccatttc ccgtatcggc gccattcact ccgtagtctt tgccgggttt tcttccaact 4680
ccttgagaga tcgtatcaac gatggggact ctaaagttgt catcactaca gatgaatcca 4740
acagaggtgg taaagtcatt gagactaaaa gaattgttga tgacgcgcta agagagaccc 4800
caggcgtgag acacgtcttg gtttatagaa agaccaacaa tccatctgtt gctttccatg 4860
cccccagaga tttagattgg gcaacagaaa agaagaaata caagacctac tatccatgca 4920
cacccgttga ttctgaggat ccattattct tgttgtatac gtctggttct actggtgccc 4980
ccaagggtgt tcaacattct accgcaggtt acttgctggg agctttgttg accatgcgct 5040
acacttttga cactcaccaa gaagacgatg tcttcacagc tggagacatt ggctggatta 5100
caggccacac ttatgtggtt tatggtccct tactatatgg ttgtgccact ttggtctttg 5160
aagggactcc tgcgtaccca aattactccc gttattggga tattattgat gaacacaaag 5220
tcacccaatt ttatgttgcc ccaactgctt tgcgtttgtt gaaaagagct ggtgattcct 5280
acatcgaaaa tcattcctta aaatctttgc gttgcttggg ttcggtcggt gaaccaattg 5340
ctgctgaagt ttgggagtgg tactctgatg aaataggtaa aaatgaaatc cccattgtag 5400
acacctactg gcaaacagaa tctggttcgc atctggtcac cccgctggct ggtggtgtca 5460
caccaatgaa accgggttct gcctcattcc ccttcttcgg tattgatgca gttgttcttg 5520
accctaacac tggtgaagaa cttaatacca gccacgcaga gggtgtcctt gccgtcaaag 5580
ctgcatggcc atcatttgca agaactattt ggatgaatca tgataggtat ctagacactt 5640
atttgaaccc ttaccctggc tactatttca ctggtgatgg tgctgcaaag gataaggatg 5700
gttatatctg gattttgggt cgtgtagacg atgtggtgaa cgtctctggt caccgtctgt 5760
ctaccgctga aattgaggct gctattatcg aagatccaat tgtggccgag tgtgctgttg 5820
tcggattcaa cgatgacttg actggtcaag cagttgctgc atttgtggtg ttgatgaaca 5880
aatctaattg gtccaccgca acagatgatg aattacaaga tatcaagaag catttggtct 5940
ttactgttag aaaagacatc gggccatttg ccgcaccaaa attgatcatt ttagtggatg 6000
acttgcccaa gacaagatct ggcaaaatta tgagacgtat tttaagaaaa atcctagcag 6060
gagaaagtga ccaactaggc gacgtttcta cattgtcaaa ccctggcatt gttagacatc 6120
taattgattc ggtcaagttg taactcgagt aagcttggta ccgcggctag ctaagtgaac 6180
ccgatgcaaa tgagacgatc gtctattcct ggtccggttt tctctgccct ctcttctatt 6240
cacttttttt atactttata taaaattata taaatgacat aactgaaacg ccacacgtcc 6300
tctcctattc gttaacgcct gtctgtagcg ctgttactga agctgcgcaa gtagtttttt 6360
caccgtatag gccctctttt tctctctctt tctttctctc ccgcgctgat ctcttcttcg 6420
aaacatcatg aataaaaaga aaaaggaaat caagaaaaaa aagccataat ttatcccaca 6480
ttttttttta ttgtcgctgt tcacaccggc gctcgacaac ccttaatata aggccgcgtt 6540
gctggcgtt 6549
<210> 7
<211> 4406
<212> DNA
<213> Synthesis
<400> 7
atggaagata tagaaggata cgaaccacat atcactcaag agttgataga ccgtggccat 60
gcaagacgta tgggccactt ggaaaactac tttgctgttt tgagtaggca gaaaatgtac 120
tcgaatttta ctgtttacgc ggaattgaat aaaggtgtta ataagagaca actaatgctt 180
gtcttgaaag tattacttca aaaatactca actcttgcgc atacaatcat tcctaagcat 240
tatcctcatc atgaagcgta ctactctagc gaagagtacc ttagtaaacc ttttccacag 300
catgatttca taaaggtgat ttctcatctt gaattcgatg acttgattat gaataatcaa 360
ccagaataca gagaagtcat ggagaaaatc tcagaacagt tcaaaaagga tgatttcaaa 420
gtcaccaata ggttaatcga attgattagc cctgtaatca tacctctggg taatccgaag 480
aggcctaatt ggagattgat ttgtttacca ggtaaggata ctgatgggtt tgaaacgtgg 540
aaaaacttcg tttatgtcac taaccactgc ggctccgacg gtgtcagtgg atcgaatttt 600
ttcaaagatt tagctctact cttttgtaaa atcgaagaaa aagggtttga ttatgatgaa 660
gagttcatcg aagatcaagt catcattgac tatgatcgag actacactga aatttctaaa 720
ttgccaaaac cgattacgga tcgtattgac tacaagccag cattgacttc attacccaaa 780
ttctttttaa caaccttcat ttatgaacat tgtaatttta aaacctccag cgaatctaca 840
cttacagcta gatatagccc ctctagtaat gctaatgcta gttacaatta cttgttgcat 900
ttcagtacta agcaagtaga acaaatcaga gctcagatca agaaaaatgt tcacgatggg 960
tgcaccctaa cacccttcat tcaagcgtgc tttcttgtag ccctgtatag actggataag 1020
ctgttcacaa aatctcttct cgagtatggg ttcgatgtgg ctattccaag caacgcaaga 1080
aggtttttac caaacgatga agagttaaga gattcttata aatacggctc caacgttgga 1140
ggttcgcatt acgcctatct aatctcctca ttcgacattc ccgaaggtga caatgacaag 1200
ttttggagtc ttgtcgaata ctactatgac cgctttttag aatcgtacga caacggtgac 1260
cacttgattg gtctgggggt cctacaactt gattttatcg ttgaaaacaa gaatatagac 1320
agccttcttg ccaactctta tttgcaccag caaagaggcg gtgcaatcat cagtaataca 1380
ggacttgtct cgcaagatac gaccaagccg tactacgttc gggatttaat cttctcgcag 1440
tctgcaggcg ccttgagatt tgcgttcggc ctaaacgttt gctccacaaa cgtgaatggt 1500
atgaacatgg acatgagcgt ggttcagggc actctacggg atcgtggcga atgggaatcg 1560
ttctgcaagc tcttctacca aaccatcggc gaatttgcgt cgctttaaat ccgctctaac 1620
cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttaatgtt ttatagttat 1680
gttagtatta agaacgttat ttatatttca aatttttcta tgttttctgt acagacgcgt 1740
gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaagtc 1800
tcatctggaa tataattccc ccctcctgaa gcaaaatgtt cctttgagcc ggaaatgttg 1860
atattccgag ttcatgtttt ccattcgcgg aggttattcc attcctaaac gagtggccac 1920
aatgaaactt caattcatat cgaccgacta tttttctccg aaccaaaaaa atagcagggc 1980
gagattggag ctgcggatga aagaggaaaa aattttttcg tagttttctt gtgcaaatta 2040
gggtgtaagg tttctagggc ttattggttc aagcagaaga gacaacaatt gtaggtccta 2100
aattcaaggc ggatgtaagg agtattggtt tcgaaagttt ttccgaagcg gcatggcagg 2160
gactacttgc gcatgcgctc ggattatctt catttttgct tgcaaaaacg tagaatcatg 2220
gtaaattaca tgaagaattc tcatgttttt tttttttttt ttttttttac ctctaaagag 2280
tgttgaccaa ctgaaaaaac ccttcttcaa gagagttaaa ctaagactaa ccatcataac 2340
ttccaaggaa ttaatcgata tcttgcactc ctgatttttc ttcaaagaga cagcgcaaag 2400
gattatgaca ctgttgcatt gagtcaaaag tttttccgaa gtgacccagt gctcatgttt 2460
tttttccgtg aaggactgac aaatatgcgc acaagatcca atacgtaatg gaaattcgga 2520
aaaactagga agaaatgctg cagggcattg ccgtgccgat cttttgtctt tcagatatat 2580
gagatgaaga atattcatca agtgctgata gaagaatacc actcatatga cgtgggcaga 2640
agacagcaaa cgtaaacatg agctgctgcg acatttgatg gcttttatcc gacaagccag 2700
gaaactccac cattatctaa tgtagcaaaa tatttcttaa cacccgaagt tgcgtgtccc 2760
cctcacgttt ttaatcattt gaattagtat attgaaatta tatataaagg caacaatgtc 2820
cccataatca attccatctg gggtctcatg ttctttcccc accttaaaat ctataaagat 2880
atcataatcg tcaactagtt gatatacgta aaatcatgtc tgctgttaac gttgcacctg 2940
aattgattaa tgccgacaac acaattacct acgatgcgat tgtcatcggt gctggtgtta 3000
tcggtccatg tgttgctact ggtctagcaa gaaagggtaa gaaagttctt atcgtagaac 3060
gtgactgggc tatgcctgat agaattgttg gtgaattgat gcaaccaggt ggtgttagag 3120
cattgagaag tctgggtatg attcaatcta tcaacaacat cgaagcatat cctgttaccg 3180
gttataccgt ctttttcaac ggcgaacaag ttgatattcc atacccttac aaggccgata 3240
tccctaaagt tgaaaaattg aaggacttgg tcaaagatgg taatgacaag gtcttggaag 3300
acagcactat tcacatcaag gattacgaag atgatgaaag agaaaggggt gttgcttttg 3360
ttcatggtag attcttgaac aacttgagaa acattactgc tcaagagcca aatgttacta 3420
gagtgcaagg taactgtatt gagatattga aggatgaaaa gaatgaggtt gttggtgcca 3480
aggttgacat tgatggccgt ggcaaggtgg aattcaaagc ccacttgaca tttatctgtg 3540
acggtatctt ttcacgtttc agaaaggaat tgcacccaga ccatgttcca actgtcggtt 3600
cttcgtttgt cggtatgtct ttgttcaatg ctaagaatcc tgctcctatg cacggtcacg 3660
ttattcttgg tagtgatcat atgccaatct tggtttacca aatcagtcca gaagaaacaa 3720
gaatcctttg tgcttacaac tctccaaagg tcccagctga tatcaagagt tggatgatta 3780
aggatgtcca acctttcatt ccaaagagtc tacgtccttc atttgatgaa gccgtcagcc 3840
aaggtaaatt tagagctatg ccaaactcct acttgccagc tagacaaaac gacgtcactg 3900
gtatgtgtgt tatcggtgac gctctaaata tgagacatcc attgactggt ggtggtatga 3960
ctgtcggttt gcatgatgtt gtcttgttga ttaagaaaat aggtgaccta gacttcagcg 4020
accgtgaaaa ggttttggat gaattactag actaccattt cgaaagaaag agttacgatt 4080
ccgttattaa cgttttgtca gtggctttgt attctttgtt cgctgctgac agcgataact 4140
tgaaggcatt acaaaaaggt tgtttcaaat atttccaaag aggtggcgat tgtgtcaaca 4200
aacccgttga atttctgtct ggtgtcttgc caaagccttt gcaattgacc agggttttct 4260
tcgctgtcgc tttttacacc atttacttga acatggaaga acgtggtttc ttgggattac 4320
caatggcttt attggaaggt attatgattt tgatcacagc tattagagta ttcaccccat 4380
ttttgtttgg tgagttgatt ggttaa 4406
<210> 8
<211> 6955
<212> DNA
<213> Synthesis
<400> 8
ggtgagcgct aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca 60
taacacagtc ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact 120
ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct agcggatgac 180
tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta tataaagtaa 240
tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga aggcaaagat 300
gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc agattgcgat 360
ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa aagaggcaga 420
agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag gtatagggtt 480
tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa tcgttgagtg 540
cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga ttgctctcgg 600
tcaagcctga attggagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa 660
agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta 720
tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc 780
ttatttagaa gtgtcaacaa cgtatctacc aacgatttga cccttttcca tcttttcgta 840
aatttctggc aaggtagaca agccgacaac cttgattgga gacttgacca aacctctggc 900
gaagaatttt aattaagagc tcagatctta tcgtcgtcat ccttgtaatc catcgatact 960
agtttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg 1020
ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat 1080
gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc 1140
cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc 1200
acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc 1260
aacagtaccc ttagtatatt ctccagtagc tagggagccc ttgcatgaca attctgctaa 1320
catcaaaagg cctctaggtt cctttgttac ttcttccgcc gcctgcttca aaccgctaac 1380
aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta 1440
tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc 1500
gaagagtatg aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat 1560
ggaaaaatca gtcaagatat ccacatgtga tgttagtaaa caaattttgg gacctaatgc 1620
ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt 1680
ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc 1740
acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct 1800
gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacaccac atatgcgtat 1860
atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgctcgg agattaccga 1920
atcatgaaaa tttcaaagaa accggaatca tgaaaaagaa catgaaaaaa aaagatgaat 1980
tgaaaagctt tatggaccct gaaaccacag ccacattaac cttcggatcc cggcagagac 2040
caatcagtaa aaatcaacgg ttaacgacat tactatatat ataatatagg aagcatttaa 2100
tagaacagca tcgtaatata tgtgtacttt gcagttatga cgccagatgg cagtagtgga 2160
agatattctt tattgatgaa tagcttgtca ccttacgtac aatcttgatc cggagctttt 2220
catgttttgc cgattaagaa ttcggtcgat gaaagaaaag gagagggcca agagggaggg 2280
cattggtgac tattgagcac gtgagtatac gtgattaagc acacaaaggc agcttggagt 2340
atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag 2400
agcacagagg ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt 2460
gtgcccaata gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta 2520
aaagcatata tgaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa 2580
cctaaggagg atgatgtggc tctggtcaat gattacggca ttgatatcgt ccaactgcat 2640
ggagatgagt cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc 2700
gtatttccca tagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt 2760
attcccttgt ttgattcaga agcaggtggg acaggtgaac atgtggattg gaactcgatt 2820
tctgactggg ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga 2880
ctgacgccag aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta 2940
agcggaggtg tggagacaaa tggtgtcata gactctaaca aaatagcaaa tttcgtcaaa 3000
aatgctaaga aatagctcga gtaagcttgg taccgcggct agcatccgct ctaaccgaaa 3060
aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata gttatgttag 3120
tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg 3180
catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg aagtaacgaa 3240
cgcagaattt tcgagttatt aaacttaaaa tacgctgaac ccgaacatag aaatatcgaa 3300
tgggaaaaaa aaactgcata aaggcattaa aagaggagcg aatttttttt taataaaaat 3360
cttaataatc attaaaagat aaataatagt ctatatatac gtatataaat aaaaaatatt 3420
caaaaaataa aataaactat tattttagcg taaaggatgg ggaaagagaa aagaaaaaaa 3480
ttgatctatc gatttcaatt caattcaatg ttaattaaga gctcagatct tatcgtcgtc 3540
atccttgtaa tccatcgata ctagtgcggc cgccctttag tgagggttga attcctacat 3600
aagaacacct ttggtggagg gaacatcgtt ggtaccattg ggcgaggtgg cttctcttat 3660
ggcaaccgca agagccttga acgcactctc actacggtga tgatcattct tgcctcgcag 3720
acaatcaacg tggagggtaa ttctgctagc ctctgcaaag ctttcaagaa aatgcgggat 3780
catctcgcaa gagagatctc ctactttctc cctttgcaaa ccaagttcga caactgcgta 3840
cggcctgttc gaaagatcta ccaccgctct ggaaagtgcc tcatccaaag gcgcaaatcc 3900
tgatccaaac ccatttactc cacgcacggc ccctagggcc tctttatgag cttgaccgag 3960
agcaatcccg cagtcttcag tggtgtgatg gtcgtctatg tgtaagtcac caatgcactc 4020
aacgattagc gaccagccgg aatgcttggc cagagcatgt atcatatggt ccagaaaccc 4080
tatacctgtg tggacgttaa tcacttgcga ttgtgtggcc tgttctgcta ctgcttctgc 4140
ctcatgttct gggaagatcg agtgctctat cgctagggga ccacccttta aagagatcgc 4200
aatctgaatc ttggtttcat ttgtaatacg ctttactagg gctttctgct ctgtcatctt 4260
tgccttcgtt tatcttgcct gctcaatgtt tagtatattc ttcgaagaaa tcacattact 4320
ttatataatg tataattcat tatgtgataa tgccaatcgc taagcataaa aaagagtcat 4380
ccgctaggtg gcataaaaaa aatgaaaatc attaccgagg cataaaaaaa tatagagtgt 4440
actagggatc caactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt 4500
agcaaccatt aatgttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa 4560
tcaaattcga tgactggaaa atgtttgtta atttcagagg tcgcctgacg catatacctt 4620
tttcaactga aaaattggga gcataaggaa aggtgagagc gccggaaccg gcttttcata 4680
tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat 4740
tatttaagga cctattgatg tttccaatag gtggttagca atcgtcttac tttctaactt 4800
ttcttacctt ttacatttca gcaatatata tatatatatt tcaaggatat accattctaa 4860
tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag 4920
ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt 4980
tcgatgatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg 5040
aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta 5100
aatggggtac cggtagtgtt agacctgaac aaggtttact cataatccgt aaagaacttc 5160
aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc 5220
caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag 5280
gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac 5340
aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac 5400
atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat 5460
tatggagcat aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac 5520
atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta 5580
ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag 5640
gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat 5700
ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc 5760
ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag 5820
aaggtaaggc cattgaagat gcagttatga aggttttgga tgcaggtatc agaactggtg 5880
atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga 5940
aaatccttgc ttaactcgag taagcttggt accgcggcta gctaagtgaa cccgatgcaa 6000
atgagacgat cgtctattcc tggtccggtt ttctctgccc tctcttctat tcactttttt 6060
tatactttat ataaaattat ataaatgaca taactgaaac gccacacgtc ctctcctatt 6120
cgttaacgcc tgtctgtagc gctgttactg aagctgcgca agtagttttt tcaccgtata 6180
ggccctcttt ttctctctct ttctttctct cccgcgctga tctcttcttc gaaacatcat 6240
gaataaaaag aaaaaggaaa tcaagaaaaa aaagccataa tttatcccac attttttttt 6300
attgtcgctg ttcacaccgg tcagtaagta tgtatacgaa cagtatgata ctgaagatga 6360
caaggtaatg catcattcta tacgtgtcat tctgaacgag gcgcgctttc cttttttctt 6420
tttgcttttt cttttttttt ctcttgaact cgagaaaaaa aatataaaag agatggagga 6480
acgggaaaaa gttagttgtg gtgataggtg gcaagtggta ttccgtaaga acaacaagaa 6540
aagcatttca tattatggct gaactgagcg aacaagtgca aaatttaagc atcaacgaca 6600
acaacgagaa tggttatgtt cctcctcact taagaggaaa accaagaagt gccagaaata 6660
acagtagcaa ctacaataac aacaacggcg gctacaacgg tggccgtggc ggtggcagct 6720
tctttagcaa caaccgtcgt ggtggttacg gcaacggtgg tttcttcggt ggaaacaacg 6780
gtggcagcag atctaacggc cgttctggtg gtagatggat cgatggcaaa catgtcccag 6840
ctccaagaaa cgaaaaggcc gagatcgcca tatttggtgt ccccgaggat ccaaatttcc 6900
aatcttctgg tattaacttc gataactacg atgatattcc agtggacgcc tctgg 6955
<210> 9
<211> 47
<212> DNA
<213> Synthesis
<400> 9
cataaacaaa caaacccgga tccatgagta aaggagaaga acttttc 47
<210> 10
<211> 38
<212> DNA
<213> Synthesis
<400> 10
caacttctgt tccatttatt tgtatagttc atccatgc 38
<210> 11
<211> 45
<212> DNA
<213> Synthesis
<400> 11
cataaacaaa caaacccgga tccatgcttt cactacgtca atcta 45
<210> 12
<211> 39
<212> DNA
<213> Synthesis
<400> 12
cttctccttt actcatctga agcagatatc tagagctac 39
<210> 13
<211> 20
<212> DNA
<213> Synthesis
<400> 13
acatccttgt cgagccttgg 20
<210> 14
<211> 44
<212> DNA
<213> Synthesis
<400> 14
ctcgaaaatt ctgcgttcgt tatgtggctc tgtcgaagac ttcc 44
<210> 15
<211> 41
<212> DNA
<213> Synthesis
<400> 15
tattgtcgct gttcacaccg tctatcagca gcagcagaca t 41
<210> 16
<211> 22
<212> DNA
<213> Synthesis
<400> 16
gtcctcggta gatcaggtca gt 22
<210> 17
<211> 44
<212> DNA
<213> Synthesis
<400> 17
ggaagtcttc gacagagcca cataacgaac gcagaatttt cgag 44
<210> 18
<211> 41
<212> DNA
<213> Synthesis
<400> 18
atgtctgctg ctgctgatag acggtgtgaa cagcgacaat a 41
<210> 19
<211> 47
<212> DNA
<213> Synthesis
<400> 19
cttgtaatcc atcgatacta gttcatatct tttcaatgac aatagag 47
<210> 20
<211> 42
<212> DNA
<213> Synthesis
<400> 20
gctctagata tctgcttcag atgtctcaga acgtttacat tg 42
<210> 21
<211> 42
<212> DNA
<213> Synthesis
<400> 21
gctctagata tctgcttcag atggaccaat tggtgaagac tg 42
<210> 22
<211> 42
<212> DNA
<213> Synthesis
<400> 22
aacttctgtt ccatgtcgac ttaggattta atgcaggtga cg 42
<210> 23
<211> 45
<212> DNA
<213> Synthesis
<400> 23
cttgtaatcc atcgatacta gtttatgaag tccatggtaa attcg 45
<210> 24
<211> 43
<212> DNA
<213> artificial sequence
<400> 24
gctctagata tctgcttcag atgtcattac cgttcttaac ttc 43
<210> 25
<211> 45
<212> DNA
<213> Synthesis
<400> 25
gctctagata tctgcttcag atgaaactct caactaaact ttgtt 45
<210> 26
<211> 46
<212> DNA
<213> Synthesis
<400> 26
aacttctgtt ccatgtcgac ttatttttta acatcgtaag atcttc 46
<210> 27
<211> 40
<212> DNA
<213> Synthesis
<400> 27
gctctagata tctgcttcag atgtcagagt tgagagcctt 40
<210> 28
<211> 44
<212> DNA
<213> Synthesis
<400> 28
aacttctgtt ccatgtcgac ttatttatca agataagttt ccgg 44
<210> 29
<211> 45
<212> DNA
<213> Synthesis
<400> 29
cttgtaatcc atcgatacta gtttattcct ttggtagacc agtct 45
<210> 30
<211> 40
<212> DNA
<213> Synthesis
<400> 30
gctctagata tctgcttcag atgaccgttt acacagcatc 40
<210> 31
<211> 47
<212> DNA
<213> Synthesis
<400> 31
cttgtaatcc atcgatacta gtttatagca ttctatgaat ttgcctg 47
<210> 32
<211> 42
<212> DNA
<213> Synthesis
<400> 32
gctctagata tctgcttcag atgactgccg acaacaatag ta 42
<210> 33
<211> 42
<212> DNA
<213> Synthesis
<400> 33
agaaaaaacc ccggatccat gggaaagcta ttacaattgg ca 42
<210> 34
<211> 40
<212> DNA
<213> Synthesis
<400> 34
tctgttccat gtcgactcac gctctgtgta aagtgtatat 40
<210> 35
<211> 47
<212> DNA
<213> Synthesis
<400> 35
cttgtaatcc atcgatacta gtttatttac ttctcttgta aaccttg 47
<210> 36
<211> 42
<212> DNA
<213> Synthesis
<400> 36
tcactaaagg gcggccgcat ggcttcagaa aaagaaatta gg 42
<210> 37
<211> 21
<212> DNA
<213> Synthesis
<400> 37
tctgtggctg cttatcccag c 21
<210> 38
<211> 46
<212> DNA
<213> Synthesis
<400> 38
gcatgaggtc gctccaattc agcctttgtc cgatgaaggt atgtaa 46
<210> 39
<211> 43
<212> DNA
<213> Synthesis
<400> 39
aggttttggg acgctcgaag cgaaactctg ttcattttgg tcg 43
<210> 40
<211> 24
<212> DNA
<213> Synthesis
<400> 40
ctgtcttgtg atcgcatact ctgc 24
<210> 41
<211> 46
<212> DNA
<213> Synthesis
<400> 41
ttacatacct tcatcggaca aaggctgaat tggagcgacc tcatgc 46
<210> 42
<211> 43
<212> DNA
<213> Synthesis
<400> 42
cgaccaaaat gaacagagtt tcgcttcgag cgtcccaaaa cct 43
<210> 43
<211> 20
<212> DNA
<213> Synthesis
<400> 43
ttgtgacacg tcagtgcagg 20
<210> 44
<211> 45
<212> DNA
<213> Synthesis
<400> 44
gcatgaggtc gctccaattc agtgttgacg ttctcaacat gatgg 45
<210> 45
<211> 42
<212> DNA
<213> Synthesis
<400> 45
gaaggttttg ggacgctcga agtaatagcg tgttacgcac cc 42
<210> 46
<211> 20
<212> DNA
<213> Synthesis
<400> 46
ccaatgcctg taacccttcg 20
<210> 47
<211> 45
<212> DNA
<213> Synthesis
<400> 47
ccatcatgtt gagaacgtca acactgaatt ggagcgacct catgc 45
<210> 48
<211> 42
<212> DNA
<213> Synthesis
<400> 48
gggtgcgtaa cacgctatta cttcgagcgt cccaaaacct tc 42
<210> 49
<211> 20
<212> DNA
<213> Synthesis
<400> 49
tgaccttcac cgacggattc 20
<210> 50
<211> 46
<212> DNA
<213> Synthesis
<400> 50
gcatgaggtc gctccaattc agccttcacc ggttagtgtt tagtaa 46
<210> 51
<211> 44
<212> DNA
<213> Synthesis
<400> 51
aggttttggg acgctcgaag gacagagatc atccttggta gaat 44
<210> 52
<211> 22
<212> DNA
<213> Synthesis
<400> 52
ctcgacaact ttcagattgc tg 22
<210> 53
<211> 46
<212> DNA
<213> Synthesis
<400> 53
ttactaaaca ctaaccggtg aaggctgaat tggagcgacc tcatgc 46
<210> 54
<211> 44
<212> DNA
<213> Synthesis
<400> 54
attctaccaa ggatgatctc tgtccttcga gcgtcccaaa acct 44
<210> 55
<211> 22
<212> DNA
<213> Synthesis
<400> 55
cgctgtcttg attcttgtga gc 22
<210> 56
<211> 43
<212> DNA
<213> Synthesis
<400> 56
gcatgaggtc gctccaattc agacttttgc ctgatccagc cag 43
<210> 57
<211> 43
<212> DNA
<213> Synthesis
<400> 57
aggttttggg acgctcgaag aagagtaacc agtgaagaca tct 43
<210> 58
<211> 22
<212> DNA
<213> Synthesis
<400> 58
cctggtggaa catcctagaa cg 22
<210> 59
<211> 43
<212> DNA
<213> Synthesis
<400> 59
ctggctggat caggcaaaag tctgaattgg agcgacctca tgc 43
<210> 60
<211> 43
<212> DNA
<213> Synthesis
<400> 60
agatgtcttc actggttact cttcttcgag cgtcccaaaa cct 43
<210> 61
<211> 20
<212> DNA
<213> Synthesis
<400> 61
gctggtcttc tggtctgtta 20
<210> 62
<211> 44
<212> DNA
<213> Synthesis
<400> 62
gcatgaggtc gctccaattc agtctcttgt tgtagtccat gacg 44
<210> 63
<211> 45
<212> DNA
<213> Synthesis
<400> 63
gaaggttttg ggacgctcga aggttagcaa tatctcgcat tatag 45
<210> 64
<211> 20
<212> DNA
<213> Synthesis
<400> 64
ttagtattcc tggagaacca 20
<210> 65
<211> 44
<212> DNA
<213> Synthesis
<400> 65
cgtcatggac tacaacaaga gactgaattg gagcgacctc atgc 44
<210> 66
<211> 45
<212> DNA
<213> Synthesis
<400> 66
ctataatgcg agatattgct aaccttcgag cgtcccaaaa ccttc 45
<210> 67
<211> 20
<212> DNA
<213> Synthesis
<400> 67
acggatgaca cagtggttag 20
<210> 68
<211> 40
<212> DNA
<213> Synthesis
<400> 68
atgaggtcgc tccaattcag agctacgctg caagctattg 40
<210> 69
<211> 50
<212> DNA
<213> Synthesis
<400> 69
gcttgagaag gttttgggac gctcgaagca taaacatggc atggcgatca 50
<210> 70
<211> 20
<212> DNA
<213> Synthesis
<400> 70
tctcaccgca tgacaagtgg 20
<210> 71
<211> 50
<212> DNA
<213> Synthesis
<400> 71
acaatagttt caatagcttg cagcgtagct ctgaattgga gcgacctcat 50
<210> 72
<211> 42
<212> DNA
<213> Synthesis
<400> 72
tgatcgccat gccatgttta tgcttcgagc gtcccaaaac ct 42
<210> 73
<211> 42
<212> DNA
<213> Synthesis
<400> 73
ggagaaaaaa ccccggatcc atggaccaat tggtgaagac tg 42
<210> 74
<211> 20
<212> DNA
<213> Synthesis
<400> 74
ggatccccgg aggccttcaa 20
<210> 75
<211> 42
<212> DNA
<213> Synthesis
<400> 75
gcatgaggtc gctccaattc agccggagag accaatagct gc 42
<210> 76
<211> 40
<212> DNA
<213> Synthesis
<400> 76
aggttttggg acgctcgaag acgccatgca tgatgcctgc 40
<210> 77
<211> 20
<212> DNA
<213> Synthesis
<400> 77
gcgcatgtga atgacagacg 20
<210> 78
<211> 42
<212> DNA
<213> Synthesis
<400> 78
gcagctattg gtctctccgg ctgaattgga gcgacctcat gc 42
<210> 79
<211> 40
<212> DNA
<213> Synthesis
<400> 79
gcaggcatca tgcatggcgt cttcgagcgt cccaaaacct 40
<210> 80
<211> 47
<212> DNA
<213> Synthesis
<400> 80
gtcaaggaga aaaaaccccg gatccatgac aatcaaggaa cataaag 47
<210> 81
<211> 49
<212> DNA
<213> Synthesis
<400> 81
gcggtaccaa gcttactcga gttatttctt tttttgagag aaaaattgg 49
<210> 82
<211> 45
<212> DNA
<213> Synthesis
<400> 82
gtcaaggaga aaaaaccccg gatccatgtc gccctctgcc gtaca 45
<210> 83
<211> 45
<212> DNA
<213> Synthesis
<400> 83
gcggtaccaa gcttactcga gttacaactt gaccgaatca attag 45
<210> 84
<211> 46
<212> DNA
<213> Synthesis
<400> 84
caaccctcac taaagggcgg ccgcatgtct cagaacgttt acattg 46
<210> 85
<211> 20
<212> DNA
<213> Synthesis
<400> 85
tagtggaggc aaggttgcat 20
<210> 86
<211> 42
<212> DNA
<213> Synthesis
<400> 86
ctcgaaaatt ctgcgttcgt tattatgccc attcaacatc cg 42
<210> 87
<211> 41
<212> DNA
<213> Synthesis
<400> 87
tattgtcgct gttcacaccg ccctggagaa gtaaatgaaa a 41
<210> 88
<211> 23
<212> DNA
<213> Synthesis
<400> 88
cggaaacatt atactcaagt cgc 23
<210> 89
<211> 42
<212> DNA
<213> Synthesis
<400> 89
cggatgttga atgggcataa taacgaacgc agaattttcg ag 42
<210> 90
<211> 41
<212> DNA
<213> Synthesis
<400> 90
ttttcattta cttctccagg gcggtgtgaa cagcgacaat a 41
<210> 91
<211> 25
<212> DNA
<213> Synthesis
<400> 91
tttggctctt gagcagtaat gtttc 25
<210> 92
<211> 25
<212> DNA
<213> Synthesis
<400> 92
atgtctgctg ttaacgttgc acctg 25
<210> 93
<211> 35
<212> DNA
<213> Synthesis
<400> 93
tagagcggat ttaaagcgac gcaaattcgc cgatg 35
<210> 94
<211> 20
<212> DNA
<213> Synthesis
<400> 94
tggaggttcg cattacgcct 20
<210> 95
<211> 54
<212> DNA
<213> Synthesis
<400> 95
gcaacgttaa cagcagacat gattttacgt atatcaacta gttgacgatt atga 54
<210> 96
<211> 46
<212> DNA
<213> Synthesis
<400> 96
aggttttggg acgctcgaag tctcatctgg aatataattc ccccct 46
<210> 97
<211> 32
<212> DNA
<213> artificial sequence
<400> 97
gcgtcgcttt aaatccgctc taaccgaaaa gg 32
<210> 98
<211> 25
<212> DNA
<213> Synthesis
<400> 98
cttcgagcgt cccaaaacct tctca 25
<210> 99
<211> 42
<212> DNA
<213> Synthesis
<400> 99
cttgtaatcc atcgatacta gtttagtttt gctggccgca tc 42
<210> 100
<211> 41
<212> DNA
<213> Synthesis
<400> 100
agtgagtcgt attacggatc cgaaggttaa tgtggctgtg g 41
<210> 101
<211> 41
<212> DNA
<213> Synthesis
<400> 101
gccacattaa ccttcggatc ccggcagaga ccaatcagta a 41
<210> 102
<211> 43
<212> DNA
<213> Synthesis
<400> 102
gcggtaccaa gcttactcga gctatttctt agcatttttg acg 43
<210> 103
<211> 44
<212> DNA
<213> Synthesis
<400> 103
cctttagtga gggttgaatt cctacataag aacacctttg gtgg 44
<210> 104
<211> 48
<212> DNA
<213> Synthesis
<400> 104
agtgagtcgt attacggatc cctagtacac tctatatttt tttatgcc 48
<210> 105
<211> 41
<212> DNA
<213> Synthesis
<400> 105
tatagagtgt actagggatc caactgtggg aatactcagg t 41
<210> 106
<211> 45
<212> DNA
<213> Synthesis
<400> 106
gcggtaccaa gcttactcga gttaagcaag gattttctta acttc 45
<210> 107
<211> 22
<212> DNA
<213> Synthesis
<400> 107
cacggcatta gtcagggaag tc 22
<210> 108
<211> 42
<212> DNA
<213> Synthesis
<400> 108
gcatgaggtc gctccaattc aggcttgacc gagagcaatc cc 42
<210> 109
<211> 44
<212> DNA
<213> Synthesis
<400> 109
tattgtcgct gttcacaccg gtcagtaagt atgtatacga acag 44
<210> 110
<211> 20
<212> DNA
<213> Synthesis
<400> 110
ccagaggcgt ccactggaat 20
<210> 111
<211> 42
<212> DNA
<213> Synthesis
<400> 111
gggattgctc tcggtcaagc ctgaattgga gcgacctcat gc 42
<210> 112
<211> 40
<212> DNA
<213> Synthesis
<400> 112
cgaaaattct gcgttcgtta cttcgagcgt cccaaaacct 40
<210> 113
<211> 40
<212> DNA
<213> Synthesis
<400> 113
aggttttggg acgctcgaag taacgaacgc agaattttcg 40
<210> 114
<211> 44
<212> DNA
<213> Synthesis
<400> 114
ctgttcgtat acatacttac tgaccggtgt gaacagcgac aata 44

Claims (7)

1. A method for constructing a recombinant strain for producing squalene, comprising the steps of: taking saccharomyces cerevisiae as an original strain, firstly reconstructing a mevalonate pathway in an online granulocyte to construct a mitochondrial engineering strain, then strengthening mevalonate synthesis in cytoplasm of the mitochondrial engineering strain to improve strain growth, and finally performing cytoplasmic engineering strategy optimization on the basis to obtain a recombinant strain of high-yield squalene through joint optimization of mitochondria and cytoplasmic double-process; wherein,,
the construction of the mitochondrial engineering strain comprises the utilization of a mitochondrial localization signal peptide to localize seven key enzyme genes from acetyl-CoA to dimethylallyl pyrophosphate in a mevalonate pathway within mitochondria of the Saccharomyces cerevisiae, wherein the seven key enzymes are: ERG10, ERG13, hmg1, ERG12, ERG8, ERG19 and IDI1;
the enhanced mevalonate synthesis includes over-expressing tmg 1 in the cytoplasm to improve mitochondrial engineering strain growth;
the cytoplasmic engineering strategy optimization comprises: the expression of the enhancing enzymes ERG10 and/or ACS1 and/or ACS2 to enhance the supply of acetyl-coa, and the expression of the attenuating enzyme ERG1 to attenuate the expression level or enzyme activity of a key enzyme in the target product consuming pathway.
2. The method of claim 1, wherein constructing a mitochondrial engineered strain comprises expressing a gene linked to a mitochondrial localization signal peptide using a GAL1-GAL10, GAL2, GAL3, or GAL7 inducible promoter.
3. The method according to claim 1, wherein the promoter for expression enhancer is a GAL1-GAL10, GAL2, GAL3 or GAL7 inducible promoter.
4. The method of claim 1, further comprising the complementation of the auxotroph of the strain, including the complementation of the four amino acid genes HIS3, LEU2, URA3 and TRP 1.
5. A recombinant strain producing squalene obtained by the construction method according to any one of claims 1 to 4.
6. Use of a recombinant strain according to claim 5 for the production of squalene, characterized in that during the fermentation culture a feed medium is added to the fermentation system, said feed medium comprising: 500-800 g/L glucose, 9g/L KH 2 PO 4 、2.5 g/L MgSO 4 、3.5 g/L K 2 SO 4 、0.28 g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
7. The use according to claim 6, characterized in that the fermentation culture process is divided into two phases: the first stage makes the thallus grow fast, and after the thallus biomass reaches certain concentration, the second stage fermentation is started; the second stage is the production stage of squalene, and a large amount of squalene synthesis is started by an induction strategy of adding an inducer galactose, so that high yield of squalene is finally obtained.
CN202111145425.8A 2021-09-28 2021-09-28 Recombinant strain for producing squalene, construction method and application thereof Active CN114015587B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111145425.8A CN114015587B (en) 2021-09-28 2021-09-28 Recombinant strain for producing squalene, construction method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111145425.8A CN114015587B (en) 2021-09-28 2021-09-28 Recombinant strain for producing squalene, construction method and application thereof

Publications (2)

Publication Number Publication Date
CN114015587A CN114015587A (en) 2022-02-08
CN114015587B true CN114015587B (en) 2023-08-18

Family

ID=80055099

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111145425.8A Active CN114015587B (en) 2021-09-28 2021-09-28 Recombinant strain for producing squalene, construction method and application thereof

Country Status (1)

Country Link
CN (1) CN114015587B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117402763B (en) * 2023-09-26 2024-03-19 广州中医药大学(广州中医药研究院) Saccharomyces cerevisiae engineering strain for producing squalene, construction method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950648A (en) * 2016-05-16 2016-09-21 浙江大学 Double-regulation strategy for realizing comprehensive utilization of acetyl coenzyme A in saccharomyces cerevisiae
KR20190080153A (en) * 2017-12-28 2019-07-08 중앙대학교 산학협력단 Recombinant yeast strains expressing the mutated squalene monoxygenase with reduced feedback regulation and method for overproducing sterol precursors using the same
CN113234610A (en) * 2021-03-05 2021-08-10 江南大学 Saccharomyces cerevisiae strain for synthesizing squalene and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950648A (en) * 2016-05-16 2016-09-21 浙江大学 Double-regulation strategy for realizing comprehensive utilization of acetyl coenzyme A in saccharomyces cerevisiae
KR20190080153A (en) * 2017-12-28 2019-07-08 중앙대학교 산학협력단 Recombinant yeast strains expressing the mutated squalene monoxygenase with reduced feedback regulation and method for overproducing sterol precursors using the same
CN113234610A (en) * 2021-03-05 2021-08-10 江南大学 Saccharomyces cerevisiae strain for synthesizing squalene and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
谢琴鼎等.植物三萜代谢途径中角鲨烯合成酶研究进展.分子植物育种.2018,第16卷(第9期),2997-3005. *

Also Published As

Publication number Publication date
CN114015587A (en) 2022-02-08

Similar Documents

Publication Publication Date Title
KR101897213B1 (en) Composition for Genome Editing Comprising Cpf1 and Use thereof
CN110129354B (en) N-acetylneuraminic acid specific biosensor and application thereof
US6562622B1 (en) Continuous in vitro evolution
US20240018564A1 (en) In situ two-phase extraction system
CN103534392A (en) Surface, anchored FC-bait antibody display system
CN108395996B (en) Classical swine fever virus subunit vaccine and preparation method and application thereof
CN109609579B (en) Genetically engineered bacterium for producing beta-carotene and construction method thereof
CN106687578B (en) Targeted mutagenesis in spirulina
US20230407344A1 (en) Fermentative production of isoprenoids
CN114015587B (en) Recombinant strain for producing squalene, construction method and application thereof
CN108624546B (en) recombinant bacillus amyloliquefaciens and construction method and application thereof
KR20150013503A (en) Surface anchored light chain bait antibody display system
WO2013082309A1 (en) Process for growing natural or engineered high lipid accumulating strain on crude glycerol and/or other sources of waste carbon for the production of oils, fuels, oleochemicals, and other valuable organic compounds
CN108285886A (en) The method that recombined bacillus subtilis resting cell produces N-acetyl-neuraminate
CN112301050A (en) Method for constructing high-yield glutathione recombinant strain and application
CN114292864B (en) Bacillus bailii mutant strain with high surfactant yield, construction method and application thereof
CN100422308C (en) High fermentation rate type recombinant alcoholic fermentation yeast, the building and expression carrier thereof
AU2015207519B2 (en) Novel cytochrome P450 polypeptide with increased enzymatic activity
CN111979134B (en) Construction and application of recombinant saccharomyces cerevisiae for synthesizing carminic acid
CN111394320B (en) Recombinant vaccinia virus expressing human tissue factor fusion protein and application thereof
CN107661496A (en) A kind of pig parvoviral immune composition and preparation method and application
KR102468650B1 (en) Recombinant vector inducing expression of T7 RNA polymerase and mRNA capping enzyme and uses thereof
CN114207125A (en) Reverse selection by suppression of conditionally essential genes
US20200385754A1 (en) Engineered ubiquitous chromatin opening elements and uses thereof
KR101875825B1 (en) Yellow fluorescent protein fragment-fusion yeast gene library and method of analysis of protein-protein interaction using the same

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
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20230906

Address after: 201403, 1st Floor, Building 34, No. 22, Lane 800, Lanfeng Road, Fengxian District, Shanghai

Patentee after: Baikaisheng (Shanghai) Biotechnology Co.,Ltd.

Address before: 200237 No. 130, Meilong Road, Shanghai, Xuhui District

Patentee before: EAST CHINA University OF SCIENCE AND TECHNOLOGY

TR01 Transfer of patent right