US20170137792A1 - Recombinant polynucleotide sequence for producing astaxanthin and uses thereof - Google Patents

Recombinant polynucleotide sequence for producing astaxanthin and uses thereof Download PDF

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Publication number
US20170137792A1
US20170137792A1 US15/353,372 US201615353372A US2017137792A1 US 20170137792 A1 US20170137792 A1 US 20170137792A1 US 201615353372 A US201615353372 A US 201615353372A US 2017137792 A1 US2017137792 A1 US 2017137792A1
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gene
seq
amino acid
promoter
acid sequence
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Jui-Jen Chang
Caroline THIA
Hao-Yeh Lin
Yu-Ju Lin
Chieh-Chen Huang
Wen-Hsiung Li
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Academia Sinica
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Academia Sinica
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Priority to US15/353,372 priority Critical patent/US20170137792A1/en
Assigned to ACADEMIA SINICA reassignment ACADEMIA SINICA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHANG, JUI-JEN, HUANG, CHIEH-CHEN, LI, WEN-HSIUNG, LIN, Hao-Yeh, LIN, YU-JU, THIA, Caroline
Publication of US20170137792A1 publication Critical patent/US20170137792A1/en
Priority to US16/200,269 priority patent/US10865397B2/en
Priority to US17/094,623 priority patent/US11781124B2/en
Abandoned legal-status Critical Current

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    • 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
    • C12N9/14—Hydrolases (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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • FIG. 17C is a line chart that depicts the growth curve of specified strains according to one example of the present disclosure.
  • FIG. 24F presents the HPLC data of the astaxanthin produced by CA6-ITS strain according to one example of the present disclosure
  • FIG. 29A are photographs that reveal the colonies of wild-type and Cz30 strains respectively treated with specified concentration of 10 deacetylbaccatin III (10 DB), which is dissolved in specified concentration of ethanol, according to one example of the present disclosure;
  • promoter is to describe a synthetic or fusion molecule or derivative, which confers, activates or enhances expression of a nucleic acid sequence in a cell, tissue or organ.
  • the first promoter is the ScPGK promoter
  • the second promoter is the ScADHI promoter
  • the third promoter is the KILac4 promoter
  • the fourth promoter is the KIADHI promoter
  • the marker promoter is the KIGapDH promoter.
  • the fifth gene cassette comprises a fifth nucleic acid sequence driven by a fifth promoter, in which the fifth nucleic acid sequence encodes a ⁇ -carotene hydroxylase.
  • the sixth gene cassette comprises a sixth nucleic acid sequence driven by a sixth promoter, in which the sixth nucleic acid sequence encodes a ⁇ -carotene ketolase.
  • the present recombinant polynucleotide sequence and/or vector can be introduced into the host cell by the method known to a skilled artisan.
  • the method for introducing the exogenous DNA and/or vector into a prokaryotic host cell includes chemical treatment (such as incubating the host cell in a solution containing divalent cations, then followed by a heat treatment), and electroporation (such as briefly treating the host cell with an electric field that creates holes in the cell membrane).
  • the thus produced astaxanthin, a precursor, and/or a derivative thereof exhibits anti-oxidation activity that renders the host cell tolerant to a stress.
  • the stress may be caused from the host cell being exposed to ethanol, butanol, UV exposure, furfural, and/or the precursor of an anticancer drug.
  • the host cell is tolerant to a precursor of an anticancer drug, 10-deacetyl baccatin III (10 DB), which gives rise to the anticancer drug, paclitaxel.
  • the tolerance of the host cell to different stresses can be evaluated by various methods, depending on the experimental purpose. For example, the tolerance can be evaluated by the measurement of colony number, cell growth, cell density, or gene expression.
  • the medium may comprises at least two components selected from the group consisting of glucose, galactose, glycerol, and fatty acid so as to efficiently induce the expression of gene cassettes within the hose cell.
  • the synthesized bkt gene had an amino acid sequence of SEQ ID NO: 8; the synthesized CrChYb gene had an amino acid sequence of SEQ ID NO: 5; the synthesized CzChYb gene had an amino acid sequence of SEQ ID NO: 6; and the synthesized HpChYb gene had an amino acid sequence of SEQ ID NO: 7.
  • the engineered yeasts were cultivated in YPG medium (1 BactoDifco-Yeast Extract, 2% BactoDifco-Peptone, 2% Merck-D(+)-Galactose) respectively at 25° C., 30° C., and 37° C. for 3 days.
  • the engineered yeasts were cultivated in YPG medium with the addition of 20% glucose, 20% galactose, or 20% glycerol.
  • the electroporation was performed (1.0 kV, 400 ⁇ , and 25 ⁇ F capacitance) using a BioRad system (GenePluserXcellTM, Bio-Rad, Hercules, Calif.) with an aluminum cuvette (2 mm).
  • the cells were spread onto YPG plates (1% BactoDifco-Yeast Extract, 2% BactoDifco-Peptone, and 2% Merck-galactose) containing G418 (200 ⁇ g/ml).
  • a recombinant polynucleotide sequence crtE-Kan-tHMG1 was constructed to comprise a crtE gene and a HMG1 gene.
  • the neomycin phosphotransferase gene essential for G418 resistance (Kan) was used as a marker gene for clone screening.
  • the Cz30 cell cultivated in glycerol which is the byproduct of the bio-diesel industry, exhibited a significant red color change, as compared with culturing in other carbon sources, indicating the potential use of Cz30 for the development of a green industry.
  • the broth of Cz30 remained in red color after being cultured for 5 days in 20% glycerol, although the relationship between glycerol metabolism and carotenoid esterification is still not clear.
  • the carotenoids were extracted from WT and Cz30 strain, and the antioxidant ability of the thus produced carotenoids was determined by use of the ABTS substrate reaction. After culturing in YPG medium at 25° C. for 72 hours, the host cells were lyophilized and the pigments in the cells were extracted by methanol. The extract of Cz30 exhibited a higher free radical scavenging capacity (72.1%) than that of WT (52.3%) ( FIG. 23A ).
  • the astaxanthin synthesis gene cassettes were introduced together with a high copy expression plasmid RS426 to the CA6-plasmid strain ( FIG. 24D ).
  • the gene cassettes would be spontaneously assembled to the plasmid in vivo; and the transformant could produce orange to red colonies.
  • the host cell With the high copy number of plasmids, the host cell has potential to express high amounts of proteins, and to convert the precursor to astaxanthin more efficiently.
  • the data of FIG. 23 revealed that the cell extract of Cz30 exhibited an antioxidant activity in comparison to that of wild-type (WT) control.
  • WT wild-type
  • the anti-stress capability of Cz30 was further confirmed by UV, furfural, ethanol, or isobutanol treatment.
  • the WT and Cz30 were separately exposed to UV for 5, 10, or 20 minutes, and then inoculated in the YPG plate with a series dilution and cultivated for 48 hours. Only some Cz30 colonies could grow on the YPG plate after being exposed to UV light for 20 minutes ( FIG. 27A ). This observation suggested that the carotenoid products of Cz30 could reduce UV damage, resulting in faster cell growth as compared to that of the WT.
  • Toxins produced during acid and steam pretreatment of lignocellulose cover a large range of substances, such as furfural and hydroxymethylfurfural from hemicellulose and cellulose, alcohols and aldehydes from lignin, and heavy metals from bioreactor. Accordingly, the second stress factor examined was furfural treatment.
  • the Cz30 strain could tolerate the treatment of 100 mM furfural, whereas the WT strain could only tolerate the treatment with 80 mM furfural.

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  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
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  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Epoxy Compounds (AREA)
  • Cosmetics (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
US15/353,372 2014-05-16 2016-11-16 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof Abandoned US20170137792A1 (en)

Priority Applications (3)

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US15/353,372 US20170137792A1 (en) 2014-05-16 2016-11-16 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof
US16/200,269 US10865397B2 (en) 2014-05-16 2018-11-26 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof
US17/094,623 US11781124B2 (en) 2014-05-16 2020-11-10 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof

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US201461994828P 2014-05-16 2014-05-16
PCT/US2015/031273 WO2015176054A2 (fr) 2014-05-16 2015-05-16 Séquence polynucléotidique de recombinaison pour la production d'astaxanthine, et utilisations de celle-ci
US15/353,372 US20170137792A1 (en) 2014-05-16 2016-11-16 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof

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US17/094,623 Active 2036-10-03 US11781124B2 (en) 2014-05-16 2020-11-10 Recombinant polynucleotide sequence for producing astaxanthin and uses thereof

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US (3) US20170137792A1 (fr)
EP (2) EP3143121A4 (fr)
JP (2) JP2017515512A (fr)
CN (2) CN106795477A (fr)
CA (2) CA2949381A1 (fr)
CL (1) CL2016002906A1 (fr)
TW (1) TWI681052B (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11124819B2 (en) * 2017-03-15 2021-09-21 The Regents Of The University Of California Genes involved in astaxanthin biosynthesis
CN114410523A (zh) * 2022-01-20 2022-04-29 中国农业科学院茶叶研究所 一种高效制备红茶菌的菌种组合及其应用

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CN110283867A (zh) * 2019-05-24 2019-09-27 华南理工大学 一种利用佐夫色绿藻生产虾青素的方法
CN117604060A (zh) * 2024-01-24 2024-02-27 逢时(青岛)海洋科技有限公司 一种基于固定化胆固醇酯酶的游离虾青素及其制备方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11124819B2 (en) * 2017-03-15 2021-09-21 The Regents Of The University Of California Genes involved in astaxanthin biosynthesis
CN114410523A (zh) * 2022-01-20 2022-04-29 中国农业科学院茶叶研究所 一种高效制备红茶菌的菌种组合及其应用

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EP3460044B1 (fr) 2024-01-24
TWI681052B (zh) 2020-01-01
TW201544593A (zh) 2015-12-01
CN106795477A (zh) 2017-05-31
CA2949381A1 (fr) 2015-11-19
JP2019150047A (ja) 2019-09-12
JP6964622B2 (ja) 2021-11-10
CL2016002906A1 (es) 2018-03-16
CA3109156C (fr) 2023-08-15
US10865397B2 (en) 2020-12-15
CA3109156A1 (fr) 2015-11-19
US11781124B2 (en) 2023-10-10
US20210071153A1 (en) 2021-03-11
EP3460044A1 (fr) 2019-03-27
EP3143121A4 (fr) 2018-02-28
JP2017515512A (ja) 2017-06-15
WO2015176054A2 (fr) 2015-11-19
US20190144841A1 (en) 2019-05-16
EP3143121A2 (fr) 2017-03-22
CN111500599A (zh) 2020-08-07
WO2015176054A3 (fr) 2016-01-07

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