WO2015105233A1 - Vecteur recombiné pour l'accroissement de la productivité de biomasse et de lipides de microalgues et son utilisation - Google Patents

Vecteur recombiné pour l'accroissement de la productivité de biomasse et de lipides de microalgues et son utilisation Download PDF

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WO2015105233A1
WO2015105233A1 PCT/KR2014/001179 KR2014001179W WO2015105233A1 WO 2015105233 A1 WO2015105233 A1 WO 2015105233A1 KR 2014001179 W KR2014001179 W KR 2014001179W WO 2015105233 A1 WO2015105233 A1 WO 2015105233A1
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microalgae
recombinant vector
promoter
lipid
ammonium
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PCT/KR2014/001179
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English (en)
Korean (ko)
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양지원
신성은
정병률
강남규
최강국
전승집
곽민수
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재단법인 탄소순환형 차세대 바이오매스 생산전환 기술연구단
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Publication of WO2015105233A1 publication Critical patent/WO2015105233A1/fr

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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

  • the present invention relates to a recombinant vector and its use to increase the biomass and lipid productivity of microalgae, and more particularly, inducible promoters derived from ammonium-free or nitrate-containing conditions and GAPDH beneath the promoter.
  • Recombinant vector comprising a glyceraldehyde-3-phosphate dehydrogenase) protein coding gene, a method of producing a microalgae transformed microalgae cells with the recombinant vector to increase the biomass and lipid productivity, the method Transformed microalgae prepared by the present invention, comprising the recombinant vector as an active ingredient, a composition for increasing biomass and lipid productivity of microalgae, a method for producing lipids by culturing the transgenic microalgae and the transformed micro It relates to a method for producing biodiesel using algae.
  • Microalgae including cyanobacteria, have been reported to produce a variety of lipids, hydrocarbons, fatty alcohols and other complex oils. Although not all microalgae are suitable for biodiesel production, some microalgal strains have shown their potential as biodiesel fuels. Chlamydomonas reinhardtii is one of the most widespread microalgal model systems, which have desirable characteristics as a biofuel source. In addition, metabolic engineering of lipid production pathways has also been widely studied using model system strains of Chlamydomonas Reinhardi.
  • 3-phosphoglycerate (PGA) obtained through the Calvin cycle in photosynthetic organisms is converted into acetyl coenzyme A (acetyl-CoA) by glycolysis in the cytoplasm and chloroplasts.
  • Acetyl coenzyme A contributes significantly to the production of fatty acids.
  • the present invention anticipates that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme may have a positive effect on the synthesis of 3-phosphoglycerin, ultimately contributing to the production of fatty acids, including an inducible promoter and a GAPDH protein coding gene.
  • a recombinant vector was prepared to transform the microalgae, and the growth and lipid content changes of the transformed microalgae were observed.
  • Korean Patent Publication No. 2013-0095517 discloses 'a novel microalga Chlamydomonas Reinhardi KNUA021 strain and fatty alcohol and fatty acid production method therefrom
  • Korean Patent No. 1107980 discloses' glgA1 gene and There is disclosed a recombinant vector comprising the same and a method for controlling the total lipid content of the bacterium by controlling the biosynthesis of the host cell transformed therefrom and the recombinant vector and its recombinant microorganism to increase the biomass and lipid productivity of the microalgae of the present invention There is no description of the use.
  • the present invention is derived from the above requirements, in the present invention transformed microalgae with a recombinant vector comprising an inducible promoter Nia1 promoter and GAPDH protein coding gene, the transgenic microalgae in a medium composition without ammonium By culturing the biomass and lipid production of microalgae was confirmed that the increase compared to the non-transformer, the present invention was completed.
  • the present invention is an inducible promoter induced in the absence of ammonium or in the condition of nitrate and recombinant recombinant characterized in that it comprises a GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein coding gene under the promoter Provide a vector.
  • GAPDH glycosyl transferase
  • the present invention provides a method for producing a transformed microalgae having increased biomass and lipid productivity compared to a non-transformer comprising transforming microalgal cells with the recombinant vector to express a GAPDH protein coding gene. do.
  • the present invention also provides a transformed microalgae with increased biomass and lipid productivity compared to the non-transformers prepared by the above method.
  • the present invention also provides a composition for increasing biomass and lipid productivity of microalgae comprising a recombinant vector as an active ingredient.
  • the present invention also provides a method for producing a lipid comprising culturing the transgenic microalgae in a culture medium.
  • step (b) extracting fatty acids from the microalgal culture of step (a);
  • step (c) it provides a method for producing biodiesel, characterized in that to produce a fatty acid ester by transesterifying the fatty acid of step (b).
  • microalgae recombinant vector comprising the Nia1 promoter and GAPDH protein coding gene
  • the biomass and lipid synthesis of the microalgae is increased.
  • Transgenic microalgae using the existing recombinant vector had a problem in that biodiesel production decreased when biomass increased compared to wild type, or biomass decreased when biodiesel production increased, but transformation using recombinant vector of the present invention
  • Microalgae not only produce a greater amount of biodiesel in the same biomass compared to wild type, but also show higher biomass increase compared to wild type under the same culture conditions, which ultimately results in high biodiesel productivity.
  • Vector is considered to be very useful industrially.
  • Figure 1 is a picture of a recombinant vector inserted Nia1 promoter and GAPDH protein coding gene of the present invention.
  • PNG gDNA genomic DNA extracted from transgenic microalgae
  • PNG vectors recombinant vectors used for transformation
  • Positive control eukaryotic 18s rRNA.
  • Figure 3 is the result of confirming the growth rate (a) and lipid content (b) in the medium without ammonium 7 of the transformants.
  • TEP general medium
  • TEP-N medium without ammonium
  • TEP normal medium
  • TEP-N medium without ammonium
  • Figure 6 is a culture of the selected transgenic microalgae and wild-type microalgae in a normal medium (TAP) and a medium without ammonium (TAP-N) and analyzed the content of fatty acid methyl ester (FAME, fatty acid methyl ester) lipid content This is the result of checking.
  • TAP normal medium
  • TAP-N medium without ammonium
  • FIG. 7 shows the results of analyzing the fatty acid methyl ester yields of the selected transformed microalgae and wild-type microalgae in a culture medium (TAP) and a medium without ammonium (TAP-N).
  • TEP general medium
  • TEP-N medium without ammonium
  • the present invention is characterized in that it comprises an inducible promoter induced in the absence of ammonium or in the presence of nitrate and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein coding gene beneath the promoter To provide a recombinant vector.
  • GAPDH glycosyl transferase
  • the inducible promoter may be a Nia1 (nirate reductase) promoter, a Cytochrome c 6 promoter, a carbonic anhydrase (CA1) promoter, or the like, preferably a Nia1 (nirate reductase) promoter May be, but is not limited thereto.
  • Nia1 promoter according to the invention may have a nucleotide sequence of SEQ ID NO: 1.
  • homologues of such promoter sequences are included within the scope of the present invention. Homologues are base sequences that vary in base sequence but have similar functional properties to the base sequence of SEQ ID NO: 1.
  • the promoter sequence has a base sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence of SEQ ID NO: 1 It may include.
  • the "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • the range of proteins includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of such proteins.
  • “Functional equivalent” means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.
  • Substantially homogeneous physiological activity means activity that increases the biomass and lipid productivity of microalgae.
  • the present invention also provides a gene encoding the GAPDH protein.
  • the gene of the present invention may include GAPDH cDNA represented by SEQ ID NO: 3 or GAPDH genomic DNA sequence represented by SEQ ID NO: 4.
  • homologues of the base sequences are included within the scope of the present invention and the details are as described above.
  • inducible promoter refers to a promoter whose activity may be increased or decreased upon external stimulation.
  • the stimulus may be a physical or chemical stimulus in nature such as temperature, light, chemicals and the like. Induction of genes under the promoter can be obtained through direct or indirect delivery of stimuli.
  • Recombinant refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form.
  • Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.
  • vector is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells.
  • carrier is often used interchangeably with “vector”.
  • the present invention is a method for producing a transformed microalgae biomass and lipid productivity is increased compared to the non-transformer comprising the step of transforming the microalgal cells with the recombinant vector to express the GAPDH protein coding gene and It provides a transgenic microalgae prepared by the above method.
  • the method for transforming microalgal cells with a recombinant vector comprises a glass bead, a gene gun, and electroporation. , Silicon carbide whisker, and the like, and may be preferably a glass bead method, but is not limited thereto.
  • Chlamydomonas Chlamydomonas
  • the wrong ah Ettlia
  • two flying it Ella Ella
  • Chlorella Chlorella
  • nanno claw drop system Nanochloropsis
  • Spirulina Spirulina
  • Chlamydomonas reinhardtii Chlamydomonas caudata caudata Wille
  • Chlamydomonas moewusii Chlamydomonas nivalis
  • Chlamydomonas perigranulata Ettlia oleoabundans
  • Nanno Rob cis Salina Nanno Rob cis salin a
  • nanno claw Rob cis Gardiner appear (Nannochlorops
  • the present invention also provides a composition for increasing biomass and lipid productivity of microalgae comprising the recombinant vector as an active ingredient.
  • the composition contains an inducible promoter induced in the absence of ammonium or nitrate as an active ingredient and a recombinant vector comprising a GAPDH protein coding gene under the promoter, and transforming the recombinant vector into microalgal cells. It can increase the biomass and lipid productivity of microalgae.
  • the present invention also provides a method for producing a lipid comprising culturing the transformed microalgae in a culture medium.
  • the culture medium may be a culture medium without ammonium, but is not limited thereto.
  • step (b) extracting fatty acids from the microalgal culture of step (a);
  • step (c) it provides a method for producing biodiesel, characterized in that to produce a fatty acid ester by transesterifying the fatty acid of step (b).
  • Chlamydomonas reinhardtii CC-124, CC-620 and CC-621 were prepared from Chlamydomonas Resource Center (USA) and vector pCr102 was purchased from Korea Research Institute of Bioscience and Biotechnology (KRIBB).
  • the microalgae were cultured using a TAP (Tris-Acetate-Phosphate) medium developed by Gorman, DS and Levine, RP in the United States, and the composition of the TAP medium is shown in Table 1 below.
  • Table 1 TAP badge composition table Ingredient Name Final concentration Ammonium Chloride (NH 4 Cl) 0.375 g / l Magnesium Sulfate Heptahydrate (MgSO 4 ⁇ 7H 2 O) 0.1 g / l Calcium Chloride Dihydrate (CaCl 2 ⁇ 2H 2 O) 0.05 g / l Dipotassium Potassium Phosphate (K 2 HPO 4 ) 108 mg / l Potassium Phosphate Monobasic (KH 2 PO 4 ) 54 mg / l Tris 2.42 g / l Glacial acetic acid 1 ml / l Trace metal solution 1 ml / l
  • TAP-N medium was used as a condition for the operation of the inducible promoter, and the composition of the TAP-N medium consisted of only the ammonium chloride component missing from the TAP medium composition table of Table 1 above.
  • E. coli with the pCr102 vector was inoculated into LB liquid medium containing 100 mg / ml ampicillin (BD bioscience, USA) and incubated at 200 rpm for agitation for 18 hours, followed by plasmid using a mini-prep kit (QIAGEN, Germany). DNA was extracted.
  • genomic DNA of Chlamydomonas Reinhardty to be used as a cloning template of the promoter and gene was prepared. Genomic DNA was prepared using the HiGene TM Genomic DNA Prep Kit (Biofact, Korea) by harvesting the Klymonas Reinhardi CC-124 cultured in TAP medium for 4 days.
  • Nia1 forward primer (5'-GG GGTACC AACCGACCAATCGATAG-3 '; SEQ ID NO: 5, Kpn I underlined digit)
  • Nia1 and reverse primer (5 ' -CCC AAGCTT CCCGGGACTAGTACTGGCAGGATTCGGC-3 '; SEQ ID NO: 6, underlined Hind III site) and GAPDH forward primer (5'-G ACTAGT ATGCAGAAGGTGCGCAG-3'; SEQ ID NO: 7, underlined Spe I site) and GAPDH reverse primer (5'-TCCC)
  • Polymerase chain reaction (PCR) was performed using CCCGGG TTATTACGCCACCCACTTC-3 ′; SEQ ID NO: 8, underlined Sma I site).
  • PCR conditions were repeated 25 times with Nia1, denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, extension at 72 ° C for 30 seconds, and for GAPDH, denaturation at 94 ° C. 25 cycles of 30 seconds, annealing 49.8 ° C. 30 seconds, extension 72 ° C. 1 minute 45 seconds were performed.
  • PCR products were prepared using a gel extraction method.
  • the prepared plasmid DNA (pCr102 vector) you wrote was treated with Kpn I and Hind III restriction enzyme, was bonded was cut out of the original psaD promoter region, using the Nia1 promoter fragment prepared by amplifying by PCR ligation kit (Takara, Japan) . Thereafter, the recombinant vector containing the Nia1 promoter was treated with Spe I and Sma I restriction enzymes, and the GAPDH protein coding gene was inserted in the same manner.
  • the autolysin obtained through the crossing of the Chlamydomonas Reinhardi CC-620 and CC-621 strains was pre-treated with the CC-124 strain, and the cell wall was digested so that DNA could easily enter.
  • Recombinant vector containing Nia1 promoter and GAPDH protein coding gene was transformed into linear DNA by treatment with Xba I restriction enzyme, and 10 ⁇ l of 100 ng / ⁇ l linear DNA was added to the CC-124 cells and 20% polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the transformants were screened by subcultured in solid and liquid medium containing antibiotics, respectively.
  • the genomic DNA was extracted from the transformant and PCR was used to confirm the presence of the inserted gene.
  • the primer information used is shown in Table 2 below.
  • the recombinant vector used for transformation was used together as a control.
  • transformant # 7 showed a 55% increase in growth rate and a 31% higher FAME content in the medium without ammonium (FIG. 3). Transformant # 7 was named PNG and further studies were performed.
  • PNG Transformants into which wild-type and GAPDH protein coding genes were introduced were cultured in ammonium-free medium (TAP) and ammonium-free medium (TAP-N), and growth rates were observed by absorbance measurement and dry weight measurement. .
  • the absorbance at 750 nm confirmed that both transformants (PNG) and wild-type grew better in TAP medium with ammonium than TAP-N medium without ammonium.
  • Transformants cultured in TAP medium maintained absorbance values similar to or slightly higher than wild type.
  • the two species showed a different pattern, while the wild type maintained a constant level after 2 days of culture without change in absorbance, whereas the transformant had a steadily increasing absorbance until 4 days after the culture.
  • the culture showed similar results to the absorbance of the wild type cultured in TAP medium (FIG. 4).
  • the growth rate confirmed by measuring the dry weight set to the same volume was also confirmed in a form similar to the absorbance measurement results (Fig. 5).
  • GC Gas chromatograph
  • both the transformants and the wild type showed 9% level of FAME content, and it was confirmed that there was no significant difference between individuals.
  • both wild-type and transformants cultured in TAP medium containing ammonium maintained 8 to 9% of FAME content during the entire incubation period, and no difference was observed between wild-type and transformants.
  • the lipid content increased over time than in the TAP medium with ammonium in both wild-type and transformants.
  • the content of FAME increased by 20% on 0.5 days, 17% on 1 day, 31% on 2 days, and 49% on 4 days, compared to wild type, depending on the time of incubation.
  • Figure 7 is a comparison of the FAME yield in the medium without ammonium, the transformant was about 29% level immediately after the medium replacement, but after 4 days of culture 140%, 14 days in the results An increase of up to 321% was confirmed (FIG. 7).
  • the FAME productivity was calculated by dividing the FAME yield by the incubation period, and it was confirmed that the transformants in the TAP-N medium condition without ammonium had much higher productivity than the other conditions every hour. 8).

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Abstract

La présente invention concerne : un vecteur recombiné comprenant un promoteur inductible, induit en l'absence d'ammonium ou en présence de nitrate, et un gène codant la protéine glycéraldéhyde-3-phosphate déshydrogénase (GAPDH) sous l'effet du promoteur ; un procédé de production de micro-algues transgéniques présentant une productivité de biomasse et de lipides augmentée par transformation de cellules de microalgues au moyen du vecteur recombiné ; des micro-algues transgéniques produites selon le procédé ; une composition contenant le vecteur recombiné en tant que principe actif pour augmenter la productivité de biomasse et de lipides dans des microalgues ; un procédé de production de lipides par culture des micro-algues transgéniques ; et un procédé de fabrication de biodiesel à l'aide des micro-algues transgéniques.
PCT/KR2014/001179 2014-01-13 2014-02-13 Vecteur recombiné pour l'accroissement de la productivité de biomasse et de lipides de microalgues et son utilisation WO2015105233A1 (fr)

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KR1020140003840A KR101567308B1 (ko) 2014-01-13 2014-01-13 미세조류의 바이오매스와 지질 생산성을 증가시키기 위한 재조합 벡터 및 이의 용도

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KR101970063B1 (ko) 2017-08-11 2019-04-17 가톨릭관동대학교산학협력단 옥수수속대 추출물을 이용한 미세조류 배양방법
KR102100650B1 (ko) 2018-06-29 2020-04-16 씨제이제일제당 주식회사 신규한 트라우즈토카이트리움 속 균주, 및 이를 이용한 다중불포화지방산 생산방법
KR102582851B1 (ko) * 2020-08-12 2023-09-26 고려대학교 산학협력단 탄산탈수효소 복합체 및 이에 의한 이산화탄소 생물학적 고정화 및 지질 생산 강화 방법
WO2023224327A1 (fr) * 2022-05-20 2023-11-23 한국생명공학연구원 Système et procédé pour augmenter l'efficacité d'édition génique de microalgues à l'aide d'autolysine

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US20100330637A1 (en) * 2008-02-23 2010-12-30 James Weifu Lee Designer Organisms for Photobiological Butanol Production from Carbon Dioxide and Water
KR20110045865A (ko) * 2009-10-28 2011-05-04 한국에너지기술연구원 ｇｌｇＡ１ 유전자와 이를 포함하는 재조합벡터와 이로부터 형질전환된 숙주세포와 이의 생합성 제어를 통한 남세균 총지질 함량 조절 방법

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DATABASE NCBI 19 April 2010 (2010-04-19), "glyceraldehyde-3-phosphate dehydrogenase Chlamydomonas reinhardtii]", accession no. P_001689871 .1 *

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