Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris

Kumar, Manu; Jeon, Jihyeong; Choi, Juyoung; Kim, Seong-Ryong

doi:10.1007/s10811-018-1396-3

Cited by 42 publications

(25 citation statements)

References 51 publications

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“…Chlorella vulgaris UTEX 395 and arctic Chlorella sp. ArM0029B 28 were grown in BG11 medium 65, including 3% glucose at 25 °C under constant illumination (50-60 μmol photons m −2 s −1 ) on a rotary shaker (150 rpm) in a multi-spin shaker (Vision Sciences, Korea) 15 . For the transformation of Chlorella, we used electroporation, as previously described 15 .…”

Section: Methodsmentioning

confidence: 99%

“…2). Those vectors were introduced into Chlorella using the electroporation method described by Kumar et al 15 .…”

Section: Construction Of Hg-csf Expression Vectors Driven By Cvndi Prmentioning

confidence: 99%

“…The number of genes assigned to each GO term was counted using in-house scripts produced by SEEDERS Co. extraction of nucleic acids and pcR. To extract nucleic acids, Chlorella cells were harvested by centrifugation, frozen quickly using liquid N, and then ground using a mixer mill MM 300 (Qiagen, Germany) as described by Kumar et al 15 . Genomic DNA was extracted from the powder using the cetyl trimethylammonium bromide method 70 .…”

Section: Short Read Mapping and Identification Of Differentially Exprmentioning

confidence: 99%

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The establishment of new protein expression system using N starvation inducible promoters in Chlorella

Shin

Choi

Jeon

et al. 2020

Sci Rep

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Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-cSf) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-cSf, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codonoptimized hG-CSF sequence, hG-cSf polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella. Chlorella is a single-celled eukaryote belonging to the green algae. It can grow phototrophically, heterotrophically, or mixotrophically to high biomass, producing different amounts of proteins and lipids 1. It grows rapidly, dividing into four cells in about 6 h. Because Chlorella synthesizes large amounts of proteins and lipids, it has been used as a raw material for biodiesel and also as a food additive 2-4. Chlorella undergoes posttranslational modifications such as protein N-glycosylation 5. The use of Chlorella for the production of valuable proteins has therefore been attempted for more than two decades 6-9. Chen et al. 10 showed expression of rabbit neutrophil peptide using a translational enhancer omega in Chlorella ellipsoidea. Kim et al. 11 produced flounder growth hormone using a 35S cauliflower mosaic virus (35S) promoter in C. ellipsoidea and showed a 25% growth increase of flounder fry. No recombinant proteins produced from Chlorella species have yet been commercialized, although the species has been studied intensively for the potential production of biodiesel. An optimized expression system for producing proteins in Chlorella is urgently needed. Recombinant protein production studies require a highly efficient method for the transformation of Chlorella. Various transformation techniques, including electroporation, PEG transformation, particle bombardment, and Agrobacterium cocultivation, have been used for the efficient transformation of Chlorella spp. 10-14. Recently, Kumar et al. 15 reported an effective method for enhancing transformation efficiency by more than 100-fold using electroporation combined with efficient protoplasting of Chlorella. Besides efficient transformation, an optimized system for the endogenous expression of proteins is indispensable. An expression system involving appropriate promoters is one of the most important factors for highefficiency production of recombinant proteins. In Chlamydomonas reinhardtii, the promoter of the heat shock protein gene HSP70a has been widely used for the expression of foreign genes in C. reinhardtii 16,17. In recent yea...

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Section: Methodsmentioning

confidence: 99%

“…2). Those vectors were introduced into Chlorella using the electroporation method described by Kumar et al 15 .…”

Section: Construction Of Hg-csf Expression Vectors Driven By Cvndi Prmentioning

confidence: 99%

Section: Short Read Mapping and Identification Of Differentially Exprmentioning

confidence: 99%

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The establishment of new protein expression system using N starvation inducible promoters in Chlorella

Shin

Choi

Jeon

et al. 2020

Sci Rep

Self Cite

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“…Nuclear transformation of C. reinhardtii may be achieved by several methods such as electroporation [ 120 ], Agrobacterium -mediated transformation [ 121 ], silicon carbide whiskers and positively-charged aminoclay nanoparticles [ 122 , 123 ] and glass beads agitation method [ 124 ]. Both electroporation and Agrobacterium -mediated transformation also succeeded in transforming algae of economic interest such as C. vulgaris , Neochloris oleoabundans and H. pluvialis [ 125 – 127 ]. The introduction of foreign genes into the nuclear genome of microalgae is generally guided by a random integration event [ 128 ].…”

Section: Main Textmentioning

confidence: 99%

Biomass from microalgae: the potential of domestication towards sustainable biofactories

et al. 2018

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Interest in bulk biomass from microalgae, for the extraction of high-value nutraceuticals, bio-products, animal feed and as a source of renewable fuels, is high. Advantages of microalgal vs. plant biomass production include higher yield, use of non-arable land, recovery of nutrients from wastewater, efficient carbon capture and faster development of new domesticated strains. Moreover, adaptation to a wide range of environmental conditions evolved a great genetic diversity within this polyphyletic group, making microalgae a rich source of interesting and useful metabolites. Microalgae have the potential to satisfy many global demands; however, realization of this potential requires a decrease of the current production costs. Average productivity of the most common industrial strains is far lower than maximal theoretical estimations, suggesting that identification of factors limiting biomass yield and removing bottlenecks are pivotal in domestication strategies aimed to make algal-derived bio-products profitable on the industrial scale. In particular, the light-to-biomass conversion efficiency represents a major constraint to finally fill the gap between theoretical and industrial productivity. In this respect, recent results suggest that significant yield enhancement is feasible. Full realization of this potential requires further advances in cultivation techniques, together with genetic manipulation of both algal physiology and metabolic networks, to maximize the efficiency with which solar energy is converted into biomass and bio-products. In this review, we draft the molecular events of photosynthesis which regulate the conversion of light into biomass, and discuss how these can be targeted to enhance productivity through mutagenesis, strain selection or genetic engineering. We outline major successes reached, and promising strategies to achieving significant contributions to future microalgae-based biotechnology.

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“…To date, suitable transformation techniques have been established only in several limited unicellular models of eukaryotic microalgae, such as Chlamydomonas reinhardtii [10,11], Nannochloropsis sp. [12,13], Chlorella [14], and Phaedodactylum tricornutum [15]. Unfortunately, no research group has reported the stable genetic transformation of filamentous eukaryotic microalgae, including those from the Tribonema genus.…”

Section: Introductionmentioning

confidence: 99%

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga

Zhang

Wang

Yang

et al. 2020

IJMS

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Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptII plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase II-encoding G418-resistant gene (nptII) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub-nptII were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

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Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris

Cited by 42 publications

References 51 publications

The establishment of new protein expression system using N starvation inducible promoters in Chlorella

The establishment of new protein expression system using N starvation inducible promoters in Chlorella

Biomass from microalgae: the potential of domestication towards sustainable biofactories

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga

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