High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica
- 64 Downloads
The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 μg mL−1 of zeocin or 5 μg mL−1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10−8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10–11 × 10−6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well.
KeywordsBiofuels Electroporation Freshwater microalgae Nannochloropsis limnetica Nuclear transformation
This work was supported by the National Key R&D Program of China (Grant No. 2018YFD0901500) and National Natural Science Foundation of China (Grant No. 91751117).
HH, taking responsibility for the integrity of the work as a whole, designed the research, analyzed the data and wrote the manuscript. YC performed the experiments and analyze the data. All authors agree on the authorship and submission of the manuscript for peer review.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
No informed consent, human or animal rights applicable.
- Sukenik A (1998) Production of eicosapentaenoic acid by the marine eustigmatophyte Nannochloropsis sp. In: Cohen Z (ed) Chemicals from microalgae. Taylor and Francis, London, pp 41–56Google Scholar
- Vieler A, Wu G, Tsai CH, Bullard B, Cornish AJ, Harvey C, Reca IB, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MS, Cavalier D, Childs KL, Clark TJ, Deshpande R, Erickson E, Ferguson AA, Handee W, Kong Q, Li X, Liu B, Lundback S, Peng C, Roston RL, Sanjaya Simpson JP, TerBush A, Warakanont J, Zäuner S, Farre EM, Hegg EL, Jiang N, Kuo MH, Lu Y, Niyogi KK, Ohlrogge J, Osteryoung KW, Shachar-Hill Y, Sears BB, Sun Y, Takahashi H, Yandell M, Shiu SH, Benning C (2012) Genome, functional gene annotation, and nuclear transformation of the Heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8(11):e1003064CrossRefGoogle Scholar
- Wang D, Ning K, Li J, Hu J, Han D, Wang H, Zeng X, Jing X, Zhou Q, Su X, Chang X, Wang A, Wang W, Jia J, Wei L, Xin Y, Qiao Y, Huang R, Chen J, Han B, Yoon K, Hill RT, Zohar Y, Chen F, Hu Q, Xu J (2014) Nannochloropsis genomes reveal evolution of microalgal oleaginous traits. PLoS Genet 10(1):e1004094CrossRefGoogle Scholar