Browsing by Author "Rey Jurado, Emma."

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    High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes
    (2019) Osorio, Hector.; Jara Lagos, Carol Andrea.; Fuenzalida Morales, Karen Andrea; Rey Jurado, Emma.; Vásquez Pérez, Luz Mónica
    Abstract Background One of the major problems in the production of lipids for biotechnological purposes using microalgae is maintaining a high productivity of these molecules without reducing cellular biomass. High production rates are usually obtained by cultivating microalgae under different stress conditions. However, many of these changes usually result in lower biomass productivity. Therefore, the optimization of the culture conditions and genetic modification techniques in these organisms is needed to generate robust new strains for profitable economic use. Results In this work, we describe a new strategy for random mutation of genomic DNA in the microalgae Nannochloropsis oceanica by insertion of a Transposome complex Tn5. This complex contains an antibiotic-resistance cassette commanded by a CMV viral promoter that allows high efficiency of transformation and the generation of mutants. This strategy, complemented with a large-scale identification and selection system for mutants, such as flow cytometry with cell selection, allowed us to obtain clonal cultures of mutants with altered phenotypes in the accumulation of intracellular lipids. The characterization of some of these mutants uncovered new genes that are likely to be involved in the regulation of lipid synthesis, revealing possible cellular responses that influence the intracellular homeostasis of lipids. Conclusion The strategies proposed here are easy to implement in different types of microalgae and provide a promising scenario for improving biotechnological applications.Abstract Background One of the major problems in the production of lipids for biotechnological purposes using microalgae is maintaining a high productivity of these molecules without reducing cellular biomass. High production rates are usually obtained by cultivating microalgae under different stress conditions. However, many of these changes usually result in lower biomass productivity. Therefore, the optimization of the culture conditions and genetic modification techniques in these organisms is needed to generate robust new strains for profitable economic use. Results In this work, we describe a new strategy for random mutation of genomic DNA in the microalgae Nannochloropsis oceanica by insertion of a Transposome complex Tn5. This complex contains an antibiotic-resistance cassette commanded by a CMV viral promoter that allows high efficiency of transformation and the generation of mutants. This strategy, complemented with a large-scale identification and selection system for mutants, such as flow cytometry with cell selection, allowed us to obtain clonal cultures of mutants with altered phenotypes in the accumulation of intracellular lipids. The characterization of some of these mutants uncovered new genes that are likely to be involved in the regulation of lipid synthesis, revealing possible cellular responses that influence the intracellular homeostasis of lipids. Conclusion The strategies proposed here are easy to implement in different types of microalgae and provide a promising scenario for improving biotechnological applications.