Abstract
BackgroundOne 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.ResultsIn 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.ConclusionThe strategies proposed here are easy to implement in different types of microalgae and provide a promising scenario for improving biotechnological applications.
Highlights
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
The expression of the antibiotic selection gene is under the transcriptional control of two promoters: the CMV viral promoter, which has been widely described in a broad range of cell types and is the most commonly used promoter in mammalian expression plasmids [42], and the EM7 promoter, a synthetic promoter based on the bacteriophage T7 promoter for expression of the Zeocin resistance factor in E. coli (Fig. 1a)
We found the highest transformation efficiency in Nannochloropsis oceanica cells (1.5 × 10−2 transformants per μg DNA), compared with those described for this type of microalgae and other microalgae models
Summary
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. Many of these changes usually result in lower biomass productivity. Fossil fuels are non-renewable sources of energy, increasingly in demand and currently insufficient to cover global energy needs [1]. Microorganisms are good candidates for the production of biodiesel because of their short life cycles, Nowadays, microalgae are considered natural factories of bioactive compounds useful for different biotechnological applications. The lipid content of Chlamydomonas, Porphyridium, Dunaliella, Isochrysis, Tetraselmis, Phaeodactylum, Nannochloropsis, Chlorella and Schizochytrium species varies between 20 and 50% of dry weight. Species of the Osorio et al Biotechnol Biofuels (2019) 12:134
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