High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes

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.

Microalgae as Sustainable Bio-Factories of Healthy Lipids: Evaluating Fatty Acid Content and Antioxidant Activity

“…Nonetheless, EPA was found in significant high abundances in other microalgae, particularly in N. oceanica, which lacks ALA. In line with our results, previous studies reported a total absence of ALA and high abundances of EPA [77][78][79][80][81][82].…”

Effect of tobacco smoke on the growth, biochemical composition, and biodiesel characteristics of two Chlamydomonas strains

“…Palmitic acid was the dominant constituent of SFAs, and showed signi cant changes upon culture treatment by tobacco smoke. It is known that palmitic acid is the most common component of the cell membrane and is the dominant SFAs [41]. Its signi cant changes in response to salinity stress have been reported previously [42,43], suggesting its possible role in acclimation to stress conditions.…”

Genetic Modification of the HSP90 Gene Using CRISPR-Cas9 to Enhance Thermotolerance in T. Suecica

“…By splicing HSP90 into a plasmid that codes for antibiotic resistance, the insertion of this plasmid can be tested by treating the host with antibiotics [33]. This is accomplished with a plasmid that contains the code for zeocin antibiotic resistance, pMOD-zeo, which will be ligated to the HSP90 HA plasmid using the restriction endonucleases NcoI-Styl and BseRI [34][35]. Using polymerase chain reaction (PCR) at 96°C, this mutant plasmid will be linearized to create free ends that can bind to T. suecica DNA.…”