Production of mutants in oleaginous microalgae to increase lipid content under variable light, salinity, and nitrogen conditions
Author: Elan Alexis Téllez Pueblas (Currículo Lattes)
Advisor Dr. Luis Fernando Fernandes Marins
Abstract
This thesis investigates the identification, cultivation strategies, and genetic manipulation of microalgae strains using random mutagenesis and targeted mutation through CRISPR/Cas9 RNP technology, with the aim of increasing lipid production—particularly neutral lipids—for potential application in biodiesel production. Initially, the strains were identified as Scenedesmus obliquus and an undescribed species of the Desmodesmus genus using molecular taxonomy techniques. Subsequently, in the random mutagenesis strategy, UV-C light irradiation was applied to induce random mutations, resulting in mutants of the Desmodesmus sp. strain with total (tM-10) and partial (tN-30) inhibition of starch synthesis. Both mutants showed a significant increase in neutral lipid levels compared to the wild type. The inhibition of starch synthesis redirected the carbon flux toward triacylglycerol (TAG) synthesis without compromising mutant growth. Additionally, cultivation strategies such as salinity adjustment, light intensity modulation, and nitrogen deficiency in two-stage cultures were validated to maximize biomass and neutral lipid production in the tN-30 mutant, demonstrating its viability under stress conditions. Fatty acid profile analyses and gene sequencing related to starch and cell wall synthesis were also performed, alongside the development of RNA extraction protocols and guide RNA validation for the Cas9 endonuclease, used in the implementation of CRISPR/Cas9 technology in both microalgal strains. The results suggest that, for the Desmodesmus sp. strain, random mutagenesis via UV light irradiation is an effective approach for generating mutants with potential for neutral lipid production, highlighting its relationship with CRISPR/Cas9 RNP technology. Overall, the findings of this thesis emphasize the importance of genetic tools in enhancing the biotechnological use of microalgae, particularly the tN-30 strain, for biodiesel production.