Exploration and exploitation of the carotenoid pathway and the photoprotection mechanisms of Nannochloropsis oceanica

Ensuring food, energy, and raw materials for a growing global population presents a formidable challenge that necessitates innovative solutions. This thesis explores the domestication and genetic engineering of the microalga Nannochloropsis oceanica as a sustainable platform for the production of high-value biotechnological compounds. In Chapter 1, the unique characteristics of the Nannochloropsis genus are introduced, emphasizing its robust lipid accumulation and potential as a green bio-factory for nutraceuticals and biofuels. Chapter 2 details the genetic modification of N. oceanica via heterologous expression of a β-carotene ketolase from Chlamydomonas reinhardtii. This modification significantly enhanced the synthesis of ketocarotenoids—particularly canthaxanthin—with up to a 10-fold increase in ketocarotenoid content under specific light conditions. The modified strains, which utilize CO₂ as the sole carbon source, demonstrate promising applications in the pigmentation sectors of fish and poultry feed. Chapter 3 addresses the scalability and phenotypic stability of the genetically modified strain. Cultivation in a 25L tubular photobioreactor under repeated batch conditions revealed that the engineered strain maintains comparable growth to the wild type while accumulating significantly higher levels of ketocarotenoids. Additionally, the strain successfully co-produces omega-3 fatty acids, notably EPA, reinforcing its potential as a dual-production system. In Chapter 4, the thesis investigates the photoprotective mechanisms in N. oceanica, focusing on the xanthophyll cycle. By employing CRISPR-Cas12-mediated genome editing to create NoZEP2 knockout mutants, the study uncovers a regulatory role for NoZEP2 in modulating non-photochemical quenching (NPQ) dynamics and pigment interconversion under varying light conditions. These findings highlight the distinct functional roles of the two zeaxanthin epoxidase genes, with NoZEP1 being essential and NoZEP2 fine-tuning the photoprotective response. An appendix further examines the role of a glycosyltransferase family 8 gene (NoGLY) in carbon partitioning and cell wall biosynthesis. While NoGLY knockout does not significantly affect growth or lipid accumulation, its overexpression suggests an influence on cell wall integrity and polysaccharide biosynthesis. Collectively, the research presented in this thesis advances our understanding of microalgal biotechnology, providing a comprehensive framework for the development of N. oceanica as a versatile platform for sustainable industrial applications.