Biosynthesis of omega-3 fatty acids in Acutodesmus obliquus

Abstract

Omega-3 fatty acids (Omega-3 FAs) are known for their tremendous advantages to the health of body and brain. Omega-3 FAs have been extensively utilized in nutraceutical and aquaculture sectors. Microalgae has been recognized as a promising alternative vegetarian source to fish; the use of microalgae can reduce the risk of exposure to mercury contamination in fish. This study aims to identify new potential microalga strain to produce omega-3 FAs by optimisation of the culture condition, random mutation, and transcriptomic study. In this study, three microalgae namely, Acutodesmus obliquus, Chlorella sp., and Chlorella vulgaris were tested. The Acutodesmus obliquus was identified as a fast-growing oleaginous microalga with a growth rate of 0.09 day-1. It was also found that Acutodesmus obliquus had a high ability of producing omega-3 FAs as it contained 47% lipid and 38% ALA. To enhance omega-3 FAs production in Acutodesmus obliquus, nine sets of optimisation experiments involving four factors (sodium acetate, sodium nitrate, sodium chloride, and dipotassium hydrogen phosphate) with three levels of concentrations each, were conducted and analysed using the Taguchi method. The optimum culture condition was determined at 0.5 g/L of sodium acetate, 0.015 g/L of sodium nitrate, 0.58 g/L of sodium chloride and 0.0002 g/L of dipotassium hydrogen phosphate. Sodium acetate was identified as the primary limiting factor to produce omega-3 FAs. It accumulates a very long chain of omega-3 FAs which is eicosatetraenoic acid (ETA; C20:4) (7.3%) and rarely found in Acutodesmus obliquus. To genetically enhance omega-3 FAs (especially ETA), an ethyl methane sulfonate (EMS) based chemical mutagenesis approach followed by selective screening using quizalofop were conducted which led to the discovery of a novel mutant strain designated as Q2-12E. Not only this mutant showed a significantly improved growth than the wild type (original strain), but it also demonstrated a two-fold higher ETA production (17%) than the wild type (8%). Since the pathways and molecular mechanisms that triggered ETA accumulation in this species are still unknown, a transcriptome analysis has been performed. Based on the results, it is primarily discovered that there is the up-regulation of elongase expression in omega-3 FAs biosynthesis pathway in the mutants of Acutodesmus species. This explains the reason for the overaccumulation of ETA in mutant over the wild type in Acutodesmus species. The pioneer transcriptome analysis conducted leads to a better understanding on ETA metabolism in this EMS-quizalofop mutant strain, and subsequently, provides a new perspective of the genetic manipulation of nutritional supplement-producing microalgae. Overall, this study has identified that Acutodesmus obliquus has rapid growth, and high lipid content under optimised nutrient concentration results in enhanced ALA and ETA production. Moreover, random mutation leads to further enhancement of ETA content due to the upregulated expression of elongase.