DL: Microbial production of omega-3 fatty acids - a model based approach

The long-chain omega-3 fatty acids EPA and DHA are essential for humans, as well as for marine fish species. The current source is fish oil. As wild fish catches cannot be further increased, continued growth of the marine aquaculture, in Norway and globally, is now seriously constrained by the availability of fish oil. New, sustainable sources of EPA and DHA are needed. Thraustochytrids are unicellular eukaryotic microorganisms, able to accumulate high levels of lipids containing high amounts of DHA. They can be cultivated at high cell concentration and are extremely promising organisms for development of economic competitive production processes for omega-3 fatty acids. Despite many years of research, there is still a lack of basic understanding of fatty acid synthesis in thraustochytrids, where DHA and saturated fatty acids are produced by two competing pathways. The AurOmega partners NTNU and SINTEF have over the last decade isolated a high number of thraustochytrid strains and characterized their lipid-producing potential. The systems biology approach of AurOmega has provided an enhanced understanding of what limits the DHA synthesis in thraustochytrids and how it can be improved. The project utilized an iterative approach applying high integration of experimental disciplines, where extensive -omics analyses and mathematical modelling is being used. A genome scale metabolic model was constructed and validated with experimental data. This model enables simulations to predict metabolic performance profiles, and complex network analysis to identify key regulatory features of DHA-synthesis, especially focusing on increasing the rate of DHA-synthesis and introduction into the storage lipids. Six mass spectrometry based analytical methods for quantifying the metabolome was developed or optimized for thraustochytrids. These methods cover central metabolic pathways and provides a high-resolution profile of the intracellular metabolic state during growth and lipid accumulation phases. Mass spectrometry-based methodology for analysis of the fatty acid composition, lipid class distribution and detection of individual lipid species have also been developed and applied on the project. These methods, in addition to proteome analyses, have been used to analyze high resolution time series from bioreactor cultivations. We have also studied the effect of inhibiting pathways directly competing with the DHA synthesis. Methods for genome editing has been established and used to study a fatty acid dehydrogenase and two enzymes involved in the synthesis of storage lipids. The acquired new knowledge can be translated into enhanced DHA production capabilities of selected thraustochytrid strains. This will form the basis for a sustainable and economically feasible industrial omega-3 fatty acid production process, thereby enabling further growth of one of the most important industries in Norway.

Målet for prosjektet var å etablere ein kunnskapsbase for DHA-syntese og lipidakkumulering i DHA-produserande thraustochytridar for deretter å kunne auke produktiviteten deira. Dei utvikla metodane for analyser av metabolittar, og ikkje minst lipidomet har stor overføringsverdi og er alt nytta i andre prosjekt. Etter prosjektet har vi vesentleg større kunnskap om lipidmetabolismen i to thraustochytride-artar og kva som kan vere dei avgrensande trinna for å få lipid med høgare innhald av DHA. Dette gjer at målet om å utnytte desse organismane til kommersiell produksjon av DHA-rik olje til ein akseptabel pris for bruk i fôr er nærare.

The long-chain omega-3 fatty acids EPA and DHA are essential for humans, as well as for marine fish species. The current source is fish oil. As wild fish catches cannot be further increased, continued growth of marine aquaculture, in Norway and globally, is now seriously constrained by the availability of fish oil. New, sustainable sources of EPA and DHA are needed. Thraustochytrids are unicellular eukaryotic microorganisms, able to accumulate high levels of lipids. They can be cultivated at high cell concentration and are extremely promising organisms for development of economic competitive production processes for omega-3 fatty acids. Despite many years of research, there is still a lack of basic understanding of fatty acid synthesis in thraustochytrids, where DHA and saturated fatty acids are produced by two competing pathways. AurOmega partners NTNU and SINTEF have over the last decade isolated a high number of thraustochytrid strains and characterized their lipid-producing potential. The systems biology approach in AurOmega will provide an enhanced understanding of what limits the DHA synthesis in thraustochytrids and how it can be improved. An iterative approach applying high integration of experimental disciplines, with extensive omics analyses and mathematical modelling will be used. The mathematical and computational analysis will be based on genome-scale metabolic reconstruction and simulations to predict metabolic performance profiles, and complex network analysis to identify key regulatory features of DHA-synthesis, with particular focus of increasing the rate of DHA-synthesis and introduction into the storage lipids. The acquired new knowledge will be translated into enhanced DHA production capabilities of selected thraustochytrid strains. This will form the basis for a sustainable and economically feasible industrial omega-3 fatty acid production process, thereby enabling further growth of one of the most important industries in Norway.