Microbial production of omega-3 fatty acids – a model based approach.

Highlights 2021

AurOmega (2017-2021) aimed to obtain a knowledge base on the biosynthesis of the omega 3 fatty acid DHA in the marine microorganisms thraustochytrids. Thraustochytrids can be cultivated at high cell concentrations and are extremely promising microorganisms for the development of economically competitive production processes for omega-3 fatty acids.

However, more basic knowledge about DHA biosynthesis and lipid accumulation is needed in order to achieve yields and productivities that are competitive with traditional DHA raw material sources as fish oil. The project comprised three main scientific areas: microbial strain construction, strain and bioprocess characterization and modeling.

Earlier in the project, a genome-scale metabolic model for our in-house strain T66 was constructed, validated, and published. Methods for high-resolution metabolite and lipid profiles of thraustochytrids have been established and published. The quantitative metabolite profiling is based on six different LC-MS/MS methods and includes challenging metabolite groups as CoAs and NADs. Such profiling provides deep insight into the central metabolism and lipid biosynthesis pathway of thraustochytrids during growth and lipid accumulation stages.

The high-resolution lipid profiling is based on supercritical fluid chromatography with MS/MS detection (published in a separate paper). The two triglyceride lipids TG 16:0_16:0_22:6 and 16:0_22:6_22:6 (16:0 palmitic acid, 22:6 – DHA) dominate in the intracellular lipid storage droplets, but we were also able to detect trace amounts of a triglyceride with DHA in all three acyl positions. In 2021 the research focuses and applying these tools to characterize and compare the type strain Aurantiochytrium limacinum SR21 with strain T66. This has challenged the previous hypothesis and generated new regarding which parts of the metabolism might be manipulated to increase the incorporation of omega 3 fatty acids into the produced triacylglycerols.

One highlight in 2021 has been to identify the enzyme that creates the first double bond in C16:0 and two enzymes that could generate cholesteryl esters. In both cases the specific function of these enzymes in fatty acid and lipid biosynthesis could not have been predicted bioinformatically, emphasizing that thraustochytrids are evolutionary distant from well-studied organisms. The results have been published in three papers.

In AurOmega, researchers with experience from the fields of synthetic biology, systems biology, bioprocess technology, analytics, and mathematical modelling cooperated and met frequently to ensure that everyone knew the challenges the other researchers were facing and contributed with their knowledge to aid in solving those challenges. It was only through this transdisciplinary approach we reached the ambitious goals of AurOmega.

Project overview

Project lead: Per Bruheim
Institution: NTNU
Partners: SINTEF
Duration: Start-up 2017

Research group

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 the 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 omega-3 fatty acid bioprocesses.

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 in the triacylglycerol storage lipids. The acquired new knowledge will be translated into enhanced DHA production capabilities of selected thraustochytrid strains and laying the foundation 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.