Novel microbes for high level production of the omega3-fatty acid DHA and astaxanthin from biomass feedstocks

Thraustochytrids are marine eukaryotic microorganisms, where some species produce high levels of the omega-3 fatty acid docosahexaenoic acid (DHA). In previous, collaborative projects at SINTEF and NTNU, a collection of thraustochytrid strains was established. Strains were identified that accumulated 60% fat of cell dry weight, where DHA accounted for more than 30%. Some of these strains additionally produced the carotenoid astaxanthin. The aim of the ThraustoEng project was to establish knowledge and technology to utilise thraustochytrids as microbial cell factories for the production of DHA and astaxanthin from sustainable raw materials. At the start of the project no complete genome sequences had been published from thraustochytrids, and methods for gene transfer in these organisms were extremely underdeveloped. Thus, the development of genetic tools, genome sequencing, and physiological studies were central goals and activities within the project. Based on the productivity of DHA and astaxanthin content, the two strains Aurantiochytrium sp. S61 and T66, were selected for genome sequencing. Approximately 12 000 genes were predicted for each of the strains. Approx. 50% of the genes could be functionally mapped in the metabolic networks of the cell, while 22% are novel. Due to the limited previous studies, it was expected that establishment of methods for gene transfer would be a challenge. Within the project, we have now established protocols for gene transfer by both conjugation and electroporation, and homologous recombination has been demonstrated. New genetic constructs for increased expression of selected genes have been generated. As an alternative approach for strain improvement, evolutionary engineering is applied. Mutant libraries have been established and new, miniaturized high-throughputhigh analytical methods are Applied for evaluation of the mutants.

Fish as food for human consumption is associated with health benefits, and one reason for this is the high content of omega3 polyunsaturated fatty acids (PUFAs), which originates from the feed. For the fish farming industry it is therefore of critical imp ortance to have access to sufficient quantities of these compounds, in particular DHA. There are several options for solving the problem, but most of them face serious economic or technical obstacles. Here we propose to study a group of heterotrophic micr oalgae, the thraustochytrids, which we believe represents currently the most realistic DHA source for the fish farming industry. Thraustochytrids are already used for production of PUFAs with a very high efficiency for direct human consumption. As a sourc e for fish feed the main problem is that the relative DHA content must be significantly increased, and here we propose innovative ways to achieve this in strains that simultaneously produce the important feed additive pigment astaxanthin. The main scienti fic challenge is that the basic understanding of the thraustochytrid biology is very limited, as for example not even genome sequences have been reported. This is obviously no longer a technical problem to overcome, so we propose that the timing is now ri ght to establish genome sequences and use 'omics technologies to obtain a detailed understanding of PUFA and carotenoid biosynthesis. Gene modification technologies are to some extent available and we will expand the potential of such methods. Evolutionar y engineering will be used to select strains with the desired phenotypes from a library of randomly distributed mutants, and inverse metabolic engineering will be used to characterize the identified mutations. Various carbon sources can be used for cultiv ation of thraustochytrids, and in this project we will investigate the potential of using raw materials that are available in large quantities, and that originate from agriculture or marine sea-weeds.