Abstract
Ashbya gossypii is a natural overproducer of riboflavin or vitamin B2, and it is used nowadays in industrial scale for its production. This filamentous fungus is suitable for metabolic engineering approaches due to the development of a large number of molecular biology techniques, its fully sequenced genome and its high similarity to the model organism Saccharomyces cerevisiae. Additionally, it has been recently proposed as a paradigm in biolipid production, because of its highly active lipid metabolism and its ability to accumulate triacylglycerol.
We here propose the use of A. gossypii for the production of ricinoleic acid and linoleic acid, two high value lipids with different industrial applications. The first is widely used as a building block for biopolymers and the second is essential for human health. Interestingly, A. gossypii mainly accumulates oleic acid, which is the common substrate for the synthesis of ricinoleic and linoleic acid. With this purpose, we manipulated A. gossypii by the heterologous expression of a gene from Claviceps purpurea, CpFAH12, an enzyme than inserts either a hydroxyl group or a double bond in the twelfth carbon of oleic acid. In our conditions, ricinoleic acid is only synthesized in modest amounts in an engineered strain that achieves high lipid production. However, linoleic acid accumulation is produced up to 20 % with respect to total fatty acids in one of the engineered strains, 15-fold the wild type amount. The genetic background of this strain consists on the simultaneous overexpression of the two ?9 desaturases identified in A. gossypii, which catalyse the insertion of the first double bond in palmitic and stearic acid, producing palmitoleic and oleic acid respectively. This strain presents an increased lipid accumulation with respect to wild type as this desaturation step is one of the rate limiting steps in lipid synthesis. Therefore, CpFAH12 major contribution in A. gossypii lipid synthesis is related to its ?12 desaturase function, enhancing linoleic acid synthesis and representing a great starting point for metabolic engineering of this fungus for producing omega-6 and omega-3 fatty acids.
