Genome Editing in Y. lipolytica Using TALENs.

TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied.

Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.

CRISPR/Cas9 is a powerful tool to edit the genome of the yeast . Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family.

An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing.

The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%.

Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering.

Background: Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest.

Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS.