Tuning Fatty Acid Profile and Yield in Pichia pastoris.

Fatty acids have been supplied for diverse non-food, industrial applications from plant oils and animal fats for many decades. Due to the massively increasing world population demanding a nutritious diet and the thrive to provide feedstocks for industrial production lines in a sustainable way, i.e., independent from food supply chains, alternative fatty acid sources have massively gained in importance. Carbohydrate-rich side-streams of agricultural production, e.g., molasses, lignocellulosic waste, glycerol from biodiesel production, and even CO2, are considered and employed as carbon sources for the fermentative accumulation of fatty acids in selected microbial hosts. While certain fatty acid species are readily accumulated in native microbial metabolic routes, other fatty acid species are scarce, and host strains need to be metabolically engineered for their high-level production. We report the metabolic engineering of Pichia pastoris to produce palmitoleic acid from glucose and discuss the beneficial and detrimental engineering steps in detail. Fatty acid secretion was achieved through the deletion of fatty acyl-CoA synthetases and overexpression of the truncated E. coli thioesterase 'TesA. The best strains secreted >1 g/L free fatty acids into the culture medium. Additionally, the introduction of C16-specific ∆9-desaturases and fatty acid synthases, coupled with improved cultivation conditions, increased the palmitoleic acid content from 5.5% to 22%.

Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris.

Gene knockout is a key technology in the development of cell factories and basic research alike. The methylotrophic yeast Pichia pastoris is typically employed as a producer of proteins and of fine chemicals, due to its ability to accumulate high cell densities in conjunction with a set of strong inducible promoters. However, protocols for genome engineering in this host are still cumbersome and time-consuming. Moreover, extensive genome engineering raises the need for a multitude of selection markers, which are limited in P. pastoris. In this chapter, we describe a fast and efficient method for gene disruption in P. pastoris that utilizes marker recycling to enable repetitive genome engineering cycles. A set of ready-to-use knockout vectors simplifies cloning procedures and facilitates quick knockout generation.