A molecular genetic toolbox for Yarrowia lipolytica

Abstract

Background Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.ResultsWe have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the “Yarrowia lipolytica Cell Atlas,” a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.ConclusionsThese molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0687-7) contains supplementary material, which is available to authorized users.

Phosphatidate phosphatase activity is induced during lipogenesis in the oleaginous yeast Yarrowia lipolytica

“…In high-glucose cells where TAG biosynthesis was induced (Figure 2a), there was an increase in the PA phosphatase activity associated with the membrane fraction (Figure 3b). A plausible explanation for this increase in PA phosphatase activity is the translocation of the Pah1 PA phosphatase from the cytosol where it is mostly found (Bredeweg et al, 2017) to the membrane fraction. In S. cerevisiae, Pah1 must translocate from the cytosol to the membrane fraction and specifically to the nuclear/endoplasmic reticulum membrane to catalyze its reaction .…”

“…In Yarrowia lipolytica, the Pah1 homologue localizes in the cytosol (Bredeweg et al, 2017), but the PA phosphatase activity is much higher in the membrane fraction (Hardman, McFalls, & Fakas, 2017).…”

“…That is because the substrate PA localizes in the membrane fraction, which means that only the membrane‐associated activity is physiologically relevant. In Yarrowia lipolytica , the Pah1 homologue localizes in the cytosol (Bredeweg et al, ), but the PA phosphatase activity is much higher in the membrane fraction (Hardman, McFalls, & Fakas, ).…”

The art of vector engineering: towards the construction of next‐generation genetic tools

“…Saccharomyces cerevisiae is a model organism with a very well-annotated genome, thus it has been essential for the evolution of the ever-growing field of genetic engineering (Nielsen et al, 2013). However, genetic tools such as vectors and integrating cassettes have been developed for non-conventional yeast as well, considering their rising importance in biotechnology, such as Pichia pastoris (Cereghino and Cregg, 2000), Kluveromyces lactis (Van Ooyen et al, 2006) and Yarrowia lipolytica (Bredeweg et al, 2017). For an expanded review of non-conventional yeasts, tools refer to Wagner and Alper (2016).…”

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing inYarrowia lipolytica

“…Genome annotation indicates this yeast contains more than 200 hydrophobic compounds assimilation pathways associated with alkane uptake, lipid oxidation and VFA detoxification (Fickers, Benetti et al 2005) et al This feature makes this yeast a superior host to utilize recalcitrant waste/toxic substrates for eco-friendly production of green chemicals . Due to its prominent industrial potential, a significant amount of work has been focused to develop genetic toolbox in this yeast, ranging from protein expression (Juretzek, Le Dall et al 2001, Nicaud, Madzak et al 2002, Bordes, Fudalej et al 2007, promoter characterization (Blazeck, Liu et al 2011, Blazeck, Reed et al 2013, gene deletions (Bredeweg, Pomraning et al 2017, Jang, Yu et al 2018, YaliBrick-based cloning (Wong, Engel et al 2017, Wong, Holdridge et al 2019), Golden-gate cloning (Celińska, Ledesma-Amaro et al 2017), Piggybac transposon (Wagner, Williams et al 2018), iterative gene integration (Gao, Tong et al 2017, Lv, Edwards et al 2019 to CRISPR/Cas9-mediated genome-editing (Schwartz, Hussain et al 2016, Wong, Engel et al 2017, Holkenbrink, Dam et al 2018) et al This genetic toolbox affords us a collection of facile genetic tools for streamlined and accelerated pathway engineering in oleaginous yeast species.…”