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

Zhong-Peng Guo,Sophie Duquesne,Florence Bordes,Christian Croux,Vinciane Borsenberger,Gilles Truan,Alain Marty

doi:10.1038/s42003-020-0936-y

Abstract

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%. Additionally, our results reveal that ylAC can be genetically maintained over multiple generations either under selective conditions or, without selective pressure, using an essential gene as the selection marker. Overall, the ylACs reported herein are game-changing technology for Y. lipolytica, opening myriad possibilities, including enzyme screening, genome studies and the use of this yeast as a previous unutilized bio-manufacturing platform.

Highlights

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
Instead of using ligation based Yeast artificial chromosomes (YACs) construction as described previously[44], we investigated whether the genes could be directly assembled in vivo using homologous recombination (HR) of Y. lipolytica
Robust method for the assembly of stable, functional artificial chromosomes capable of driving gene expression, we explored the use of Y. lipolytica-specific artificial chromosome (ylAC) for large pathway engineering

Summary

Introduction

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. Alone at the bottom of the hemiascomycetous yeast tree, Yarrowia lipolytica is phylogenetically distant from the industrial work-horse Saccharomyces cerevisiae and all other well-studied yeast species[1] This unconventional obligate aerobe, can feed on a wide range of carbon sources (including lipids, n-alkanes and glycerol), accumulate high cellular content of lipids and secrete large amounts of organic acids, such as citric acid, isocitric acid, and α-ketoglutaric acid[2,3] and even proteins[4]. These desirable physiological traits and its “generally recognized as safe” (GRAS) status[5], have made Y. lipolytica a strain of interest for the detergents, food, pharmaceutical and environmental industries[4,6,7]. Sequential genome insertions are generally hampered by decreasing transformation efficiencies and progressive loss of strain fitness[27]

Methods

Results

Conclusion