Identification of a Yarrowia lipolytica acetamidase and its use as a yeast genetic marker

Maureen Hamilton,Vasiliki Tsakraklides,A Joe Shaw,Andrew L Consiglio,Kyle Macewen

doi:10.1186/s12934-020-1292-9

Abstract

BackgroundYarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks. Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. The potential to combine multiple genetic manipulations depends on the availability of multiple or recyclable selectable markers.ResultsWe found that Y. lipolytica has the ability to utilize acetamide as the sole nitrogen source suggesting that the genome contains an acetamidase gene. Two potential Y. lipolytica acetamidase gene candidates were identified by homology to the A. nidulans acetamidase amdS. These genes were deleted in the wild-type Y. lipolytica strain YB-392, and deletion strains were evaluated for acetamide utilization. One deletion strain was unable to grow on acetamide and a putative acetamidase gene YlAMD1 was identified. Transformation of YlAMD1 followed by selection on acetamide media and counterselection on fluoroacetamide media showed that YlAMD1 can be used as a recyclable genetic marker in Saccharomyces cerevisiae and Ylamd1Δ Y. lipolytica.ConclusionsThese findings add to our understanding of Y. lipolytica nitrogen utilization and expand the set of genetic tools available for engineering this organism, as well as S. cerevisiae.

Highlights

Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks
As there are no known acetamidase genes in Y. lipolytica, we searched for the gene(s) responsible for growth on acetamide
A. nidulans is a filamentous fungus and its amdS gene was functional in the yeast S. cerevisiae that shares many selection markers with Y. lipolytica

Summary

Introduction

Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. Yarrowia lipolytica is a non‐conventional yeast of interest to the biotechnology industry that is rapidly emerging as a valuable host for the production of a variety of lipid and non-lipid biochemicals [1]. It is a dimorphic, oleaginous yeast that has been approved by the US Food and Drug Administration for use in manufacturing processes that are generally regarded as safe (GRAS) [2]. Hamilton et al Microb Cell Fact (2020) 19:22 only the uracil auxotrophy marker is counterselectable and lends itself to marker recycling through the use of 5′-fluoroorotic acid

Methods

Results

Conclusion