Combinatorial Metabolic Engineering of Alkane Biosynthesis in the Osmotolerant Yeast Debaryomyces hansenii CBS 767

Abstract

ABSTRACT Debaryomyces hansenii is a yeast with diverse potential applications like producing oleochemicals from biomass. Yet, combinatorial metabolic engineering in D. hansenii is limited because, like many nonconventional organisms, key genomic data and genetic elements are missing. Here, we report phenotypic characterization, genomic integration loci, and modular genetic parts that enable combinatorial metabolic engineering of alkane biosynthesis in D. hansenii . Phenotypic characterization revealed that D. hansenii produces lipids when grown on components of lignocellulosic and algal biomass in standard and saline media. Notably, D. hansenii produced 57.25% more lipids than Yarrowia lipolytica Po1f when grown on glucose. Next, genomic integration loci were identified and gene expression elements (using known homology to S. cerevisiae ) were derived from the D. hansenii CBS767 genome. We derived 23 promoters and 24 terminators, recoded known selection markers and reporters, designed genomic integration plasmids, and made all compatible with a modular cloning standard. Flow cytometry measurements show that the promoters span an expression range of three orders of magnitude and the terminators span one order of magnitude. To demonstrate combinatorial metabolic engineering, the parts collection was used to assemble 18 alkane biosynthesis pathways. With no other genetic modifications, the best strain produced 38.31 mg/L and 1.47 mg/L of heptadecane from glucose and arabinose, respectively. This work establishes D. hansenii as a cell factory for synthesizing oleochemicals from lignocellulose derived sugars while providing a blueprint for leveraging phenotyping, genomics, and modular parts collections with automation platforms to develop cell factories from nonconventional organisms. Furthermore, the genetic parts collection now enables functional genetics for various applications in Debaryomyces yeasts.