Combinatorial metabolic engineering of alkane biosynthesis in the osmotolerant yeast Debaryomyces hansenii CBS 767

Abstract

Debaryomyces hansenii is a promising yeast with diverse potential applications, one of which is producing oleochemicals from inexpensive biomass. Yet, combinatorial metabolic engineering in D. hansenii is not possible 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 together enable combinatorial metabolic engineering of an alkane pathway 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. We designed genomic integration loci and derived gene expression elements from a resequenced D. hansenii CBS767 genome. While characterizing the integration sites, we also optimized the transformation procedure, validated selection markers, and determined fluorescent reporters. Using homology to known elements in S. cerevisiae, we derived 23 promoters and 24 terminators and made them compatible with a modular cloning standard so pathway constructs could be made with automated liquid handling. Flow cytometry measurements show that the promoters span an expression range of three orders of magnitude and the terminators span one order of magnitude. The new part collection was used to construct a genomically integrated combinatorial library of 18 alkane biosynthesis pathways. With no other genetic modifications, the best strain produced 38 mg/L of heptadecane. This is the highest titer observed in a microbe with no other modifications besides the biosynthetic genes. Thus, this work establishes D. hansenii as a cell factory for synthesizing oleochemicals from biomass while providing a blueprint for leveraging phenotyping, genomics, modular parts collections, and automated liquid handling for making cell factories from nonconventional organisms. Furthermore, the genetic parts collection now enables functional genetics for various applications in Debaryomyces yeasts.