Genome and metabolic engineering in non-conventional yeasts: Current advances and applications

Abstract

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

Keywords: CRISPR, Clustered regularly interspaced short palindromic repeats; CRISPR-Cas9; DSB, double strand break; HR, homologous recombination; Hansenula polymorpha; Kluyveromyces lactis; Kluyveromyces marxianus; NHEJ, nonhomologous end-joining; PAM, protospacer adjacent motif; Pichia pastoris; Scheffersomyces stipitis; TALEN, transcription activator-like effector nucleases; Yarrowia lipolytica; sgRNA, short (or single) guide RNA.

Fig. 1

Schematic diagram of the generation and utilization of auxotrophic markers for engineering yeast. Random mutagenesis of host DNA or homologous recombination of a cassette that inactivates an essential gene for nutrient synthesis can be used to produce stable auxotrophic strains. The presence of an auxotrophy allows more advanced genome editing and pathway engineering tools to be applied in the yeast species of interest. Shown here are 1) targeted and random integration using a selectable marker (bottom, left), 2) HisG/lacZ-mediated marker recovery (bottom, middle), 3) Cre-lox-mediated marker recovery (bottom, middle), and 4) Markerless editing by CRISPR-Cas9 (bottom, right).

Fig. 2

CRISPR-Cas9-mediated genome editing. The Cas9-sgRNA complex scans DNA until finding a complementary sequence. Upon binding, the endonuclease domains cleave both DNA strands 3 bases upstream of the PAM sequence. The double strand break is then repaired either by homologous recombination (HR) if an appropriate homology donor is present, or by nonhomologous end-joining (NHEJ). Repair via HR allows for precise genome editing at the target site, while NHEJ introduces short insertions or deletions.