Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System.

A mutant excision+/integration- piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.

Improving ionic liquid tolerance in Saccharomyces cerevisiae through heterologous expression and directed evolution of an ILT1 homolog from Yarrowia lipolytica.

Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.