P61: Genome shuffling of the pentose-fermenting yeast Pachysolen tannophilus for improved tolerance and fermentation in lignocellulosic hydrolysates

The economics of lignocellulosic biomass conversion can be enhanced significantly if all the sugars in potential hydrolysates are efficiently fermented to the desired product(s). Pretreatment followed by enzyme hydrolysis of lignocellulosic substrates yield fermentable sugars as well as sugar and lignin degradation products that inhibit subsequent fermentation. An efficient lignocellulosic biomass conversion process requires an ethanol producing organism able to utilize pentoses and tolerate inhibitors generated in the pretreatment process. Pentose-fermenting yeasts such as Pachysolen tannophilus can ferment both glucose and xylose to ethanol. Previous work demonstrated that random UV mutagenesis and selection could be used to obtain strains of P. tannophilus with enhanced tolerance to inhibitors in pulping waste liquor. These improved hardwood spent sulfite liquor (HWSSL) and acetic acid-tolerant UV mutant populations were subjected to genome shuffling followed by selection to obtain recombinants with further improved tolerance to hydrolysate inhibitors and HWSSL. Genome shuffling uses recursive recombination to enhance the genetic diversity of mutant populations with improved traits, allowing for the creation of new recombinants with improved phenotypes. Genome shuffled strains showed improved tolerance to HWSSL (80% v/v), acetic acid (1.10% w/v) and hydroxymethylfurfural (0.60% w/v) as compared to the best UV mutant (70% v/v, 0.80% w/v and 0.50% w/v, respectively) and WT (50% v/v, 0.60% w/v and 0.50% w/v, respectively). The fermentation performance of the genome shuffled strains was assessed in HWSSL and chemically defined medium. Strains with enhanced tolerance to inhibitors in HWSSL are of interest for bioconversion of mixed sugars in lignocellulosic hydrolysates to ethanol.