Genetic Improvement of the Pentose-Fermenting Yeast Pachysolen tannophilus for Fermentation of Lignocellulosic Substrates

An efficient lignocellulosic bioconversion process requires robust inhibitor-tolerant yeast strains capable of fermenting all sugars in non-detoxified lignocellulosic hydrolysates to desired product(s). Pentose-fermenting yeasts such as Pachysolen tannophilus can ferment both glucose and xylose to ethanol, but performs poorly in the presence of hydrolysate inhibitors. We initiated a strain development program to improve the properties of P. tannophilus by non-recombinant means. A blend of random mutagenesis coupled with genome shuffling was used to obtain improved strains of P. tannophilus with enhanced ethanol production and tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to HW SSL-tolerant (UHW301, UHW303 and UHW303) UV mutants (60 and 70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70–0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to acetic acid-tolerant (UAA301, UAA302 and UAA303) UV mutants, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp–1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates, and this information can guide engineering strategies aimed at developing strains for efficient bioethanol production.