Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains

Abstract

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved 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 the HW SSL UV mutant (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 the acetic acid UV mutant, 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.

Second Generation Bioethanol Production: On the Use of Pulp and Paper Industry Wastes as Feedstock

“…Harner et al (2015) used genome shuffling to obtain Pachysolen tannophilus mutants with higher tolerance to inhibitors in HSSL. Among the genome shuffled strains, GHW301 produced the highest amount of ethanol, 7.4-8.5 g•L −1 •h −1 , in the different media containing HSSL (70-100 % v/v) [132]. Pereira et al (2015), using an evolutionary engineering strategy, obtained a population of S. stipitis with increased tolerance to E. globulus SSL inhibitors.…”

Improvement of Candida parapsilosis by genome shuffling for the efficient production of arabitol from l-arabinose

“…Zhang and Geng [ 16 ] used a modified method of genome shuffling to combine S. cerevisiae cells with the P. stipitis genome to obtain isolates with better ethanol productivity. Another xylose-fermenting yeast, Pachysolen tannophilus , was genome shuffled to improve its tolerance to inhibitors in hardwood spent sulfite liquor [ 17 ]. The ethanologenic yeast Candida krusei was modified with this method to obtain fusants with a higher acetic acid tolerance and an improved ability to produce ethanol from xylose [ 18 ].…”