P131: Generation and Characterization of Acetate Tolerant Mutants in Zymomonas mobilis for Enhanced Utilization of Lignocellulosic Hydrolysate

Zymomonas mobilis is an ethanologen that shows great promise in the realm of cellulosic ethanol or advanced biofuels. However, high concentrations of acetate present in dilute acid pretreated corn stover hydrolysate is inhibitory to recombinant Z. mobilis strains resulting in poor fermentation, particularly in xylose.  To overcome these hurdles, we sought to identify mutants capable of tolerating elevated concentrations of acetate and to identify the genetic features associated with these mutants through multiple approaches of a transposon-mediated mutant library construction, lab-directed evolution, as well as next-generation sequencing (NGS)-based genome resequencing and directional mRNA-Seq. Briefly, a transposition vector containing an antibiotic resistant gene marker was used for the construction of a knockout library in Z. mobilis strain 8b (a xylose and glucose utilizing recombinant) by randomly inserting the DNA containing the antibiotic marker gene into the genome.  We achieved high-level transposon insertion coverage of the genome in our library of ~20,000 clones which correlates to an insertion event occurring approximately every 110 base pairs.  Selective pressure on the library population was conducted in pH-controlled fermenters with medium containing xylose as the sole carbon source and increasingly higher concentrations of acetate. Acetate-tolerant mutants were isolated and showed not only improved performance in acetate-containing medium but achieved higher growth rates in xylose alone. Multiple genes have been identified as candidate targets to improve xylose utilization and acetate tolerance of Z. mobilis. The genetic features of these mutants and their potential mechanism(s) for improved fermentation performance in the presence of hydrolysates will be discussed.