2-06: Identification and Characterization of the Genetic Loci in Zymomonas mobilis conferring Tolerance to Pretreated Corn Stover Hydrolysate

The existence of multiple inhibitors within the pretreated corn stover hydrolysate will restrain the growth of biocatalysts such as Zymomonas mobilis leading to low ethanol yield and/or prolonged fermentation process. Development of robust biocatalysts is crucial for economic biofuel production. In this study, a transposon-based overexpression construct was created and used to build a mutant library of Z. mobilis 33C (an engineered strain capable of utilizing glucose, xylose, and arabinose). About 30,000 mutant isolates were pooled and hydrolysate-tolerant candidates were selected on agar plates containing 40% pretreated corn stover hydrolysate. Five candidates were further confirmed and selected by continuously transferring the single colony onto 40% hydrolysate agar plate eight times to stabilize the phenotype of hydrolysate tolerance. Bioscreen C and agar plate assay were used to investigate the tolerance of these five candidates to the major individual inhibitors of acetate, furfural, or ethanol. The genetic location of each mutant was further identified through chromosomal sequencing. Four mutants had similar genetic insertional location within the intergenic region, while another one was inserted into a structural gene. The introduction of either one of these two genetic elements into parental strains of both 33C and 8b (parental strain of 33C) through electroporation transformation also helped 33C and 8b tolerate to hydrolysate. Our study therefore has identified and confirmed two genetic loci conferring hydrolysate tolerance to Z. mobilis, which can be further combined and explored for more robust hydrolysate strain engineering and the understanding of hydrolysate tolerance mechanisms.