S169: The use of classical and systems biology tools to identify genes that overcome biocatalyst inhibition

The application of classical and systems biology tools is a powerful approach to identify genes that overcome biocatalyst inhibition, which will be highlighted using examples from Zymomonas mobilis and Clostridium thermocellum. An acetate tolerant Z. mobilis mutant (AcR) was previously created and mutations in genome were identified and confirmed through the combination of microarray-based comparative genome sequencing (CGS), next-generation 454-pyroresequencing, and Sanger sequencing. Transcriptomics studies were conducted to compare gene expression profiles between wild-type ZM4 and the acetate tolerant mutant AcR under selective conditions. Microarray analysis showed that nhaA expression was significantly increased (>16-fold) in strain AcR compared to ZM4 under all conditions tested and this gene was downstream of 1,461-bp deletion in AcR. Genetic studies showed the deletion in AcR enhanced the nhaA expression and led to enhanced sodium acetate tolerance in strain AcR. C. thermocellum is a candidate industrial biocatalyst for cellulosic fuel ethanol production but the wild-type is relatively sensitive to ethanol compared to current industrial yeast. In another example, a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE) that augments C. thermocellum ethanol tolerance was identified using a similar approach. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant strain. Carbon flow is also affected in strains containing the mutated gene. The simplicity of the genetic basis for mutant phenotypes observed here informs rational engineering of microbial strains for biofuels production. The importance of accurate genome annotations is also discussed.