RNAseq of engineered S. cerevisiae strains for isobutanol production

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketoacid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses indicated that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2-null strain harboring a cytosolic localized isobutanol pathway outperformed the strain with the mitochondrial localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol. Overall design: Strains were grown in MMAS 10% glucose media for 48 hours under anaerobic conditions in triplicate (replicate cultures were started from different colonies, grown on the same day), originally for a total of 36 samples to be analyzed by RNAseq. Three samples were lost during extraction steps, leading to only 33 samples sent for sequencing. Y795 is the parental strain used as a reference. Samples for RNAseq were collected at 4 (T3), 10 (T6), and 26 (T14) hours post inoculation to represent the different phases of cell growth: early-exponential phase, mid-exponential phase, and early stationary phase. The engineered strain descriptions are as follows: mIBAILV5 or Y797, a strain harboring a mitochondria localized isobutanol pathway with a NADPH-dependent Ilv5p ketoacid reductoisomerase (KARI); cIBAILV5 or Y812, a strain harboring a cytosolic localized isobutanol pathway with a NADPH-dependent Ilv5p KARI; and cIBAIlvC6E6 or Y799, a strain harboring a cytosolic localized isobutanol pathway with a NADH-dependent IlvC6E6p KARI.