Metabolic and evolutionary responses of to genetic interventions aimed at improving ethanol production.

Background: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving that occurred spontaneously in some of the engineered strains.

Results: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of , and and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in or a mutation in produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively.

Conclusions: Modifications in the organic acid fermentative pathways in caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in or a disruption in . Strains with deletions in and pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.