Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyper-Producer Clostridium saccharoperbutylacetonicum strain N1-4

While a majority of academic studies concerning acetone, butanol, ethanol (ABE)-production by Clostridium have focused on C. acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end-product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyper-producer C. saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021 (C. s. N1-4). Following initial failures, we found the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I) which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency 1.1 × 106 cfu/μg DNA), we performed targeted gene deletions in C. s. N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knock-outs of key genes in the native ABE metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case, an engineered strain (pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (adh11 and adh5) displayed 8.5- and 11.8-fold respective increases in batch ethanol production.