Deletion of glyceraldehyde‐3‐phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1‐4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production

Abstract

Summary The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1‐4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde‐3‐phosphate (GAP) to 3‐phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN , demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4‐fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

The Industrial Fermentation Process and Clostridium Species Used to Produce Biobutanol

“…The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for increased refinements for the improvement of the N1-4 strain. Efficient CRISPR-Cas9 genome engineering systems are now available and have paved the way for elucidating the solvent production mechanism in this hyper-butanol-producing species and for engineering strains with desirable enhanced butanol-producing features, allowing for potential improvements in solvent titres and energy metabolism [80,81]. Examples of the application of these new technologies are the enhancement of sucrose metabolism through the deletion of a transcriptional repressor gene to increase solvent production [82] and the deletion of glyceraldehyde-3-phosphate dehydrogenase to increase the ATP pool and accelerate solvent production [80].…”

“…Efficient CRISPR-Cas9 genome engineering systems are now available and have paved the way for elucidating the solvent production mechanism in this hyper-butanol-producing species and for engineering strains with desirable enhanced butanol-producing features, allowing for potential improvements in solvent titres and energy metabolism [80,81]. Examples of the application of these new technologies are the enhancement of sucrose metabolism through the deletion of a transcriptional repressor gene to increase solvent production [82] and the deletion of glyceraldehyde-3-phosphate dehydrogenase to increase the ATP pool and accelerate solvent production [80]. A hyper-butanol producer N1-4 strain was converted into a hyper-butyrate producer for butyrate production using glucose and lignocellulosic sugar substrates [83].…”