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

Monaghan, Taylor I., Baker, Joseph A., Krabben, Preben, Davies, E. Tim, Jenkinson, Elizabeth R., Goodhead, Ian. B, Robinson, Gary K., Shepherd, Mark (2022) Deletion of glyceraldehyde-3-phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1-4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production. Microbial Biotechnology, 15 (5). pp. 1574-1585. E-ISSN 1751-7915. (doi:10.1111/1751-7915.13990) (KAR id:91762)

Abstract

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.

Item Type: Article
DOI/Identification number: 10.1111/1751-7915.13990
Additional information: For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
Uncontrolled keywords: Applied Microbiology and Biotechnology, Biochemistry, Bioengineering, Biotechnology
Subjects: Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Biotechnology and Biological Sciences Research Council (https://ror.org/00cwqg982)
Depositing User: Mark Shepherd
Date Deposited: 29 Nov 2021 11:30 UTC
Last Modified: 04 Mar 2024 18:02 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/91762 (The current URI for this page, for reference purposes)

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