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  • Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii
Title: Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii
Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GS CcpA ). As negative controls, the ribozyme M1GS CcpA–Sc (constructed with a scrambled GS CcpA ) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GS CcpA ( C. beijerinckii _M1GS CcpA ) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii _M1GS CcpA–Sc produced 50% more solvent than C. beijerinckii _pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GS CcpA–Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GS CcpA–Sc was renamed M1GS DisA . Compared to C. beijerinckii _M1GS CcpA and _pMTL500E, C. beijerinckii _M1GS DisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii _M1GS DisA . Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii .  more » « less
Award ID(s):
1803022
PAR ID:
10285329
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Frontiers in Bioengineering and Biotechnology
Volume:
9
ISSN:
2296-4185
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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