Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii

Abstract

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 (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA–Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA–Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA–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, M1GSCcpA–Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA 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_M1GSDisA. 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.

Effects of Clostridium beijerinckii and Medium Modifications on Acetone–Butanol–Ethanol Production From Switchgrass

“…Optical density was determined at 600 nm using a DU ® 800 spectrophotometer (Beckman Coulter Inc., Brea, CA, United States) to estimate the changes in the growth of the C. beijerinckii strains during fermentation. Gas chromatography was conducted to quantify the concentrations of acetone, butanol, ethanol, acetic, and butyric acids using a 7890A system (Agilent Technologies 7890, Agilent Technologies Inc., Wilmington, DE), according to a previously described method ( Ujor et al, 2021 ). Sugar concentrations (glucose, xylose, and arabinose) were analyzed using HPLC (Waters, Milford, MA, United States) equipped with an evaporative light scattering detector (Waters, Milford, MA, United States) according to a previous method ( Ujor et al, 2021 ).…”

“…Gas chromatography was conducted to quantify the concentrations of acetone, butanol, ethanol, acetic, and butyric acids using a 7890A system (Agilent Technologies 7890, Agilent Technologies Inc., Wilmington, DE), according to a previously described method (Ujor et al, 2021). Sugar concentrations (glucose, xylose, and arabinose) were analyzed using HPLC (Waters, Milford, MA, United States) equipped with an evaporative light scattering detector (Waters, Milford, MA, United States) according to a previous method (Ujor et al, 2021). The concentrations of the LDMICs were quantified using HPLC according to the method of Agu et al, 2016. The ABE yield was determined by dividing the total grams of ABE produced by the total grams of glucose or sugars utilized during fermentation, while ABE productivity was calculated by dividing the maximum amount of ABE (g/L) produced by the corresponding fermentation time (h).…”

Glycerol Utilization as a Sole Carbon Source Disrupts the Membrane Architecture and Solventogenesis in Clostridium beijerinckii NCIMB 8052

“…To inoculate TGY, a total of 2 mL of spore suspension (200 µL in each 1.5 mL tube) was heat-shocked at 75 • C for 10 min in a compact digital dry bath/block heater (ThermoFisher Scientific, Waltham, MA, USA), cooled on ice for 3 min, and then inoculated into 100 mL of TGY broth, which was then grown overnight. After 12 h, the fermentation medium was inoculated with the preculture (6%, v/v; [20]). Fermentation was conducted in a P2 medium (100 mL) containing 60 g/L glycerol or glucose (as a control where appropriate) and yeast extract (1 g/L) in loosely capped 250-mL Pyrex culture bottles.…”

“…beijerinckii preculture was cultivated in TGY broth as described above. Afterwards, the preculture was used to inoculate P2 medium supplemented with glucose (60 g/L) or glycerol (60 g/L), with and without the acetate (2.2 g/L) addition, and then the cultures were grown anaerobically at 35 ± 1 • C. Samples were collected at 12 h and 24 h and cells were harvested by centrifugation at 13,000× g for 5 min at 4 • C. Total RNA was extracted from the samples as per the procedures described by Ujor et al [20] and Gangwar et al [30]. After centrifugation, cell pellets were washed with an ice-cold 25 mM Tris-Cl buffer in water and re-suspended to reach an optical density (OD) of ~1.0 (at 600 nm).…”

Ultrasound-assisted extraction of emodin from Rheum officinale Baill and its antibacterial mechanism against Streptococcus suis based on CcpA

“…According to the previous studies, catabolite control protein A (CcpA) played an important role in bacterial resistance, virulence gene expression, and biofilm formation [15] , [16] , [17] . CcpA belongs to LacI/GaIR family that regulates various metabolic pathways, such as starch metabolism [18] , glucose metabolism [19] , arabinose metabolism [20] and the phosphotransferase system mediated by phosphoenolpyruvate [21] . Moreover, CcpA can participate in the regulation of carbon overflow metabolism and control the flow of carbon entering tricarboxylic acid cycle when the carbon source is abundant in environment [22] .…”