Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum

Background The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. Results Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. Conclusions In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

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 (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.

Comparison of different lignocellulosic biomass for Solvent production by Clostridium beijerinckii strains

The aim of the present study was to compare the effect of lignocellulosic sugar hydrolysate, obtained after acid pretreatment of different lignocellulosic biomass on production of solvents by two different Clostridium beijerinckii strains. C. beijerinckii C-01 produced solvents via the isopropanol-butanol-ethanol (IBE) pathway and C. beijerinckii B-17 via the acetone-butanol-ethanol (ABE). The hydrolysate was obtained after acid pretreatment of rice-husk, rice-straw and sugarcane-bagasse. Solvent production was observed from all the three substrates by both the strains. However, the highest solvent production of 9.2 gL− 1 similar to medium supplemented with commercial sugars was observed in sugarcane-bagasse hydrolysate by C. beijerinckii B-17. Degradation products i.e. 5-Hydroxy-methyl-furfural, levulinic acid, furfural and formic acid were found in different concentrations during acid pretreatments. The highest amount of formic acid (3.5 gL− 1), along with significant amounts of levulinic acid and furfural, was found to be present in rice-husk hydrolysate. The study shows that rice-straw and sugarcane-bagasse obtained after acid pretreatment are better substrates in comparison to rice-husk for butanol production by Clostridium beijerinckii strains.