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The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered C. beijerinckii NCIMB 8052 for the fermentation of LB-derived hydrolysates to acetone–butanol–ethanol (ABE). The engineered microbial strain ( C. beijerinckii _SDR) was produced by the integration of an additional copy of a short-chain dehydrogenase/reductase (SDR) gene ( Cbei_ 3904) into the chromosome of C. beijerinckii NCIMB 8052 wildtype, where it is controlled by the constitutive thiolase promoter. The C. beijerinckii _SDR and C. beijerinckii NCIMB 8052 wildtype were used for comparative fermentation of non-detoxified and detoxified hydrothermolysis-pretreated switchgrass hydrolysates (SHs) with and without (NH 4 ) 2 CO 3 supplementation. In the absence of (NH 4 ) 2 CO 3 , fermentation of non-detoxified SH with C. beijerinckii _SDR resulted in the production of 3.13- and 2.25-fold greater quantities of butanol (11.21 g/L) and total ABE (20.24 g/L), respectively, than the 3.58 g/L butanol and 8.98 g/L ABE produced by C. beijerinckii _wildtype. When the non-detoxified SH was supplemented with (NH 4 ) 2 CO 3 , concentrations were similar for butanol (9.5 compared with 9.2 g/L) and ABE (14.2 compared with 13.5 g/L) produced by C. beijerinckii _SDR and C. beijerinckii _wildtype, respectively. Furthermore, when C. beijerinckii _SDR and C. beijerinckii _wildtype were cultured in detoxified SH medium, C. beijerinckii _SDR produced 1.11- and 1.18-fold greater quantities of butanol and ABE, respectively, than when there was culturing with C. beijerinckii _wildtype. When the combined results of the present study are considered, conclusions are that the microbial strain and medium modifications of the fermentation milieu resulted in greater production of fuels and chemicals from non-detoxified LB hydrolysates. 

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 . 

Abstract In situdetoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression ofCbei_3974 andCbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, inClostridium beijerinckiiNCIMB 8052 (Cb), resulting in two strains:Cb_3974 andCb_3904. Expression ofCbei_3974 led to 2-fold increase in furfural detoxification relative toCb_3904 andCb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures ofCb_3974 relative toCb_3904 andCb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation,Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared toCb_3904 andCb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains ofCbtested. Overall,Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development ofCb_3974 represents a significant progress towards engineering solventogenicClostridiumspecies that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.