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null (Ed.)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
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Abstract The local compositional heterogeneity associated with the short‐range ordering of Mg and Nb in PbMg1/3Nb2/3O3(PMN) is correlated with its characteristic relaxor ferroelectric behavior. Fully ordered PMN is not prepared as a bulk material. This work examines the relaxor behavior in PMN thin films grown at temperatures below 1073 K by artificially reducing the degree of disorder via synthesis of heterostructures with alternate layers of Pb(Mg2/3Nb1/3)O3and PbNbO3, as suggested by the random‐site model. 100 nm thick, phase‐pure films are grown epitaxially on (111) SrTiO3substrates using alternate target timed pulsed‐laser deposition of Pb(Mg2/3Nb1/3)O3and PbNbO3targets with 20% excess Pb. Selected area electron diffraction confirms the emergence of (1/2, 1/2, 1/2) superlattice spots with randomly distributed ordered domains as large as ≈150 nm. These heterostructures exhibit a dielectric constant of 800, loss tangents of ≈0.03 and 2× remanent polarization of ≈11 µC cm−2at room temperature. Polarization–electric field hysteresis loops, Rayleigh data, and optical second‐harmonic generation measurements are consistent with the development of ferroelectric domains below 140 K. Temperature‐dependent permittivity measurements demonstrate reduced frequency dispersion compared to short range ordered PMN films. This work suggests a continuum between normal and relaxor ferroelectric behavior in the engineered PMN thin films.