Comparative analysis of high butanol tolerance and production in clostridia

2016, was the 100 years anniversary from launching of the first industrial acetone-butanol-ethanol (ABE) microbial production process. Despite this long period and also revival of scientific interest in this fermentative process over the last 20 years, solventogenic clostridia, mainly Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum and Clostridium pasteurianum, still have most of their secrets. One such poorly understood mechanism is butanol tolerance, which seems to be one of the most significant bottlenecks obstructing industrial exploitation of the process because the maximum achievable butanol concentration is only about 21 g/L. This review describes all the known cellular responses elicited by butanol, such as modifications of cell membrane and cell wall, formation of stress proteins, extrusion of butanol by efflux pumps, response of regulatory pathways, and also maps both random and targeted mutations resulting in high butanol production phenotypes. As progress in the field is inseparably associated with emerging methods, enabling a deeper understanding of butanol tolerance and production, progress in these methods, including genome mining, RNA sequencing and constructing of genome scale models are also reviewed. In conclusion, a comparative analysis of both phenomena is presented and a theoretical relationship is described between butanol tolerance/high production and common features including efflux pump formation/activity, stress protein production, membrane modifications and biofilm growth.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D₂O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.