Increased membrane fluidity, which causes cofactor leakage and loss of membrane potential, has long been documented as a cause for decreased cell growth during exposure to ethanol, butanol, and other alcohols. Reinforcement of the membrane with more complex lipid components is thus thought to be beneficial for the generation of more tolerant organisms. In this study, organisms with more complex membranes, namely, archaea, did not maintain high growth rates upon exposure to alcohols, indicating that more complex lipids do not necessarily fortify the membrane against the fluidizing effects of alcohols. In the presence of alcohols, shifts in lipid composition to more saturated and unbranched lipids were observed in most of the organisms tested, including archaea, yeasts, and bacteria. However, these shifts did not always result in a decrease in membrane fluidity or in greater tolerance of the organism to alcohol exposure. In general, organisms tolerating the highest concentrations of alcohols maintained membrane fluidity after alcohol exposure, whereas organisms that increased membrane rigidity were less tolerant. Altered lipid composition was a common response to alcohol exposure, with the most tolerant organisms maintaining a modestly fluid membrane. Our results demonstrate that increased membrane fluidity is not the sole cause of growth inhibition and that alcohols may also denature proteins within the membrane and cytosol, adversely affecting metabolism and decreasing cell growth.

Many industrial processes used to produce chemicals and pharmaceuticals would benefit from enzymes that function under extreme conditions. Enzymes from extremophilic microorganisms have evolved to function in a variety of extreme environments, and bioprospecting for these microorganisms has led to the discovery of new enzymes with high tolerance to nonnatural conditions. However, bioprospecting is inherently limited by the diversity of enzymes evolved by nature. Protein engineering has also been successful in generating extremophilic enzymes by both rational mutagenesis and directed evolution, but screening for activity under extreme conditions can be difficult. This review examines the emerging synergy between bioprospecting and protein engineering in developing extremophilic enzymes. Specific topics include unnatural industrial conditions relevant to biocatalysis, biophysical properties of extremophilic enzymes, and industrially relevant extremophilic enzymes found either in nature or through protein engineering. 77 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Further ANNUAL REVIEWS T opt : optimum activity temperature 78 Liszka et al. Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. www.annualreviews.org • Enzymes Under Extreme Conditions 79 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only.greater potential for success. The answer to this question is not obvious and may depend strongly on the conditions required, the desired enzymatic reaction, and the type of enzyme used. EXTREME CONDITIONS RELEVANT TO INDUSTRIAL PROCESSES Conditions Found in NatureMicroorganisms exist in very different environments, and their enzymes and proteins have adapted to extreme temperatures, pressures, alkalinity/acidity, and/or osmolarity. Many of these extreme conditions mimic those found in industrial processes that currently employ enzymes or stand to benefit from them. Nature, therefore, is an abundant source of enzymes and proteins tolerant to extreme conditions. Extreme temperatures. Performing enzyme reactions at elevated temperatures has several potential advantages including higher substrate solubility, faster reaction rates, reduced risk of system contamination, lower solution viscosity, and increased solvent miscibility. However, there are many examples of enzymes used in processes that operate at lower temperatures as well, such as cold-active hydrolytic enzymes used in laundry detergents or for cleaning animal hides, proteases used for cleaning contact lenses, and pectinases for clarifying and extracting fruit juices (9). Mesophilic enzymes are less effective at...

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe 2؉ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the ␦-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.The underground corrosion of metal pipes used for gas or water and the generation of sulfide during digestion of domestic and agricultural wastes have been the economic and environmental processes that have historically driven the desire to understand the metabolism of sulfate-reducing bacteria (SRB) (16). The SRB have been a particular problem for the petroleum industry due to their roles in metal corrosion, petroleum souring, and the health hazards of hydrogen sulfide. In contrast, SRB can be advantageous for bioremediation processes. A variety of studies (6,15,17,33,39) have...

Despite extensive studies on microbial and enzymatic lignocellulose degradation, relatively few Archaea are known to deconstruct crystalline cellulose. Here we describe a consortium of three hyperthermophilic archaea enriched from a continental geothermal source by growth at 90 °C on crystalline cellulose, representing the first instance of Archaea able to deconstruct lignocellulose optimally above 90 °C. Following metagenomic studies on the consortium, a 90 kDa, multidomain cellulase, annotated as a member of the TIm barrel glycosyl hydrolase superfamily, was characterized. The multidomain architecture of this protein is uncommon for hyperthermophilic endoglucanases, and two of the four domains of the enzyme have no characterized homologues. The recombinant enzyme has optimal activity at 109 °C, a halflife of 5 h at 100 °C, and resists denaturation in strong detergents, high-salt concentrations, and ionic liquids. Cellulases active above 100 °C may assist in biofuel production from lignocellulosic feedstocks by hydrolysing cellulose under conditions typically employed in biomass pretreatment.