Alcohol-based liquid fuels feature significantly in the political and social agendas of many countries, seeking energy sustainability. It is certain that ethanol will be the entry point for many sustainable processes. Conventional ethanol production using maize- and sugarcane-based carbohydrates with Saccharomyces cerevisiae is well established, while lignocellulose-based processes are receiving growing interest despite posing greater technical and scientific challenges. A significant challenge that arises from the chemical hydrolysis of lignocellulose is the generation of toxic compounds in parallel with the release of sugars. These compounds, collectively termed pre-treatment inhibitors, impair metabolic functionality and growth. Their removal, pre-fermentation or their abatement, via milder hydrolysis, are currently uneconomic options. It is widely acknowledged that a more cost effective strategy is to develop resistant process strains. Here we describe and classify common inhibitors and describe in detail the reported physiological responses that occur in second-generation strains, which include engineered yeast and mesophilic and thermophilic prokaryotes. It is suggested that a thorough understanding of tolerance to common pre-treatment inhibitors should be a major focus in ongoing strain engineering. This review is a useful resource for future metabolic engineering strategies.

The annotation of novel plant genes is frequently based on sequence and structural similarity to known protein motifs. Understanding the biological function of these genes is dependent on identifying conditions under which they are activated, however. The resurrection plant, Xerophyta humilis is a good model system for identifying and characterizing genes which are important for desiccation tolerance. Desiccation induced-1 (dsi-1VOC), a previously uncharacterized plant gene, is up-regulated during desiccation in leaves, roots, and seeds in X. humilis. The X. humilis desiccation induced-1 gene, Xhdsi-1VOC, shares structural homology with the vicinal oxygen chelate (VOC) metalloenzyme superfamily. Proteins in this superfamily share little sequence similarity, but are characterized by a common βαβββ structural fold. A number of plant orthologues of XhDsi-1VOC have been identified, including Arabidopsis thaliana At1g07645, which is currently annotated as a glyoxalase I-like gene, and many ESTs derived from seed cDNA libraries. Xhdsi-1VOC and its orthologues do not, however, contain the glutathione and zinc binding sites conserved in glyoxalase I genes. Furthermore, expression of Xhdsi-1VOC in yeast failed to rescue a yeast glyoxalase I mutant. Messenger RNA transcripts for At1g07645 accumulate during seed maturation, but are not induced by water loss, salt or mannitol stress in vegetative tissue in Arabidopsis. It is concluded that dsi-1VOC is a seed-specific gene in desiccation-sensitive plants that is activated by water loss in vegetative tissues in the resurrection plant X. humilis and plays an important role in allowing plant tissues to survive loss of 95% of their relative water content.

Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.

Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple approach that combines magnetic bead capture with subtractive hybridization for the rapid and direct recovery of full-length target ORFs. When compared with other PCR-based techniques, a higher degree of specificity is achieved through the use of larger gene fragments during hybridization followed by several high-stringency washes. Together with the recent advances in environmental nucleic acid extraction techniques, this approach should allow for the further exploration of the metagenomic sequence space.

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