Fatty Acid-Derived Biofuels and Chemicals Production in Saccharomyces cerevisiae

Volatile energy costs and environmental concerns have spurred interest in the development of alternative, renewable, sustainable and cost-effective energy resources. Advanced biofuels have potential to replace fossil fuels in supporting high-power demanding machinery such as aircrafts and trucks. Microbial biosynthesis is generally considered as an environmental friendly refinery process, and fatty acid biosynthesis is an attractive route to synthesize chemicals and especially drop-in biofuels due to the high degree of reduction of fatty acids. The robustness and excellent accessibility to molecular genetics make the yeast S. cerevisiae a suitable host for the production of biofuels, chemicals and pharmaceuticals, and recent advances in metabolic engineering as well as systems and synthetic biology allow us to engineer the yeast fatty acid metabolism and modification pathways for production of advanced biofuels and chemicals

Hypothermic cardiopulmonary bypass alters oxygen/glucose uptake in the pediatric brain

AbstractObjectives: Neurologic morbidity related to cardiac surgery has been recognized as a major morbidity. A variety of causes related to cardiopulmonary bypass, including microemboli, nonpulsatile flow, hemodilution, and inflammatory mediation, have been proposed. Because oxygen and glucose are the predominant metabolic substrates for the brain, we sought to examine the uptake of these substrates by the pediatric brain during hypothermic cardiopulmonary bypass. Methods: Eleven children (median age 5 months, range 1 day-17 years) undergoing a variety of cardiac surgical procedures with the use of hypothermic cardiopulmonary bypass were studied. Cerebral arteriovenous differences for oxygen, glucose, and lactate were obtained before, during, and after bypass. On the basis of the predictable stoichiometric relationship for the oxidation of glucose, the relationship of substrate uptake was expressed as the oxygen/glucose index. Oxygen/glucose index (%) = (arteriovenous oxygen difference [μmol/mL]/arteriovenous glucose difference [μmol/mL] × 6) × 100 Results: All children survived with no obvious neurologic sequelae. During cooling on cardiopulmonary bypass, the oxygen/glucose indexes fell significantly from prebypass values (53% ± 19% at 28°C and 54% ± 25% at 24°C vs 117% ± 70%; P <.05, analysis of variance). This decline resulted from decreased oxygen uptake with stable glucose uptake (P <.05). Although oxygen and glucose uptake both increased with rewarming, the net effect was only a slight increase in oxygen/glucose index (62% ± 16%). Postbypass oxygen/glucose index exceeded prebypass values (149% ± 83%). Conclusions: Hypothermic cardiopulmonary bypass alters the relationship between oxygen and glucose uptake in the pediatric brain. The relationship of these findings to bypass-related neurologic morbidity remains to be explored. (J Thorac Cardiovasc Surg 2001;121:366-73

Geology of Rum Cay, Bahamas: A Field Trip Guide

See other Smith authored Field Trip Guides of Gerace Research Centre

Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast Kluyveromyces marxianus

It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 \ub0C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as Kluyveromyces marxianus can grow at temperatures of 45 \ub0C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits

Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology

Production of 10-methyl branched fatty acids in yeast

Background: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses