␣-Synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human ␣-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of ␣-synuclein. Mitochondrial imported ␣-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type ␣-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial ␣-synuclein with A53T mutation as compared with wild-type ␣-synuclein. Importantly, ␣-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of ϳ14-kDa ␣-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of ␣-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and ␣-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of ␣-synuclein with complex I. These results provide evidence that mitochondrial accumulated ␣-synuclein may interact with complex I and interfere with its functions. Parkinson disease (PD)2 is associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta.
Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.
BackgroundThe main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction.ResultsIn this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/wbiomass H2SO4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum.ConclusionsThe novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.
Although first-generation fuel ethanol is produced in Brazil from sugarcane-based raw materials with high efficiency, there is still little knowledge about the microbiology, the biochemistry and the molecular mechanisms prevalent in the non-aseptic fermentation environment. Learning-by-doing has hitherto been the strategy to improve the process so far, with further improvements requiring breakthrough technologies. Performing experiments at an industrial scale are often expensive, complicated to set up and difficult to reproduce. Thus, developing an appropriate scaled down system for this process has become a necessity. In this paper, we present the design and demonstration of a simple and effective laboratory-scale system mimicking the industrial process used for first generation (1G) fuel ethanol production in the Brazilian sugarcane mills. We benchmarked this system via the superior phenotype of the Saccharomyces cerevisiae PE-2 strain, compared to other strains from the same species: S288c, baker's yeast, and CEN.PK113-7D. We trust that such a system can be easily implemented in different laboratories worldwide, and will allow a better understanding of the S. cerevisiae strains that can persist and dominate in this industrial, non-aseptic and peculiar environment.
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