With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors-that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.

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Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as ''overflow metabolism'' or ''the Crabtree effect.'' The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.alternative oxidase ͉ Crabtree effect ͉ NADH oxidase ͉ redox metabolism R edox homeostasis is a fundamental requirement for sustained metabolism and growth in all biological systems. The intracellular redox potential is primarily determined by the NADH/NAD ratio and to a lesser extent by the NADPH/NADP ratio. In Saccharomyces cerevisiae, Ͼ200 reactions involve these cofactors spread over a large spectrum of cellular functions (1). Because NADH is a highly connected metabolite in the metabolic network (1), any change in the NADH/NAD ratio leads to widespread changes in metabolism (2). NADH is generated primarily in the cytosol by glycolysis and in the mitochondria by the tricarboxylic acid (TCA) cycle. Because the NADH/NAD redox couple cannot traverse the mitochondrial membrane in S. cerevisiae and other eukaryotic cells (3), distinct mechanisms oxidize NADH to NAD in the cytosol and mitochondria. Cytosolic NADH is oxidized by two external (cytosolic) mitochondrial membrane-bound NADH dehydrogenases encoded by NDE1 and NDE2 genes with catalytic sites facing the cytosol (4). Additionally, glycerol-3-phosphate dehydrogenases (encoded by GPD1 and GPD2) oxidize cytosolic NADH with concomitant glycerol formation when the NADH formation rate surpasses its oxidation rate (5). Mitochondrial NADH is oxidized by one internal mitochondrial membrane-bound NADH dehydrogena...

SUMMARY Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.

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OverviewIndustrial cultivation media, such as molasses, wort, agricultural waste and lignocellulose hydrolysates, contain a mixture of metabolizable carbohydrates. These carbohydrates are taken up by cells in a certain order with intermittent lag phases due to a set of mechanisms controlled by glucose, referred to hereafter as glucose control. Thus, the presence or uptake of glucose has a negative impact upon the metabolism of other sugars. Glucose repression reduces the transcription rate of repressible genes, and is the most thoroughly investigated mechanism of glucose control (Fig. 1).In this review the different mechanisms of glucose control in Saccharomyces cereuisiae are first discussed, focusing on the mechanism of glucose repression. Next, the function of the regulatory protein Migl in the signalling cascade of glucose repression is explained. The impact of Migl-mediated glucose repression on metabolic functions is then discussed. It will be made clear that glucose repression is not confined to the above-mentioned example of mixed sugar metabolism. The last part of the review summarizes the effects glucose repression has on single metabolic functions. These components are then linked to a holistic view with the aim of understanding the effects of Migl on overall metabolism. Against this background possible physiological impacts of deletion/disruption of the MZGl gene are discussed.Glucose control in 5. cerevisiae

Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals.