Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose.L-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.
In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase, S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0.10 h ؊1 , in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1⌬ mutant and unaffected in an nde2⌬ mutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1⌬ nde2⌬ double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1⌬ nde2⌬ mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D ؍ 0.10 h ؊1 ) of an nde1⌬ nde2⌬ mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h ؊1 under various nutrient-limited conditions (anaerobically glucose-or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h ؊1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactoselimited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8 -HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14 C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zerotrans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.
Metabolic fluxes may be regulated ''hierarchically,'' e.g., by changes of gene expression that adjust enzyme capacities (Vmax) and/or ''metabolically'' by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the Ϸ10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50 -20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels.gene-expression cascade ͉ glycolysis ͉ posttranscriptional regulation ͉ regulation analysis ͉ systems biology T he 1990s have witnessed a revolution in molecular cell biology. Nucleotide sequences of complete genomes were elucidated, and new techniques enabled genome-wide analysis of mRNA and protein concentrations and accurate estimates of metabolic flux distributions (1). The central dogma of molecular biology is that DNA encodes mRNA and mRNA encodes proteins, which in turn fulfill the many functions in the cell. Therefore, a strong correlation was anticipated among mRNA concentrations, protein concentrations, and metabolic fluxes. However, subsequent gene-expression studies led to the paradoxical conclusion that correlations between mRNA levels and protein levels (2, 3), between mRNA and in vivo fluxes (4, 5), and between enzyme activities and fluxes (6, 7) were far from perfect.There are several explanations for the lack of correlation between the different levels of gene expression. Clearly defined and strictly controlled cultivation methods are required to obtain highquality datasets (8, 9). Furthermore, there should be a time delay between changes at the mRNA level and the corresponding changes of protein concentrations and enzyme activities. However, even in steady-state chemostat cultures, in which the cells grow in a constant environment for prolonged periods of time, mRNA levels, protein concentrations/activities, and fluxes correlated poorly (4, 6, 10). A remaining explanation might be that much of the regulation of gene expression is posttranscriptional. Indeed, regulatory mechanisms that affect translation, protein degradation, posttranslational modification of proteins, and enzymes directly have been documented extensively. High-throughput measurements of translation rates and protein turnover in Saccharomyces cerevisiae showed that these varied significantly between proteins and conditions (11-13). Posttranslational mo...
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substratespecificity decarboxylase activity.
NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1⌬ mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanolacetaldehyde shuttle is essential for respiratory growth of the ndi1⌬ mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3⌬ mutant (0.22 h ؊1 ) was substantially reduced compared to that of the wild-type strain (0.33 h ؊1 ). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3⌬ ndi1⌬ mutant, contributing to residual redox-shuttle activity in this strain.
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