458 research outputs found
Finite-size corrections to the free energies of crystalline solids
No full textWe analyze the finite-size corrections to the free energy of crystals with a fixed center of mass. When we explicitly correct for the leading (ln N/N) corrections, the remaining free energy is found to depend linearly on 1/N. Extrapolating to the thermodynamic limit (N → ∞), we estimate the free energy of a defect-free crystal of particles interacting through an r–12 potential. We also estimate the free energy of perfect hard-sphere crystal near coexistence: at ρσ3 = 1.0409, the excess free energy of a defect-free hard-sphere crystal is 5.918 89(4)kT per particle. This, however, is not the free energy of an equilibrium hard-sphere crystal. The presence of a finite concentration of vacancies results in a reduction of the free energy that is some two orders of magnitude larger than the present error estimate
Reverse engineering of industrially relevant phenotypes in yeast: An integrated approach
No full textReverse engineering is the study of discovering the structure, function and operation of a device or system with the express aim to reconstruct its key functionalities. This principle is applied to many disciplines, from military, through computer engineering, to health, but also in metabolic engineering. In this context, reverse metabolic engineering examines a particular functionality or phenotype of a cell or culture and subsequently aims to reconstruct it, with the aid of targeted genetic modification, in another cell or culture. Even with increasing knowledge on targeted metabolic engineering, microbial production platforms for fuels and chemicals are often obtained by non-targeted approaches, such as mutagenesis or evolutionary engineering. Reverse engineering of the interesting traits of these microbial platforms not only provides the potential to implement and combine them in other hosts, but also allows for the protection of the resulting intellectual property. The major challenge in reverse metabolic engineering is the elucidation of the molecular mechanisms underlying the phenotype of the strains of interest. In this thesis, various techniques were evaluated for their application in reverse metabolic engineering of a diverse range of industrially relevant phenotypes in the yeast Saccharomyces cerevisiae. Simultaneously, the different analytical methods that were used in these studies were evaluated for their individual and combined contributions.BiotechnologyApplied Science
Engineering of aromatic amino acid metabolism in Saccharomyces cerevisiae
No full textSaccharomyces cerevisiae is a popular industrial microorganism. It has since long been used in bread, beer and wine making. More recently it is also being applied for heterologous protein production and as a target organism for metabolic engineering. The work presented in this thesis describes how S. cerevisiae may be used as a metabolic-engineering platform to produce aromatic compounds such as phenylalanine or its catabolites, phenylethanol and phenylacetate. In this thesis two research lines were followed: The first research line focused on the molecular identity and substrate specificity of 2-oxo-acid decarboxylase in S. cerevisiae whereas the second line of research investigated the elimination of feedback inhibition steps in the phenylalanine biosynthetic pathway in S. cerevisiae. The impact of feedback inhibition on the aromatic biosynthesis pathway was quantified by analyzing intra- and extracellular concentrations of relevant aromatic compounds in glucose-limited chemostat cultures of wild-type and engineered strains.Applied Science
Analysis of the hybrid genomes of brewing yeasts
No full textOne of the best guarded secrets of brewers is represented by the brewing yeast employed in beer fermentation, due to its profound impact upon the specific flavour profile of the final product. The current research tackles the genome diversity of lager brewing strains as well as their impact on important phenotypic traits. Furthermore, the development of a new recyclable dominant marker is described. This opened the way to unravel the molecular mechanism of cross-genome transcriptional regulation within a lager brewing strain. To prove the inherited genotypic and phenotypic advantages of the hybrid nature of bottom fermenting yeasts, an "artificial" hybrid was constructed between a haploid S.cerevisiae (italic) strain of the CEN.PK family and a haploid strain derived from the S. eubayanus (italic) type strain CBS12357, using mass mating. The heterosis (hybrid vigor) of the offspring proved the beneficial combination between the cold tolerance from one parent and the strong fermentative metabolism of sugars from the other parent. The research in this thesis brings some layers of transparency into the opaqueness of the genomes of lager brewing strains.BiotechnologyApplied Science
Engineering synthetic glycolytic pathways in Saccharomyes cerevisiae
No full textIn the past few decades, concerns on climate change have contributed to the development of biotechnological alternatives for petrochemical processes. In order to make these processes economically competitive, there has been an intensive search for improved, more efficient production organisms. In the past decade, genetic engineering of microorganisms has undergone a tremendous development. The radical modification or replacement of a key route in metabolism requires a strategy in which the essential cellular function of this route is not interrupted while it is being replaced. In this thesis, such a strategy for radical reprogramming of metabolism is developed and applied to the sugar metabolism (glycolysis) of baker's yeast (Saccharomyces cerevisiae). The application of such a large number of changes in the yeast genome implied the exploration of a new scale of genetic modification and demanded the further development of several modification techniques. The results presented in this thesis demonstrate that it is possible to integrally replace an essential metabolic pathway, which is essential for the viability of the cell. This research therefore provides a basis for a new, modular approach for metabolic engineering, in which entire metabolic routes can be quickly replaced and optimized. The ability to quickly replace the yeast glycolysis by alternative routes supplies also a wonderful tool for fundamental research into the regulation of the glycolytic "flux". This may eventually contribute to the further development of S. cerevisiae to a more efficient industrial platform for the production of chemicals from sugars. In addition, the present study provides an illustration of the rapid developments and new possibilities in the field of genetic modification.BiotechnologyApplied Science
Functional genomics of beer-related physiological processes in yeast
No full textSince the release of the entire genome sequence of the S. cerevisiae laboratory strain S288C in 1996, many functional genomics tools have been introduced in fundamental and application-oriented yeast research. In this thesis, the applicability of functional genomics for the improvement of yeast in beer-related processes is investigated. To this end, genome-wide analysis was focused on a range of nutritional conditions that are typically encountered in beer fermentation. Ultimately, this should provide the brewer with the information and knowledge required to understand and improve yeast quality and fermentation performance. Fusel alcohols are derived from amino acid catabolism via a pathway that was first proposed a century ago by Ehrlich. In the past decade, many efforts have contributed to a better understanding of the genes involved in this pathway and how their expression is regulated. A ‘centenary’ review of the Ehrlich pathway is given in this thesis. Our growing understanding of the key components of the Ehrlich pathway and their regulation will aid in the design of strains that exhibit specific flavor profiles in foodstuffs as well as in the metabolic engineering of yeast strains for the production of individual Ehrlich pathway products. Transcriptomics can also be used as diagnostic tool for industrial fermentations. For example, the prediction of Zn bioavailability in wort is not always accurate and research has therefore focused on the identification of molecular markers for Zn deficiency. For this purpose, we investigated the transcriptional responses S. cerevisiae at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation in Zn-limited cultures was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under these conditions. In industrial fermentations, the ability to accumulate substantial amounts of storage carbohydrates is an important criterion in the selection and development of new strains, as they contribute to cellular robustness. An analysis of storage carbohydrate metabolism in anaerobic chemostat cultures grown at a fixed specific growth rate under five different nutrient-limitation regimes revealed that storage carbohydrate accumulation is not a general response to nutrient limitation. Over the conditions tested, glycogen accumulation was most pronounced under nitrogen limited conditions. Although the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4, the main regulatory control of glycogen biosynthesis was post-translational. The hop plant, Humulus lupulus, contains an exceptionally high content of secondary metabolites, the hop iso-?-acids, which possess a range of beneficial properties including antiseptic action. By applying transcriptome analysis and phenotype screening of the S. cerevisiae gene deletion collection we found that yeast tolerance to hop iso-?-acids involves two major processes: active export of iso-?-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-?-acids. Iso-?-acids were also shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator. The co-localization of iso-?-acids and zinc in the vacuole has important implications for the maintenance and quality of brewing strains. Especially upon serial re-pitching of yeast, hop acid stress in the vacuole may result in decreased viability of the yeast culture.BiotechnologyApplied Science
Molecular responses of Saccharomyces cerevisiae to near-zero growth rates
No full textBiotechnologyApplied Science
Transcriptomics and quantitative physiology of ß-lactam producing Penicillium chrysogenum
No full textWith an over 1000-fold improvement in specific productivity since its discovery, penicillin is one of the very successful examples of industrial biotechnology. Although classical strain improvement programmes have been a major contributor to this success, the wish for a more rational approach towards improvements has driven the work described in this thesis. As most ß-lactam biosynthesis routes share the first steps in their pathway, the production of penicillin-G in chemostat cultivations of P. chrysogenum has been chosen as a model system. Compared to common laboratory organisms such as bakers' yeast, the information on P. chrysogenum is relatively limited, which also reduces the range of possibilities for a rational approach. The majority of the work described in this thesis can therefore be assigned to the analysis phase of the metabolic engineering cycle. In the first half of the thesis more classical techniques are employed to investigate two important aspects of ß-lactam production: NADPH metabolism and the unexpectedly high energy demand of penicillin production. The work presented in the second half of this thesis was made possible by the availability of the genome sequence of P. chrysogenum. A whole series of transcriptome studies of chemostat based cultivations of P. chrysogenum was set up with the aim to identify key factors involved in penicillin production. Using this experimental design it was possible to explore transcriptional responses towards ß-lactam production and side chain catabolism at a genome-wide scale. Each of these studies has provided putative targets for metabolic engineering of ß-lactam production by Penicillium chrysogenum.BiotechnologyApplied Science
Towards fermentation of galacturonic acid-containing feedstocks with Saccharomyces cerevisiae
No full textThe ambition to reduce our current dependence on fossil transportation fuels has driven renewed interest in bioethanol. Pectin-rich feedstocks like sugar beet pulp and citrus peel, which are currently sold as cattle feed, are promising raw materials for the production of bioethanol. This thesis explores the challenges related to the fermentation of pectin-rich hydrolysates with Saccharomyces cerevisiae. Galacturonic acid is a major constituent of pectin-rich hydrolysates. Achieving efficient conversion of this compound is desired. This requires introduction of a heterologous pathway for galacturonate metabolism. In this project, we functionally expressed uronate isomerase and tagaturonate dehydrogenase, the first two enzymes of a bacterial catabolic pathway. Introduction of the entire 5-enzyme catabolic route did not result in a S. cerevisiae strain capable of galacturonic acid fermentation. Galacturonic acid will therefore remain present in the fermentation medium. It was shown that the presence of galacturonic acid negatively affects fermentation characteristics in aerobic chemostat cultivations. In addition, it was demonstrated that, especially at low pH, galacturonic acid has a drastic impact on cellular viability and galactose, arabinose and xylose consumption. As long as galacturonic acid is not consumed by S. cerevisiae, these inhibitory effects of galacturonic acid will remain a key issue in the yeast-based production of bioethanol and other products from pectin-rich feedstocks.Department of BiotechnologyApplied Science
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