Cellular Processes and Mechanisms in Saccharomyces cerevisiae Influencing Anaerobic Xylose Fermentation

In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses) extracted from the raw material. The aim of the work presented in this thesis has been to increase the ethanol productivity of recombinant xylose-fermenting strains of the yeast Saccharomyces cerevisiae during batch fermentation of a glucose/xylose mixture. A parameter that has a big influence on productivity is cellular growth and the yeast strains currently used today grow rather poorly on xylose. Many of the signals cells use to regulate growth originate from changes in the concentrations of metabolites inside the cells. To increase our knowledge of xylose metabolism the dynamic changes in intracellular metabolite concentrations were measured during batch fermentation of a glucose/xylose mixture using LC-MS/MS. This study gave meaningful insights about important intracellular signals, biological phenomena and mechanism. The analysis of the metabolite data pointed toward limitations in the folding of proteins inside the ER, which might be the underlying cause of the slow growth on xylose. Another important factor is the regulation of expression of genes required for sugar transport and those related to fermentative metabolism. Hexokinase 2 (Hxk2p) is an important bi-functional protein that acts both as a catalytic enzyme and a global transcription factor. This protein plays a role in the regulation of the above mentioned genes and becomes inactivated in the presence of xylose. As a consequence it loses its’ regulatory function. In an effort to improve repression signals during xylose fermentation this protein was engineered to become immune towards inactivation by xylose. By combining methods for protein and genetic engineering with fermentation technology a mutation in the gene was identified which increased the catalytic activity by 64% in the presence of xylose. The new variant allowed faster glucose consumption in the presence of xylose, but had no obvious impact on xylose fermentation. These results indicate that Hxk2p does not act alone and other proteins are involved in the regulation. These proteins remain to be identified. This thesis describes the cellular processes required for balanced anaerobic microbial growth and the intracellular signals that regulate them. The aim has been to identify biochemical mechanisms that limit anaerobic growth of recombinant S. cerevisiae strains on xylose

Development and Application of Fluxomics Tools for Analyzing Metabolisms in Non-Model Microorganisms

Decoding microbial metabolism is of great importance in revealing the mechanisms governing the physiology of microbes and rewiring the cellular functions in metabolic engineering. Complementing the genomics, transcriptomics, proteinomics and metabolomics analysis of microbial metabolism, fluxomics tools can measure and simulate the in vivo enzymatic reactions as direct readouts of microbial metabolism. This dissertation develops and applies broad-scope tools in metabolic flux analysis to investigate metabolic insights of non-model environmental microorganisms. 13C-based pathway analysis has been applied to analyze specific carbon metabolic routes by tracing and analyzing isotopomer labeling patterns of different metabolites after growing cells with 13C-labeled substrates. Novel pathways, including Re-type citrate synthase in tricarboxylic acid cycle and citramalate pathways as an alternate route for isoleucine biosynthesis, have been identified in Thermoanaerobacter X514 and other environmental microorganisms. Via the same approach, the utilizations of diverse carbon/nitrogen substrates and productions of hydrogen during mixotrophic metabolism in Cyanothece 51142 have been characterized, and the medium for a slow-growing bacterium, Dehalococcoides ethenogenes 195, has been optimized. In addition, 13C-based metabolic flux analysis has been developed to quantitatively profile flux distributions in central metabolisms in a green sulfur bacterium, Chlorobaculum tepidum, and thermophilic ethanol-producing Thermoanaerobacter X514. The impact of isotope discrimination on 13C-based metabolic flux analysis has also been estimated. A constraint-based flux analysis approach was newly developed to integrate the bioprocess model into genome-scale flux balance analysis to decipher the dynamic metabolisms of Shewanella oneidensis MR-1. The sub-optimal metabolism and the time-dependent metabolic fluxes were profiled in a genome-scale metabolic network. A web-based platform was constructed for high-throughput metabolic model drafting to bridge the gap between fast-paced genome-sequencing and slow-paced metabolic model reconstruction. The platform provides over 1,000 sequenced genomes for model drafting and diverse customized tools for model reconstruction. The in silico simulation of flux distributions in both metabolic steady state and dynamic state can be achieved via flux balance analysis and dynamic flux balance analysis embedded in this platform. Cutting-edge fluxomics tools for functional characterization and metabolic prediction continue to be developed in the future. Broad-scope systems biology tools with integration of transcriptomics, proteinomics and fluxomics can reveal cell-wide regulations and speed up the metabolic engineering of non-model microorganisms for diverse bioenergy and environmental applications

Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003

Study of the differences in the fermentative metabolism of S. cerevisiae, S. uvarum and S. kudriavzevii species

Tesis por compendio[ES] Saccharomyces cerevisiae, además de ser un importante organismo modelo en biología, es indiscutiblemente la especie de levadura más utilizada en procesos fermentativos industriales, incluyendo el sector enológico. Su capacidad de fermentar en concentraciones elevadas de azúcares, tolerar concentraciones altas de etanol y soportar la adición de sulfitos, son algunos de los factores que explican su éxito en fermentaciones vínicas. El metabolismo fermentativo de S. cerevisiae en condiciones enológicas se conoce bien gracias a una amplia bibliografía científica. En cambio, aún se sabe poco sobre el metabolismo de las especies de Saccharomyces criotolerantes, S. uvarum y S. kudriavzevii, quienes han suscitado recientemente el interés del sector vitivinícola por sus buenas propiedades fermentativas a bajas temperaturas, tales como la producción de vinos con mayor contenido en glicerol y alta complejidad aromática, llegando a veces a reducir su contenido en etanol. En este contexto, esta tesis pretende ampliar nuestros conocimientos sobre el metabolismo fermentativo de S. uvarum y S. kudriavzevii en condiciones enológicas, profundizando en el entendimiento de las diferencias existentes con el de S. cerevisiae, así como entre cepas de S. cerevisiae de distintos orígenes. Para ello, hemos utilizado varias técnicas ómicas para analizar la dinámica de los metabolomas (intra- y extracelulares) y/o transcriptomas de cepas representativas de S. cerevisiae, S. uvarum y S. kudriavzevii a alta (25 °C) y baja (12 °C) temperatura de fermentación. También, hemos desarrollado un modelo metabólico a escala de genoma que, junto a un análisis de balance de flujos, es capaz de cuantificar los flujos a través del metabolismo del carbono y del nitrógeno de levaduras en cultivo de tipo batch. Así, el conjunto de estos trabajos nos ha permitido identificar rasgos metabólicos y/o transcriptómicos relevantes para el sector enológico en estas especies. También se aporta nueva información sobre las especificidades de redistribución de flujos en la red metabólica de levaduras del género Saccharomyces acorde a la especie y las fluctuaciones ambientales que ocurren durante una fermentación vínica.[CAT] Saccharomyces cerevisiae, a més de ser un important organisme model en biologia, és indiscutiblement l'espècie de llevat més utilitzat en processos fermentatius industrials, incloent el sector enològic. La seua capacitat de fermentar grans concentracions de sucres, tolerar concentracions altes d'etanol i suportar l'addició de sulfits, són alguns dels factors que expliquen el seu èxit en fermentacions víniques. D'aquesta manera, el metabolisme fermentatiu de S. cerevisiae en condicions enològiques està ben descrit i es beneficia d'una àmplia bibliografia científica. En canvi, poc se sap encara sobre el metabolisme de les espècies de Saccharomyces criotolerants, S. uvarum i S. kudriavzevii, els qui han recentment suscitat l'interés del sector vitivinícola per les seues bones propietats fermentatives a baixes temperatures, com ara la producció de vins amb major contingut en glicerol, alta complexitat aromàtica i arribant a vegades a reduir el seu contingut en etanol. En aquest context, aquesta tesi pretén ampliar els nostres coneixements sobre el metabolisme fermentatiu de S. uvarum i S. kudriavzevii en condicions enològiques, aprofundint en l'enteniment de les diferències existents amb el de S. cerevisiae, així també com entre ceps de S. cerevisiae de diferents orígens. Per a això, hem utilitzat diverses tècniques omiques per a analitzar la dinàmica dels metabolomes (intra- i extracelul·lars) i/o transcriptomes de ceps representatius de S. cerevisiae, S. uvarum i S. kudriavzevii a alta (25 °C) i baixa (12 °C) temperatures de fermentació. També, hem desenvolupat un model metabòlic a escala del genoma que, al costat d'una anàlisi de balanç de fluxos, és capaç de quantificar els fluxos a través del metabolisme carbonat i nitrogenat de llevats en cultius de tipus batch. Així, el conjunt d'aquests treballs ens ha permés identificar trets metabòlics i/o transcriptómics rellevants per al sector enològic en aquestes espècies. També aporta nova informació sobre les especificitats de redistribució de fluxos en la xarxa metabòlica de llevats del gènere Saccharomyces concorde a l'espècie i les fluctuacions ambientals ocorrent durant una fermentació vínica.[EN] Saccharomyces cerevisiae, besides being an important model organism in biology, is undoubtedly the most widely used yeast species in industrial fermentation processes, including the winemaking sector. Its ability to ferment at high levels of sugars, tolerate high ethanol concentrations and withstand the addition of sulfites are some of the factors explaining its success in wine fermentation. Accordingly, the fermentative metabolism of S. cerevisiae under oenological conditions is well described and benefits from a large scientific literature. In contrast, little is known about the metabolism of the cryotolerant Saccharomyces species, S. uvarum and S. kudriavzevii, which have recently attracted the interest of the wine industry for their good fermentative properties at low temperatures, such as the production of wines with higher glycerol content, high aromatic complexity and sometimes even reduced ethanol content. In this context, this thesis aims to expand our knowledge on the fermentative metabolism of S. uvarum and S. kudriavzevii under oenological conditions, deepening our understanding of the existing differences with that of S. cerevisiae, as well as between S. cerevisiae strains of different origins. For this purpose, we have used several omics techniques to analyze the dynamics of the (intra- and extracellular) metabolomes and/or transcriptomes of representative strains of S. cerevisiae, S. uvarum and S. kudriavzevii at high (25 °C) and low (12 °C) fermentation temperatures. Also, we have developed a genome-scale metabolic model that, together with a flux balance analysis, is able to quantify fluxes through carbon and nitrogen metabolism of yeast in batch culture. Taken together, this work has allowed us to identify metabolic and/or transcriptomic traits relevant to the oenological sector in these species. It also provides new information on the specificities of flux redistribution in the metabolic network of Saccharomyces yeasts according to the species and environmental fluctuations occurring during wine fermentation.The present work has been carried out at the Department of Food Biotechnology of the IATA (CSIC). Romain Minebois was funded by a FPI grant (REF: BES-2016-078202) and supported by projects AGL2015-67504-C3-1R and RTI2018-093744-BC31 of the Ministerio de Ciencia e Inovación awarded to Amparo Querol.Minebois, RCM. (2021). Study of the differences in the fermentative metabolism of S. cerevisiae, S. uvarum and S. kudriavzevii species [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/176018TESISCompendi

The relationship of glycolytic/gluconeogenic intermediates in brewing yeast (Saccharomyces uvarum) fermentations to growth

Bibliography: pages 215-264.The objective of this study has been to understand the metabolic interrelationship between yeast growth, regulation of glycolytic/gluconeogenic flux and accumulation of glycosyl donors for polysaccharide synthesis in brewing yeast (Saccharomyces uvarum) fermentations. Loss of fermenting power of a brewing yeast population may be created by a condition that inhibits growth by limiting amino acid formation and protein synthesis. In commercial strains of S. uvarum this loss may be transitory, or, if not corrected, may ultimately lead to yeast degeneration. The potential industrial impact is realised for fermentation systems which may limit yeast growth, eg. continuous systems, use of pressure and, particularly, systems utilizing immobilised cells