Metabolic engineering has enabled the advancement of biotechnology over the past few decades through the use of cells as biochemical factories. Cellular factories have now provided many new safe and sustainable routes to fuels, pharmaceuticals, polymers, and specialty chemicals. Many of these successes have been achieved using the yeast Saccharomyces cerevisiae, which has been used and shaped by humans for millennia for the production of food and drink. Consequently, S. cerevisiae is one of the most studied eukaryotic organisms in existence, and has established genetic tools for engineering efforts. However, despite the many achievements in metabolic engineering of S. cerevisiae, it is still significantly more difficult to engineer than its prokaryotic counterpart, Escherichia coli. As a result, there is an unmet need to further develop genetic tools in yeast and to do so in the context of metabolic pathway engineering. The work presented here addresses this need through the study and engineering of heterologous gene expression. First, a new biosynthetic pathway is engineered for the production of muconic acid in yeast. Muconic acid is a dicarboxylic acid that can serve as a platform chemical for the production of several bio-polymers. The final muconic acid producing strain was developed through significant metabolic engineering efforts and reached a titer of nearly 141 mg/L muconic acid, a value nearly 24-fold higher than the initial strain. Second, a new method of engineering promoters is presented that allows for increased expression of native promoters and the de novo design of synthetic promoters. The highest expression synthetic promoter is within the top 6th percentile of native yeast promoters. Third, a study of native and synthetic terminators for heterologous gene expression is completed for the first time. This study demonstrates that terminators can tune heterologous expression by as much as an order of magnitude. Fourth, a comparative study of plasmid components dissects the different contributions to plasmid burden, copy number, and gene expression level. Collectively, this work represents a significant step forward in the metabolic engineering of yeast through the establishment of a new pathway and the study and engineering of new tools for heterologous gene expression. / text

RISE Bioeconomy is working with the biorefinery concept. That is, for the company, to utilize all wood derivatives in effective processes, resulting in unique products. RISE Bioeconomy is investigating the possibilities of producing holocellulose through an oxidative delignification process of wood, using peracetic acid (PAA). The product, high-purity holocellulose, can be used for the production of for example cellulose nanofibers, a product that in recent years have increased in interest. The PAA-delignification process relies on oxidative treatment with peracetic acid, where the lignin is solubilized in water through the formation of carboxylic groups in the lignin, yielding cellulose of very low lignin content and minimal degradation of the cellulose. In addition to the holocellulose, a solution containing water, acetic acid, salts and highly oxidized lignin is obtained. The PAA-delignification has yet only been performed in lab-scale, however, the aim is to scale up the process. There has not yet been any further utilization of the oxidized lignin and it has been proposed that esterification of the carboxylic groups with an alcohol is a possible valorization route. Due to the up-scaling of the PAA-delignification, it is of large interest to find a valorization route for the lignin by-product to fulfill the biorefinery concept. This master thesis focuses on evaluating esterification of an oxidized lignin, with the aim to propose a process suitable for up-scaling. The lab trials for the PAA-delignification of wood yielded limited amounts of lignin and due to difficulties while isolating this lignin, it was not partially characterized until months into the project. Therefore, model compounds were initially used to evaluate the process setup. Compounds with muconic acid type structures are some of the expected products from PAA-delignification of wood and muconic acid was therefore used as a model compound. In addition to this, vanillic acid was used to represent the phenolic lignin compounds. Gas Chromatography/Mass Spectrometry (GC/MS) analysis of the vanillic acid esterification was shown to be a suitable method for analyzing the conversion of this reaction. Due to the difficulties with the analysis of the model compounds, no results regarding the reaction procedures were obtained. As a “proof-of-concept”, a kraft lignin was oxidized, isolated and then esterified. Two different methods of esterification were performed, with and without a molecular sieve. It was assumed that the amount of carboxylic groups would increase after oxidation and decrease after a successful esterification. The amount of carboxylic groups after each process was analyzed with Phosphorus Nuclear Magnetic Resonance (31P-NMR). For the process without molecular sieve, the 31P-NMR analysis indicated that esterification of the lignin had occurred, to a limited extent. This was confirmed by Fourier Transform Infrared Spectrometry (FTIR). The process with molecular sieve showed no indication of esterification of the lignin. The project did prove the concept of esterifying an oxidized lignin and led to several different proposals for further work in the area. Due to the very wide scope of this project and several unexpected obstacles, this project did not fully answer the research questions given.

Os limites de exposição ocupacional ao benzeno, um agente carcinogênico, vêm diminuindo drasticamente nos últimos anos. Por outro lado, a concentração de benzeno em ambientes não ocupacionais tem aumentado devido à emissão biogênica e antropogênica, como exaustão de motores a gasolina e fumaça de cigarro. Indicadores Biológicos de Exposição (IBE) são utilizados como ferramentas importantes na avaliação da exposição humana ao benzeno. Com a diminuição dos limites de exposição, se faz necessário o desenvolvimento de metodologias analíticas com sensibilidade adequada para a determinação de IBE em fluidos biológicos que se correlacionem com baixas concentrações de benzeno absorvido pelo organismo. A utilização do fenol urinário como IBE ao benzeno, embora reconhecida mundialmente, tem a desvantagem de não apresentar boa correlação com a concentração de benzeno ambiental quando esta é menor do que 10 ppm (32 mg/m3). Os ácidos trans,trans-mucônico e S-fenilmercaptúrico, metabólitos do benzeno encontrados na urina, estão entre os compostos mais estudados como IBE ao benzeno. Neste trabalho, o ácido trans,trans-mucônico foi determinado na urina de indivíduos expostos ao benzeno utilizando as técnicas de Eletroforese Capilar (CE) e HPLC, ambas com detecção no UV. Na determinação por HPLC foi adaptada uma metodologia da literatura utilizando coluna analítica com fase reversa. Na determinação por CE foi proposta uma metodologia empregando duas condições analíticas alternativas: uma que utiliza um capilar especial com cela ótica de alta sensibilidade e a outra que utiliza um capilar comum, mas com adição de um modificador orgânico ao eletrólito. As duas condições apresentaram grandes vantagens, como análise rápida (15 minutos) e baixo limite de detecção (25 µg/L). Foram analisadas amostras de urina de indivíduos fumantes e não fumantes onde a sensibilidade da metodologia proposta foi suficiente para diferenciar estatisticamente os dois grupos avaliados. / The occupational exposure limits for benzene, a well-known carcinogenic agent, showed a drastic and continuous decrease in the last few years. In the other hand, benzene concentrations in non-occupational environments are increasing due to biogenic and anthropogenic emissions, like motor fuelled exhaustion and cigarette smoke. Biological markers of exposure are used like a powerful aid to evaluate human exposure for benzene. With the decrease of the exposure limits, the development of analytical methodologies is needed to accomplish adequate sensitivity for the exposure markers determination founded in biological fluids, in order to establish a correlation between the marker and the benzene concentration absorbed by organism. One of the biological marker of exposure for benzene recognized worldwide is urinary phenol, but the exposure for benzene at concentrations smaller than 10 ppm (32 mg/m3) usually is not correlated with urinary phenol concentration. The benzene metabolites trans,trans-Muconic and S-phenylmercapturic acids found in urine have been largely evaluated as biological markers of exposure. At the present work, trans,trans-muconic acid in urine from exposed individuals was determined by Capillary Electrophoresis (CE) and HPLC using UV detection. For HPLC, a pre-established method from literature using reversed phase column was utilized. A new analytical method was proposed using CE in two different conditions: one utilized a special capillary with high sensitivity optical cell and the other utilized a common capillary and an organic modifier added to the electrolyte. Both conditions showed interesting advantages, such as short-time analysis (15 min) and lower limit of detection (L.O.D = 25 µg/L). Urine samples from smokers and non-smokers individuals were analyzed and the proposed method allowed statistical differentiation between these groups.

Os limites de exposição ocupacional ao benzeno, um agente carcinogênico, vêm diminuindo drasticamente nos últimos anos. Por outro lado, a concentração de benzeno em ambientes não ocupacionais tem aumentado devido à emissão biogênica e antropogênica, como exaustão de motores a gasolina e fumaça de cigarro. Indicadores Biológicos de Exposição (IBE) são utilizados como ferramentas importantes na avaliação da exposição humana ao benzeno. Com a diminuição dos limites de exposição, se faz necessário o desenvolvimento de metodologias analíticas com sensibilidade adequada para a determinação de IBE em fluidos biológicos que se correlacionem com baixas concentrações de benzeno absorvido pelo organismo. A utilização do fenol urinário como IBE ao benzeno, embora reconhecida mundialmente, tem a desvantagem de não apresentar boa correlação com a concentração de benzeno ambiental quando esta é menor do que 10 ppm (32 mg/m3). Os ácidos trans,trans-mucônico e S-fenilmercaptúrico, metabólitos do benzeno encontrados na urina, estão entre os compostos mais estudados como IBE ao benzeno. Neste trabalho, o ácido trans,trans-mucônico foi determinado na urina de indivíduos expostos ao benzeno utilizando as técnicas de Eletroforese Capilar (CE) e HPLC, ambas com detecção no UV. Na determinação por HPLC foi adaptada uma metodologia da literatura utilizando coluna analítica com fase reversa. Na determinação por CE foi proposta uma metodologia empregando duas condições analíticas alternativas: uma que utiliza um capilar especial com cela ótica de alta sensibilidade e a outra que utiliza um capilar comum, mas com adição de um modificador orgânico ao eletrólito. As duas condições apresentaram grandes vantagens, como análise rápida (15 minutos) e baixo limite de detecção (25 µg/L). Foram analisadas amostras de urina de indivíduos fumantes e não fumantes onde a sensibilidade da metodologia proposta foi suficiente para diferenciar estatisticamente os dois grupos avaliados. / The occupational exposure limits for benzene, a well-known carcinogenic agent, showed a drastic and continuous decrease in the last few years. In the other hand, benzene concentrations in non-occupational environments are increasing due to biogenic and anthropogenic emissions, like motor fuelled exhaustion and cigarette smoke. Biological markers of exposure are used like a powerful aid to evaluate human exposure for benzene. With the decrease of the exposure limits, the development of analytical methodologies is needed to accomplish adequate sensitivity for the exposure markers determination founded in biological fluids, in order to establish a correlation between the marker and the benzene concentration absorbed by organism. One of the biological marker of exposure for benzene recognized worldwide is urinary phenol, but the exposure for benzene at concentrations smaller than 10 ppm (32 mg/m3) usually is not correlated with urinary phenol concentration. The benzene metabolites trans,trans-Muconic and S-phenylmercapturic acids found in urine have been largely evaluated as biological markers of exposure. At the present work, trans,trans-muconic acid in urine from exposed individuals was determined by Capillary Electrophoresis (CE) and HPLC using UV detection. For HPLC, a pre-established method from literature using reversed phase column was utilized. A new analytical method was proposed using CE in two different conditions: one utilized a special capillary with high sensitivity optical cell and the other utilized a common capillary and an organic modifier added to the electrolyte. Both conditions showed interesting advantages, such as short-time analysis (15 min) and lower limit of detection (L.O.D = 25 µg/L). Urine samples from smokers and non-smokers individuals were analyzed and the proposed method allowed statistical differentiation between these groups.