Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular / La producción de biocombustibles como fuente de energía alternativa a los combustibles fósiles busca presentar nuevas maneras de obtención que sean renovables y amigables con el ambiente. Dentro de estos biocombustibles surgen los alcoholes de fusel, como el butanol, el cual, al ser comparado con el etanol, presenta ventajas en su uso como combustible. Por otro lado, Saccharomyces cerevisiae es una levadura que puede producir isobutanol a partir del catabolismo de valina, posee resistencia a altas concentraciones de alcoholes y condiciones favorables para el escalamiento y producción, lo que la hacen un modelo para la obtención de butanol; Sin embargo, una de las limitantes de este proceso, es su baja producción, por lo que existe un gran interés en lograr un aumento de ésta. Este trabajo se enfocó en obtener, mediante ingeniería metabólica en S. cerevisiae, cepas sobre productoras de isobutanol. Para esto se utilizó la ruta de Ehrlich, la cual naturalmente se realiza dentro de la mitocondria, exceptuando las dos últimas reacciones. Se seleccionaron las enzimas que catalizan estas dos reacciones, alcohol deshidrogenasa (LlADHARE1) y α-cetoisovalerato deshidrogenasa (SkARO10), provenientes de Lactococcus lactis y Saccharomyces kudriavzevii respectivamente, y, mediante la fusión del péptido señal de citocromo C oxidasa se destinaron a la mitocondria. Utilizando Gibson Assembly se ensamblaron estos genes a promotores y terminadores nativos de S. cerevisiae. La transformación se realizó por recombinación homóloga in vivo mediante electroporación con el fin de obtener transformantes estables. La integración de la construcción de interés se realizó en el cromosoma V en el gen URA3. La medición de la producción de alcoholes se realizó mediante HPLC. De este trabajo se obtuvieron finalmente cepas transformadas con el gen SkARO10 de S. kudriavzevii con una producción de isobutanol de 19 mg/L, logrando de esta manera observar que la trasformación con el gen α-cetoisovalerato deshidrogenasa con expresión mitocondrial logra incrementar la producción de butanol al doble. Se propone que, para incrementar aún más la producción, es necesario integrar el gen de una enzima del tipo alcohol deshidrogenasa y utilizar una cepa industrial con producción basal de isobutanol mayor. / Production of biofuels as an alternative energy source to fossil fuels looks for renewable and environmentally friendly alternatives. Fusel alcohols, such as butanol, have advantages as a fuel compared to ethanol. Saccharomyces cerevisiae is a yeast that has the biosynthetic route for isobutanol, high alcohol-resistance and favourable conditions for scaling-up and production. These features make S. cerevisiae a promising cell-factory for the production of butanol; however, this yeast naturally produces low quantities of this alcohol, which is a limitation to the process. Thus, there is a great interest in developing strains that over-produce isobutanol. The aim of this work was to obtain over-producer strains of isobutanol, by means of metabolic engineering of S. cerevisiae using the Ehrlich pathway, which occurs inside the mitochondria except the last two reactions. The enzymes that catalyze these two reactions were selected, alcohol dehydrogenase (LlADHARE1) and α-ketoisovalerate dehydrogenase (SkARO10) from Lactococcus lactis and Saccharomyces kudriavzevii respectively, and fused to the cytochrome C oxidase signal peptide for sorting to mitochondria. The Gibson Assembly technique was used to assemble these genes to native promoters and terminators from S. cerevisiae. To obtain stable transformants, the transformation was carried out by in vivo homologous recombination by electroporation. The construct was integrated in the chromosome V in URA3 gene. Transformed strains with gene SkARO10 from S. kudriavzevii produced 19 mg/L of isobutanol, indicating that transformation with the gene α-ketoisovalerate dehydrogenase with mitochondrial sorting increases production of butanol two-fold. Additionally, to reach higher levels of butanol production, integration of alcohol dehydrogenase, is needed, in addition to use an industrial strain with a higher basal production of isobutanol.

Une pénurie de C4 s'est produite au cours des dernières années en partie à cause de la révolution naissante du gaz de schiste. Le faible coût et la grande abondance de cette source d'énergie nouvellement découverte sont rapidement devenues un facteur de changement pour l'industrie chimique. Bien que la concurrence avec cette source d'énergie non renouvelable ne soit pas une tâche facile, ses lacunes, telles qu'une production minimale en C3 et en C4, créent de nouvelles opportunités pour les molécules biosourcées. Dans ce travail, nous rapportons la conversion nouvellement découverte du (bio) isobutanol en n-butènes sur un catalyseur zéolitique. Ce catalyseur conduit à un rendement exceptionnel en butènes linéaires, ce qui est paradoxal car la FER est un catalyseur bien connu pour l'isomérisation squelettale des butènes. Bien que la recherche sur le dernier ait débuté il y a 30 ans, aucune conclusion définitive n'a encore été faite sur le mécanisme dominant : une réaction de monomoléculaire ou un mécanisme de "carbon pool". Acquérir des connaissances plus approfondies sur ce sujet est alors nécessaire pour expliquer la sélectivité inattendue en n-butène de cette réaction.Un criblage de catalyseurs a confirmé la sélectivité supérieure de FER pour les n-butènes (81%). Ceci est une augmentation significative par rapport aux catalyseurs de déshydratation couramment utilisés, alumine  (1%) et MFI (23%). Comme la sélectivité en n-butène pour les deux zéolites est significativement plus élevée que pour l’alumine un premier indice est que l'environnement confiné et cristallin de la porosité de la zéolite est crucial pour la sélectivité du n-butène. Cependant nous avons démontré que l'isobutanol est incapable d'accéder aux micropores de la structure FER; sa déshydratation doit donc se produire sur la surface externe de la zéolithe, en particulier sur ses sites acides de Brønsted.Nous avons déterminé l'identité du site actif responsable de l'isomérisation. En raison des similitudes avec la réaction d'isomérisation squelettale de du butène 1 à l'isobutène, un mécanisme de "carbon pool" a dû être envisagé. Cependant, aucun lien n'a été trouvé entre ces espèces et l’évolution de la sélectivité du n-butène, ne permettant pas de conclure quant à la participation de ces espèces carbonées comme sites actifs d'isomérisation. Par la suite, nous avons cherché à déterminer si les n-butènes sont formés directement ou via un mécanisme de type râteau dans lequel l'isobutène serait un intermédiaire. Nous avons établi que les constantes de vitesse pour l'isomérisation individuelle des butènes sont très inférieures à la constante de vitesse globale, excluant ainsi le mécanisme de type râteau. Par conséquent, le site actif d'isomérisation doit être identique ou situé à proximité du site actif de déshydratation pour éviter la désorption des espèces intermédiaires de surface sous forme d’isobutène. Nous avons ensuite démontré la nécessité d'une densité minimale d'acidité de Brønsted externe pour une sélectivité élevée en n-butène, également valable pour différentes zéolites, (10 MR ZSM-5 et ZSM-22 et 8 MR Erionite). Ces résultats nous ont ainsi amener à conclure que la sélectivité exceptionnelle des zéolithes de type FER en n-butène est le résultat de leur forte densité de sites acide de Brønsted externes. Cependant, une trop forte densité a un effet indésirable en terme de désactivation.L’ensemble de ces résultats nous permet de conclure que le catalyseur idéal doit présenter une densité de site d'acide de Brønsted externe très spécifique, suffisamment importante pour produire une sélectivité élevée en n-butène tout en évitant une désactivation excessive. Alors que les sites acides de Brønsted internes ne semblent pas jouer le rôle de site actifs pour la transformation de l'isobutanol en butènes linéaires, il n’est pas exclus qu’elle ait un impact sur le taux de formation des espèces carbonées. / A C4-shortage has been arising during the recent years partly as a result of the nascent shale gas revolution. The low cost and vast abundance of this newly discovered energy source rapidly became a game changer for the chemical industry. Although competing with this non-renewable energy source is not an easy task, its shortcomings such as a minimal C3 and C4 production, are creating new opportunities for bio-based molecules. In this work, we report on the newly discovered conversion of (bio)isobutanol to n-butenes over a zeolitic (ferrierite, FER structure) catalyst. It displays an exceptional yield in linear butenes. However, this is remarkable since FER is a well-known catalyst for the skeletal isomerisation of n- towards iso-butene. Although research on the n- to iso-butene skeletal isomerisation started 30 years ago, no firm conclusion has yet been made on the prevailing mechanism: a monomolecular reaction or a carbon pool mechanism. Acquiring deeper knowledge on this topic is required to explain the unexpected n-butene selectivity of this reaction. A screening of catalysts confirmed the superior selectivity of FER for n-butenes (~ 80 %). This is a significant increase compared to the commonly used dehydration catalysts, γ-alumina (~ 1 %) and MFI (~ 25 %). Since n-butene selectivity for both zeolites is significantly higher, a first hint is found that the confined and crystalline zeolite environment should be crucial for n-butene selectivity. However, we have demonstrated that isobutanol is unable to access the micropores of the FER structure; its dehydration must therefore occur on the external surface, in particular its acidic Brønsted sites.We have determined the identity of the active site responsible for isomerisation. Due to similarities with the 1- to isobutene skeletal isomerisation reaction, a carbon pool mechanism had to be considered. However, no correlation was found, hence allowing to exclude the carbonaceous species as possible isomerisation active sites. Next, we have investigated whether the n-butenes are formed directly or via a rake-type mechanism where isobutene is an intermediate. We have established that the rate constants for the individual butene isomerisation are lower than the over-all rate constant of iso-butanol transformation to n-butenes, thus excluding the rake-type mechanism. Hence, either dehydration-isomerisation occur in the same site or isomerisation occurs on a site located in the immediate neighbouring of the dehydration site in order to avoid the desorption of intermediate species as isobutene. With this respect, we have subsequently demonstrated the requirement of a minimal external Brønsted acid density for a high n-butene selectivity. Strikingly, the latter held also for different zeolites, (10 MR ZSM-5 and ZSM-22 and 8 MR Erionite) which suggests a general trend relating the n-butene selectivity to the density of external Brønsted acid sites with the existence of a “critical threshold” of external BAS density beneath which the selectivity towards n-butene is severely degraded. We concluded that the exceptional n-butene selectivity of FER is a result of its high external Brønsted acid density. However, an adverse effect was observed on isobutanol conversion as a high density of external sites is also linked to a faster deactivation. Additionally, we have determined that the selectivity for a secondary set of products, octenes, decreased with increasing the external Brønsted sites density; isolated external Brønsted acid sites should therefore function as active sites for octene formation.Finally, we conclude that the ideal catalyst should contain a very specific external Brønsted acid site density, sufficiently dense to produce high n-butene selectivity while avoiding excessive deactivation. While internal Brønsted acidity does not function as active sites for the one-step transformation, it is not excluded that they can impact the rate of carbonaceous species formation

The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker ́s yeast were grown in YPD media containing isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be investigated further, to be able to engineer a suitable producer of the biofuel butanol.

Today, most transportation fuels are derived from crude oil. However, fossil fuels are limited resources and contribute to climate change, and are therefore not considered as sustainable. Biofuels are highly relevant candidates for replacing fossil fuels and research has gone into butanol as a biofuel. It has a high energy density, is less hygroscopic and can be blended up to 85% with gasoline. The yeast Saccharomyces cerevisiae is considered a good host for bio- butanol production; it produces small amounts of isobutanol naturally through the Ehrlich pathway, is easy to manipulate genetically and can therefore be engineered to produce higher titres of butanol. End-product toxicity, however, is a problem that needs to be solved to make butanol production in S. cerevisiae more effective, since the organism cannot tolerate higher concentrations of butanol than 2%. Four different S. cerevisiae strains were cultivated in 1.5%, 2%, 3% and 4% isobutanol by spot tests and in liquid media to evaluate their tolerance. Gene expression was measured for genes RPN4, RTG1 and ILV2 to examine their up-regulation and relevance in butanol tolerance. S. cerevisiae strain Saflager 34/70 was determined as the most tolerant strain and was able to grow in 2% liquid isobutanol and 3% isobutanol on agar plates. A three-fold up-regulation of RPN4, a transcription factor involved in the regulation of proteasome gene expression, was observed. These results contribute to the progress of genetic engineering of butanol host organisms, which is needed to create a more effective production of butanol as a biofuel.

This research conducted exhaust tests in an HDDE (heavy-duty diesel engine) using pure diesel fuel mixed with 10 to 30% isobutanol under the condition of U.S. Transient Cycle. Characteristics of 18 carbonyls emissions were investigated and compared with those using pure diesel. Results showed that the brake power (BP) and brake thermal efficiency (BTE) were decreased with increasing isobutanol mixtures (10 to 30%). Brake specific fuel consumption (BSFC) was increased for isubutanol ¡Ø 10%, but was decreased for isubutanol above 10%. The regulated emissions of CO, PM and NOx were decreased, but CO2 and THC were increased, due to variations of cetane number and heating value. Total carbonyls emission concentrations with pure diesel fuel were 893.25 £gg/m3, with emission factors being 52.57 mg/bhp-hr or 218.44 mg/L-fuel. When 10 to 30% isobutanol mixture was added, total carbonyls concentrations ranged from 1108.21 to 2622.27 £gg/m3, with emission factors being 268.83 to 610.94 mg/L-fuel, or 68.93 to 175.25 mg/bhp-hr. The ozone formation potential of diesel engine with pure diesel fuel was 7132.72 g-O3/m3.When 10 to 30% isobutanol mixture was used, total ozone formation potential ranged from 8764.39 to 20168.73 g-O3/m3. Total carbonyls emissions were increased with increasing isobutanol contents. In summary, addition 10% isobutanol was an optimal blend, since both fuel saving and reductions of pollutant emissions can be achieved.

Pollution and environmental concerns require further improvements in engine technologyas well as research of alternative fuels. Users of handheld products are directlyexposed to the exhaust gas and thus to the occurring emissions, which cancause significant damage. Pursuing to reduce emissions is therefore a vital task.The European commission proposed a renewable energy directive, that claims abiofuel share of at least 6,8% by 2030 and a reduction of first generation fuel butan increasing of second generation fuels. Ethanol is up to now the most commonsource of renewable alternatives in gasoline blends. Isobutanol has several advantagesover ethanol and can be produced as a second generation biofuel.This thesis aims to get a better understanding on how isobutanol influences theemission and combustion in small two-stroke engines and furthermore what effectdifferent isobutanol blends with neat gasoline and alkylate have on the engine performance.Different isobutanol blends with neat gasoline as well as alkylate fuel have beenused and analyzed.This thesis has via an experimental study shown, that hydrocarbon as well as carbonmonoxide emission increased with increasing isobutanol percentage. Hence, nitrogenoxides emission decreased with isobutanol. Lower cylinder and exhaust temperatureswere measured with isobutanol blends. Through cylinder pressure measurements,the mass fraction burned, mass fraction burned 50% and rapid burningangle could be analyzed. It has shown that isobutanol blends reach a mass fractionburned of 50% slightly later and have a greater rapid burning angle.

The diminishing of fossil fuels and growing concerns towards climate change have intensified biofuel production from renewable resources. Recently, progresses are made in microbial production of biofuels. Among various biofuels, isobutanol is gaining an increasing attention due to its high energy content and suitable chemical and physical properties, enabling it to be a suitable substitution of fossil fuel. In this study, instead of using heterotrophic microorganisms, we performed metabolic engineering of Synechocystis PCC 6803 (Synechocystis) for isobutanol production under autotrophic condition. After introduced 2-keto acid pathway, Synechocystis is able to produce isobutanol when provided with water, carbon dioxide and solar energy. When cultivated in an optimal condition (50 μmol photons m-1s-2 and adjusted pH to 7-8 with HCl), the engineered strain pEEK2-ST was able to produce 425 mg L-1 in-flask isobutanol titer and 911 mg L-1 cumulative isobutanol titer, respectively, in 46 days. There should be bottlenecks existing in 2-keto acid pathway based on the similar isobutanol production of strain pEEK2-ST with and without pyruvate addition. However, the attempt to identify potential bottlenecks of upstream genes by overexpressing ST and one of the three upstream genes failed, instead what we conclude is that the isobutanol production is tightly correlated to Kivd (ST) expression level. Thus, more strategies will be employed for identifying bottlenecks successfully and further improvement of isobutanol production in the future. In conclusion, this study demonstrates the importance of cultivation condition on isobutanol production in Synechocystis.