2-phenylethanol (2PE) is a valuable fragrance compound which gives a rose-like aroma. As such 2PE is one the highest used fragrances globally. While 2PE is predominantly produced from petrochemical resources, there is a growing market for a naturally derived alternative for food products. 2PE from natural sources is priced so highly due to limited supply from rose petals. Recently, a few reports have demonstrated the production in yeasts through both the de novo production from glucose and ex novo biosynthesis with L-phenylalanine as a precursor. While these are promising most of the yeasts used can only produce low titres under optimal conditions, and the fermentation still appears to be too expensive. In this investigation the wine yeast M. pulcherrima was selected to be assessed for 2PE production. M. pulcherrima is known to produce 2PE in small titres in wine production though has yet to be explored as a platform for this product. M. pulcherrima has several advantages as a yeast platform, in that it produces a range of antimicrobials which can ward off invasive species, allowing for less sterile control in any large scale fermentation. M. pulcherrima was demonstrated to be able to produce 2PE in high titres in the batch mode through de novo synthesis of glucose, producing up to 1 g/L in shake flasks on the lab scale. Arabitol was also observed in the fermentation broth and was produced up to 20 g/L. The fermentation was then scaled up to 2L in batch mode. From these experiments, up to 700 mg/L of 2PE was produced. This is substantially more than any other yeast in the literature to date. Though when xylose or glycerol was present then 2PE production was severely limited. M. pulcherrima was also demonstrated to be able to produce 2PE by bioconversion from phenylalanine up to 1.5 g/L. This 2PE concentration is suggested to be threshold of toxicity to M. pulcherrima by the toxicity study. The production of 2PE could be increased substantially by introducing an absorbent into the process. Liquid solvents and solid adsorbents were assessed to increase 2PE production, used as in-situ 2PE adsorbents. Oleyl alcohol was found to be a good solvent for in-situ extractive solvent in M. pulcherrima culture and increase the production to 3.3 g/L which is higher than 2PE tolerance threshold of the yeast. Activated carbon was also found to be an excellent 2PE adsorbents, with maximum Langmuir adsorption capacity up to 0.807 g/g. 2PE synthesis with activated carbon as an in-situ adsorbent can increase 2PE production to 14 g 2PE/L. Finally, the process was scaled to 2L and run in batch, continuous and semi-continuous modes. This study demonstrates that not only is M. pulcherrima a viable organism to produce 2PE but it has the potential to be scaled up and run in a more cost effective semi-continuous mode when coupled to a continuous extraction technique.

abstract: Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2019

The pine weevil (Hylobius abietis L.) is one of the two topmost economically important insect pests in Swedish conifer forests. The damage increase in areas were the silvicultural practice is to use clear cuttings were the insects gather and breed. During egglaying the female protects her offspring by creating a cave in roots and stumps were she puts her egg and covers it with frass, a mixture of weevil feces and chewed bark. Adult pine weevils have been observed to feed on the other side of the egg laying site and antifeedant substance has been discovered in the feces of the pine weevil. We think it is possible that microorganisms present in the frass contribute with antifeedant/repellent substances. Little is known about the pine weevils associated bacteria community and their symbiotic functions. In this thesis the bacterial community is characterized in gut and frass both from pine weevils in different populations across Europe as well as after a 28 day long diet regime on Scots pine, silver birch or bilberry. Volatile substances produced by isolated bacteria as well as from a consortium of microorganisms were collected with solid phase micro extraction (SPME) and analyzed with GC-MS. The main volatiles were tested against pine weevils using a two-choice test. Wolbachia, Rahnella aquatilis, Serratia and Pseudomonas syringae was commonly associated with the pine weevil. 2-Methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol were found in the headspace from Rahnella aquatilis when grown in substrate containing pine bark. 2-Methoxyphenol and 3-methyl-1-butanol, phenol and methyl salicylate were found in pine feces. Birch and bilberry feces emitted mainly linalool oxides and bilberry emitted also small amounts of 2-phenylethanol. A second part of the thesis discusses the role of fungi in forest insect interactions and the production of oxygenated monoterpenes as possible antifeedants. Spruce bark beetles (Ips typhographus L.) aggregate with the help of pheromones and with collected forces they kill weakened adult trees as a result of associated fungi growth and larval development. A fungi associated with the bark beetle, Grosmannia europhoides, was shown to produce de novo 2-methyl-3-buten-2-ol, the major component of the spruce bark beetle aggregation pheromone. Chemical defense responses against Endoconidiophora polonica and Heterobasidion parviporum were investigated using four clones of Norway spruce with different susceptibility to Heterobasidion sp. Clone specific differences were found in induced mono-, sesqui and diterpenes. A number of oxygenated monoterpenes which are known antifeedants for the pine weevil were produced in the infested areas. / <p>QC 20160601</p>

Zugl.: Bonn, Univ., Diss.

Peu d’enzymes de synthèse de composés odorants sont connues chez le genre Rosa. Ce travail de thèse a permis l’identification de quelques-unes de ces protéines grâce à la technologie des puces à ADN, à l’analyse de l’expression des gènes par RT-PCR quantitative en temps réel (qPCR) et à l’analyse des parfums par chromatographie en phase gazeuse (CPG). Une puce confrontant les ADNc d’une rose parfumée à ceux d’une rose non parfumée a permis de corréler l’expression d’un gène, codant pour une Nudix hydrolase, très fortement exprimé dans la rose parfumée, avec la présence des monoterpènes dans le parfum de nombreux cultivars de rosiers. La caractérisation d’un rosier dont l’expression de ce gène est fortement réduite par ARN interférants, a permis de confirmer le rôle de celui-ci dans la synthèse des monoterpènes. La phénylacétaldéhyde synthase (PAAS) est une autre enzyme participant à la synthèse du parfum. Trois allèles de cette protéine ont précédemment été mis en évidence. Les résultats de qPCR et de CPG dans une population hybride ont permis de montrer que l’allèle a1 est le seul à pouvoir induire la synthèse et l’émission de 2-phényléthanol. Les activités respectives des différentes isoformes ont été testées in vitro chez la levure et in planta dans des feuilles de tabac et des cals de rosier : ces expériences montrent que les trois isoformes ont des activités comparables. L’absence de synthèse de 2-phényméthanol chez les plantes présentant les isoformes a2 et a3 réside donc dans la très faible expression de leurs allèles, induisant probablement une faible concentration de l’isoforme dans les cellules / Very few enzymes responsible for the biosynthesis of scent compounds in the genus Rosa are known so far. This PhD thesis aims to identify some of these proteins with DNA microarray technology, gene expression analysis by real-time quantitative PCR (qPCR) and scent analysis by gas chromatography (GC). An array comparing cDNA from a scented rose to those of a non-scented one, showed a correlation between expression of a yet-unknown gene, encoding a Nudix hydrolase, highly expressed in the scented rose, and the presence of monoterpenes in the scent of many rose cultivars. Characterization of a rose cultivar, in which expression of this gene has been decreased by RNA interference, confirmed its role in monoterpene synthesis. The phenylacetaldehyde synthase (PAAS) is another enzyme implicated in scent biosynthesis. Three alleles of this protein had been previously described. qPCR and GC experiments in a hybrid population showed that the a1 allele is the only one able to induce 2-phenylethanol biosynthesis. The respective activities of the different isoforms were tested in vitro in yeast, and in planta in tobacco leaves and rose calli: these experiments showed that the three isoforms have comparable activities. The lack of 2-phenylethanol production in plants having a2 and a3 isoforms is thus due to the very low expression of their respective alleles, probably inducing very low isoform concentration in cells

Previous research of colonies formed by yeast Saccharomyces cerevisiae growing on glycerol agar medium revealed two major cell types of U and L cells that are formed within these colonies. This colonial cell differentiation seem to be caused by communication among yeast cells as well as whole colonies and affected by changes in the environment (for example changes in nutrients). Studies of U and L cells showed that U cells are more resistant against biological, chemical and physical stresses than L cells. The aim of this thesis was to isolate U and L cell types and investigate their resistance against selected low molecular weight chemical substances produced in Ehrlich pathway. Ehrlich pathway was discovered in 1907 and was classified as amino acid catabolic pathway in yeast S. cerevisiae. The low molecular intermediates are formed in Ehrlich pathway which are called fusel (original name from German) alcohols and acids. These chemical substances are widely used in food industry and cosmetics especially because of their aroma. Several studies provided indications that these chemical substances may affect development of colonies and important yeast functions such as switching to the pseudohypfal growth of S. cerevisiae cells. Some chemical substances of the Ehrlich pathway were selected and their effects on...

Las esmectitas como hectorita o saponita, son silicatos laminares. Tienen múltiples aplicaciones (catálisis, nanocomposites, adsorbentes) e interesa tener un material reproducible y sin impurezas. Los métodos convencionales de síntesis comportan tratamientos largos o a altas temperaturas. La introducción del microondas puede disminuir ambos. El objetivo de esta tesis es estudiar la síntesis de hectorita y saponita con microondas y su aplicación como soportes catalíticos de Ni en la hidrogenación de óxido de estireno para la obtención de 2-feniletanol (componente principal de la esencia de rosas) y en la preparación de composites con poliuretano. Se obtienen hectoritas y saponitas reproducibles. Cuando se emplean como soportes de catalizadores de Ni se obtienen conversión y selectividad total hacia el 2-feniletanol. Cuando se utilizan las arcillas obtenidas en preparación de composites sus propiedades son comparables a los de la bibliografía. / Smectites like hectorite or saponite, are phyllosilicates. They can be applied for many purposes (catalysis, nanocomposites, adsorbents) but a reproducible, pure material is necessary. Classical methods of synthesis involve long synthesis times at high temperatures. The use of microwaves can be an interesting alternative. The scope of this thesis is to study the synthesis of hectorites and saponites with microwaves and their application as supports of Ni catalysts in the hydrogenation of styrene oxide to obtain 2-phenylethanol (main component of rose oil) and in the preparation of composites with polyurethane. We obtained reproducible hectorites and saponites. When used as supports of Ni catalysts we obtained total conversion and selectivity to 2-phenyethanol. When obtained clays were used in the preparation of polyurethane composites, their properties were comparables to those of the bibliography

This thesis describes the synthesis of novel ligands that include enantiomerically pure acetal and oxazoline moieties. These ligands are utilised in a number of metalmediated asymmetric syntheses. All asymmetric acetals and pyridine based acetals are synthesised in good yield in a single step from their corresponding enantiomerically pure diols. C2 symmetric bisacetals are investigated as ligands in the organolithium and Grignard additions to benzaldehyde with promising results. C2 symmetric bisacetals and pyridine based acetals are tested for their ability to induce asymmetry in copper(l) catalysed cyclopropanation of styrene using ethyl diazoacetate and the lanthanide(lII) catalysed Diels-Alder cycloaddition involving Danishefsky's diene with little success. Enantiomerically pure phosphinooxazoline ligands are available in good yield in two steps from their corresponding enantiomerically pure aminoalcohols. Enantiomerically pure acetal substituted pyridines and phosphinooxazoline ligands are considered in the rhodium (I) catalysed hydrosilylation of ketones. Reaction conditions for the more successful phosphinooxazoline ligands are optimised. Using these ligands a range of enantiomerically enriched alcohols is presented in good yield and enantiomeric excess. Novel phosphinooxazoline ligands are applied to the palladium(O) catalysed allylic substitution reaction with excellent enantioselectivities of the substitution product.