Since the 1970's there has been industrial interest in using microorganisms as microcapsules. The encapsulation of actives (e.g. flavours, drugs, perfumes) is a necessary process for pharmaceutical and food companies because the precious and often expensive ingredients must be protected from degradation and also released in a specific site or under a specific stimulus. Saccharomyces cerevisiae, baker's yeast, represents a first choice microorganism for the encapsulation of active ingredients. It is biodegradable and biocompatible with human digestion and skin, and can be produced in an easy and cheap way. A major part of this project has been dedicated to the development of robust methods of extraction and quantification of hydrophobic substances loaded inside yeast cells, which have been subsequently combined with an indirect, fluorescence-based method for the evaluation of the rate of loading of hydrophobic substances in the same cells. In particular, it has been found that this process reaches a limit in the maximal loading capacity of intact yeast cells, most likely reflecting the maximal volume of the lipid droplet organelles in which loaded hydrophobes accumulate. With the new on-line (fluorescence-based) and off-line (chromatography-based) methods developed here it has been established that the loading process fundamentally follows a diffusion model, in which the solubility in water determines the permeation of substances through the cell wall and ultimately their uptake by yeast cells. However, treating yeast cells with organic solvents like DMSO - a new approach introduced in Prof. Tirelli's lab to enhance the encapsulation of hydrophobes - completely changes the chemical-physical parameters of the encapsulation process. In DMSO-treated cells, substances are loaded fundamentally in response to their hydrophobicity. Conversely, once loaded, the same substances are released with a rate that is inversely proportional to their hydrophobicity, as observed by applying a novel approach to measure the release of hydrophobes encapsulated in yeast cells, either in the absence of presence of DMSO-treatment. In conclusion, the new evidence reported here clarifies basic aspects of hydrophobe encapsulation in intact yeast cells and will thus help improving future applications of these microcapsules as a valid, inexpensive and biocompatible drug delivery system.

A search for members of the genus Leucospor idium (Ustilaginaceae) in and near southern British Columbia has yielded 147 isolates of L. scottii, and a single isolate of an undescribed species with apparent affinities in the genus. L. scottii was primarily found on decaying marine vegetation and driftwood, but isolates were also obtained from stream foam, snow, a decaying turnip root, bark mulch, and rain-derived stem flow over the trunk of a living tree. The species predominated in laboratory incubations of marine algal materials collected in the winter, spring, and late autumn. The majority of isolates obtained directly from natural substrates were also found during periods of cold seasonal conditions. It is suggested that low temperature is characteristic of L. scottii habitats. Locally-obtained L. scottii strains are all heterothallic, and are completely interfertile with mating test strains originating from the southern hemisphere. Morphological and known physiological and biochemical characters of the local strains are similar to those previously described for the species. However, mating studies of local isolates have revealed that the tetrapolar incompatibility system of L. scottii is distinguished by the possession of multiple alleles at the A locus. Similar modifications of the tetrapolar system are known in Tremella and other heterobasidiomycete genera, but have not previously been reported in species of the Leucosporidium-Rhodosporidium group. The strain thought to belong to an undescribed species of Leucosporidium was obtained from a sample of filamentous green algae growing in a vernal pond near a peat bog. The fungus is homothallic, and is morphologically and physiologically distinct from other Leucosporidium and Rhodosporidium species. Of the six known species of Leucosporidium, five (L. antarcticum, L. friqidum, L. gelidum, L. nivalis, and L. stokesii) were not found during the course of the present study. All five species are obligately psychrophilic and may not be able to remain established within the study area. However, a survey of local L. scottii isolates showed that 5 out of 147 isolates (3.4%) were obligately psychrophilic. / Science, Faculty of / Botany, Department of / Graduate

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The synthesis of a new thiamine analogue, 2'-hydroxythiamine, is reported. Kinetic studies with thiamine pyrophosphate analogues and apopyruvate decarboxylase (EC 4.1.1.1) from brewer's yeast, gave the values of 2.3 X 10^-5 M as the K_m for thiamine pyrophosphate and 2.0 X 10^-5 M as the K_m for 2'-ethylthiamine pyrophosphate. The V_max for the latter was 14% that of thiamine pyrophosphate. Inhibitor constants, K_i, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme. All values are given for the pyrophosphate esters: tetrahydrothiamine, 0.65 X 10^-5 M; oxythiamine, 2.0 X 10^-5 M; 2'-n-butylthiamine, 4.5 X 10^-5 M; 2'-methoxythiamine, 7.0 X 10^-5 M; pyrithiamine, 7.8 X 10^-5; thiochrome, 15. X 10^-5 M; 2'-desmethylthiamine, 22. X 10^-5 M; 2'-hydroxythiamine, 38. X 10^-5 M. None of the inhibitors exhibited coenzyme activity. A hydrophobic interaction of the 2'-methyl group of thiamine pyrophosphate with the apoenzyme is suggested from these studies. The formation of a fluorescent complex at pH 6.7 between apopyruvate decarboxylase and thiochrome pyrophosphate was detected and found to be dependent upon Mg(II) ions. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate function in binding to the protein. The shift in the fluorescence emission spectrum of thiochrome pyrophosphate toward lower wavelengths upon complex formation with the apoenzyme, coincided with the behavior of thiochrome in solvents of decreasing dielectric constant. This latter observation suggests involvement of the thiochrome pyrophosphate with a hydrophobic region of the enzyme. A study of the pH dependency of the enzyme-coenzyme complex indicated considerable recombination of apoenzyme and coenzyme at alkaline pH, where dissociation of the coenzyme usually takes place. A rationale for the interpretation of the pH-behavior of the enzyme-coenzyme complex is offered. An amino acid analysis of a highly purified sample of pyruvate decarboxylase, considered to be essentially homogeneous, is reported. Assuming a molecular weight of 175,000 for the enzyme, a total of 1317 amino acid residues were calculated, of which 52.1% fall into the non-polar category. the half-cystine content was calculated as 10.3%, and the proline content, as 4.6%. The specific volume was calculated as 0.737 ml per g. A single low-angle X-ray diffraction study gave a value of 35.5 ± 1.5 A for the radius of gyration of pyruvate decarboxylase. Assuming a spherical shape, a diameter of 91.6 ± 4.0 A was calculated.

Synthetic biology consists of the design and construction of customized cell-based systems, and metabolic engineering is its co-discipline that aims to engineer these cells into biological factories for the production of drugs, chemical commodities and fuels. Together, these two disciplines continue to provide various innovative solutions to current problems of humanity in the areas of medicine, agriculture and energy. In this dissertation, we use synthetic biology and metabolic engineering approaches to explore the potential of engineered live yeasts as therapeutic platforms for treating inflammatory bowel disease (IBD). The vast majority of microbial-based therapeutics at the moment have focused on bacteria instead of yeasts, and all of these engineered live bacterial platforms use either proteins or peptides as therapeutic agents of choice. This dissertation seeks to enhance yeast’s beneficial properties to humans by genetically engineering them to produce TAN-1612, a small molecule tetracycline with therapeutic potential. We choose tetracyclines as our small molecule therapeutic agent because these compounds are one of the most impactful natural products that humanity has benefited from due to its significant antimicrobial and anti-inflammatory properties. We genetically engineer strains of baker’s yeast Saccharomyces cerevisiae and the probiotic yeast Saccharomyces cerevisiae var boulardii to produce in situ the fungal tetracycline TAN-1612, a natural product with anti-inflammatory properties (instead of anti-microbial so as to not disturb the gut microbiome), and to study the molecular mechanisms involved in their potential beneficial effects for IBD. Our engineered live yeast therapeutics would provide an effective, safe, and cheap alternative to treating IBD and other gastrointestinal tract disorders compared to the currently available but costly and laborious therapies. In Chapter 1, we review key milestones in the fields of synthetic biology and metabolic engineering that have enabled and inspired the generation of both engineered live microbial-based systems and small molecules as the therapeutic agents for the potential treatment of a wide array of human diseases such IBD, cancer, and pathogenic infections. In Chapter 2, we develop synthetic biology and metabolic engineering approaches for designing, building, and testing of the biosynthetic pathway of TAN-1612 in genetically engineered yeasts such as S. cerevisiae and S. boulardii. These approaches enable the production of TAN-1612 in yeasts with titers as high as ~61 mg/L which represent a 100-fold improvement from previous reported yeast strains. These engineering approaches hold great potential to advance the heterologous biosynthesis of other small molecule therapeutics in yeasts. In Chapter 3, we explore the role of TAN-1612 as an anti-inflammatory agent, inhibitor of tetracycline inactivating enzymes, and inducer of gene expression with the goal of identifying its best therapeutic or biological application that can be leveraged for the development of engineered live yeast-based systems for the in situ treatment of IBD. Advances in DNA synthesis and sequencing technologies have spurred the high-throughput construction of microbial strains for numerous applications in synthetic biology and metabolic engineering. Breakthrough technologies in our abilities to screen and select target molecule biosynthesis, however, are needed in order to realize the potential of both of these disciplines for drug discovery and production. Current state-of-the-art methods such as liquid/gas chromatography – mass spectrometry (LC/GC – MS) are applicable to screen or select a variety of target molecules but their throughput remains low (~102 samples/day). Other screening or selection methods available are highly dependent on the molecule of interest and generally inapplicable to other compounds. Therefore, in Chapter 4 we propose that the Fluorescence Polarization (FP) assay can be readily adapted as a general, medium-throughput (~104 samples/day) screen for synthetic biology and metabolic engineering applications. As a proof-of-principle, we develop an FP assay to detect the immunosuppressant polyketide FK506, use this assay to detect FK506 biosynthesized from Streptomyces tsukubaensis cultures in microtiter plates, and finally apply this FP assay to generate S. tsukubaensis strains with increased production of FK506. Lastly, we outline the experimental steps necessary to adapt the FP to screen different classes of natural products beyond polyketides such as FK506 or tetracyclines. The fungal polyketide TAN-1612 is an attractive chemical scaffold for the generation of novel tetracycline-based therapeutics using metabolic engineering approaches. Libraries of > 108 are usually required to generate chemical compound diversity to identify drugs with significant therapeutic activities. Compared to screening methods, genetic selections allow the detection of target molecules at a much higher throughput (> 108 samples/day) because the population of cells can be cultured and assayed in one-pot manner. Thus, in Chapter 5 we establish the yeast three hybrid (Y3H) assay as a general, high-throughput selection technology to detect tetracycline derivatives. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating producer and non-producer yeast strains of TAN-1612. The Y3H assay can be used for the heterologous biosynthesis of tetracycline analogues in yeasts especially because the Y3H would enable the production and detection of these derivatives in the same yeast cell.

Understanding the signaling mechanisms that determine juvenile-to-mature transition and bud fate is vital for controlling tree reproduction. FD-like proteins also appear to be important for initiating reproductive development. In this study, phylogenetic analysis showed that three FD-like genes (FDL1, FDL2, and FDL3) are present in the poplar genome. FDL1 and FDL2 are products of a recent whole genome duplication event while FDL3 escaped such duplication. Yeast two-hybrid assays demonstrated that FT1 and FT2 proteins interact with FDL3 protein, but not with FDL1 or FDL2 protein. Analysis of the expression levels of FD-like transcripts in Populus deltoides via quantitative real-time PCR showed that FDL3 abundantly expresses in the shoot apex where it probably interacts with FT1 in late winter and early spring. Following the duplication event, FDL1 and FDL2 appear to have diverged in function as they express in a number of tissues in the fall, winter, and spring.

Wastewater has been shown to contain the necessary nutritive requirements for the growth of microorganisms. The term, oleaginous, has been given to a classification of microorganisms know to produce up to twenty percent of the weight as oil. This study is designed to examine the potential accumulation of lipids within an oleaginous consortium grown on synthetic wastewater. Potential of the fluorescent stain, Nile red, as a lipid detector is also emphasized. Percentages of extractables greater than thirtyive percent were achieved within the oleaginous consortium using a nitrogen-limited medium. Low pH was found to increase the percentages of extractables. Xylose was shown to be a more optimal carbon source for accumulation than glucose. Nile red was shown to bind to intracellular inclusions and may be useful in monitoring lipid accumulation in industrial settings.

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No description available.

Carotenoids in W. dermatitids were found to be associated with membranes of lipid globules and/or proteins dispersed in the lipids in the yeast-phase of the organism. The lipid globules increase in size and the pigment concentrations increase with age of the cell. Electron micrographs show these organelles to be surrounded by a single unit membrane. The free carotenoids are extractable with ethyl ether from pigmented fractions of osmotically ruptured protoplasts only after the sample has been treated with acetone, indicating the pigment is non-covalently bound, presumably to a protein.