Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2016. / Microbial spoilage has been reported in various food products and this has led to increased food, fruit and beverage losses, thereby threatening economic growth, food safety and security. Furthermore, statistics have shown that more than 30% of agricultural produce in developing countries, mostly in Africa, is lost owing to microbial spoilage. Beverages, food and fruits are predominant contributors to the South African export market. In recent years, contamination of these products resulting in spoilage has been a problem, although partial spoilage control has been achieved using chemical preservatives such as dimethyl dicarbonate, sodium benzoate, potassium sorbate, and sulphur dioxide (SO2). However, prolonged exposure to these chemical preservatives can cause human health problems such as skin and/or eyesight damage, muscle and stomach pain, cardiovascular disease and the impairment of brain function. To mitigate such health concerns, biologically benign alternatives are deemed suitable, providing the rationale for this study.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: QUANTITATIVE YEAST PHYSIOLOGY AND NITROGEN METABOLISM DURING HETEROLOGOUS PROTEIN PRODUCTION By Johann F. Görgens The physiology and nitrogen metabolism of the yeast, Saccharomyces cerevisiae, during heterologous xylanase production in a defined medium was quantified by the comparison of isogenic yeast strains, whereby several potential limitations in the production of the heterologous xylanase could be identified. The presence of global sensing and regulatory mechanisms, by which the yeast is able to actively regulate both heterologous gene expression and the physiological response to the process, was also investigated. The deleterious effects of heterologous xylanase production on the physiology of the recombinant host were disproportionately large with respect to the amount of foreign protein produced. The cellular processes involved in this response were identified by the transcriptional profiling of isogenic recombinant strains, in a novel analytical approach to investigating foreign protein production by S. cerevisiae. Heterologous gene expression affected a combination of cellular processes and induced the yeast stringent stress response. The corresponding loss of metabolic functionality resulted in the disproportionate physiological effects of foreign protein production, similar to previous observations in recombinant Escherichia coli, and a possible reduction in attainable production levels. Reducing the propensity of recombinant gene expression to introduce metabolic stress may therefore increase production levels of foreign proteins by yeast. The metabolic vitality of transformed strains was also reduced by the presence of multiple copies of active, plasmid-based PGK1-promoters in the cell without expression of the heterologous gene. The negative effect was caused by an increase in the biosynthetic and glycolytic capacity of the strain at the expense of other processes. Production levels of heterologous xylanase were influenced by expression vector selection and the presence of auxotrophic mutations in transformed strains of S. cerevisiae. The increased transcription levels obtained with the multicopy plasmidbased YEp-type expression system, compared to the integrative YIp-type expression system, resulted in higher levels of xylanase production. Heterologous xylanase production thus did not saturate the secretory capacity of the host strain. The genetic stability of the autoselective YEp-type expression system in long-term chemostat culture was also demonstrated. High levels of heterologous xylanase production by transformed S. cerevisiae strains containing auxotrophic markers required the stabilisation of nitrogen metabolism via saturation of yeast cells with an excess of imported amino acids. By the removal of excessive auxotrophic markers, high levels of xylanase production by a prototrophic transformant in defined medium without amino acid addition could be obtained. Heterologous xylanase production by the prototrophic transformant was further enhanced by increasing the availability of preferred amino acids or succinate in the defined medium, indicating an additional requirement for metabolic precursors and building blocks for foreign protein synthesis. Comparable levels of heterologous xylanase production were obtained in high cell density cultures of the alternative yeast, Pichia stipitis, by the proper induction of the native ADH2-promoter, the control of oxygenation, and addition of an amino acid mixture to the defined medium, indicating the presence of generic limitations in transcription, nutrient availability and the yeast biosynthetic capacity for foreign protein production by various yeasts. The presence of global sensing and regulatory mechanisms was confirmed by the physiological response of S. cerevisiae to heterologous protein production, which included the downregulation of biosynthesis and growth, and the induction of various processes involved in the stringent stress response. Additionally, heterologous xylanase production was actively regulated on a posttranscriptional level by the auxotrophic transformants in response to the level of amino acid availability. The biosynthetic capacity for foreign protein production by both recombinant S. cerevisiae and P. stiptis was also regulated in response to the physiological state of the yeast and the availability of nutrients. The presence of these regulatory mechanisms complicated the manipulation of cellular biosynthesis at will. / AFRIKAANSE OPSOMMING: KWANTITATIEWE GIS-FISIOLOGIE EN -STIKSTOF METABOLISME GEDURENDE HETEROLOË PROTEÏEN PRODUKSIE Deur Johann Ferdinand Görgens Die fisiologie en stikstof-metabolisme van die gis, Saccharomyces cerevisiae, gedurende heteroloë xilanase produksie in ‘n gedefiniëerde medium is gekarakteriseer deur isogeniese gis-rasse te vergelyk, waardeur verskeie moontlike beperkings in die produksie van die heteroloë xilanase uitgewys kon word. Die teenwoordigheid van globale sensoriese- en beheer-meganismes, wat die gis in staat stel om beide heteroloë geen uitdrukking en die fisiologiese respons op die proses aktief te reguleer, is ook ondersoek. Die nadelige effekte van heteroloë xilanase produksie op die fisiologie van die rekombinante gasheer-organisme was uitermatig groot in vergelyking met die hoeveelheid vreemde proteïen wat geproduseer is. Die sellulêre prosesse verantwoordelik vir hierdie respons is identifiseer deur die transkripsionele profiele van isogeniese rekombinante rasse te vergelyk, in ‘n nuwe analitiese benadering tot die bestudering van vreemde proteïen produksie deur S. cerevisiae. Heteroloë geen uitdrukking het ‘n kombinasie van sellulêre prosesse geaffekteer en die gis se algemene voedingstres-respons geaktiveer. Die gepaardgaande verlies aan metaboliese funksie het die uitermatige fisiologiese effek van vreemde proteïen produksie veroorsaak, soortgelyk aan vorige waarnemings met rekombinante Escherichia coli. Die haalbare produksie-vlakke is moontlik ook verlaag deur hierdie respons. ‘n Verlaging van die geneigdheid van rekombinante geen uitdrukking om metaboliese stres te veroorsaak, mag dus die produksievlakke van vreemde proteïene in gis verbeter. Die metaboliese groei-potensiaal van die getransformeerde rasse is ook verlaag deur die teenwoordigheid van etlike aktiewe kopieë van plasmied-gebaseerde PGK1-promotors in die sel, sonder uitdrukking van die heteroloë geen, deur ‘n toename in die biosintetiese en glikolitiese kapasiteit ten koste van die ander sellulêre prosesse. Die produksievlakke van heteroloë xilanase is deur die keuse van uitdrukkings-sisteem en die teenwoordigheid van autotrofiese mutasies in die getransformeerde rasse van S.cerevisiae beïnvloed. Die verhoogde transkripsie vlakke wat met die multi-kopie, plasmied-gebaseerde YEp-tipe uitdrukkingsisteem, eerder as die geïntegreerde YIp-tipe sisteem, verkry is, het tot verhoogde xilanase produksie gelei. Heteroloë xilanase produksie het dus nie die uitskeidingskapasiteit van die gasheer versadig nie. Die genetiese stabiliteit van die autoselektiewe, YEp-tipe uitdrukkingsisteem in langtermyn chemostaat-kulture is ook gedemonstreer. Hoë vlakke van xilanase produksie deur getransformeerde S. cerevisiae rasse met autotrofiese merkers het die stabilisering van die stikstof metabolisme, deur die versadiging van die sel met ingevoerde aminosure, vereis. Die verwydering van oormatige autotrofiese merkers het tot hoë vlakke van xilanase produksie deur die prototrofiese transformant in gedefinieerde medium sonder aminosuur byvoeging gelei. Heteroloë xilanase produksie deur die prototrofiese transformant kon verder verbeter word deur die byvoeging van voorkeur-aminosure of suksinaat tot die gedefinieerde medium, en ‘n addisionele behoefte aan metaboliese voorloper-molekules en bou-blokke vir vreemde proteïensintese het dus bestaan. Vergelykbare vlakke van heteroloë xilanase produksie is in kulture met hoë sel-digthede van die alternatiewe gis, Pichia stipitis, verkry deur die doeltreffende induksie van die eiesoortige ADH2-promotor en die byvoeging van ‘n aminosuur-mengsel tot die gedefinieerde medium, wat die teenwoordigheid van generiese beperkinge in transkripsie, voedingstof-beskikbaarheid en biosintetiese kapasiteit van die gis vir vreemde proteïen produksie deur verskeie giste uitgewys het. Die teenwoordigheid van globale sensoriese- en beheer-meganismes is bevestig deur die fisiologiese respons van S. cerevisiae tot heteroloë proteïen produksie, wat die afwaartse regulering van biosintese en groei, en die induksie van verskeie prosesse betrokke by die algemene voedingstres-respons, ingesluit het. Heteroloë xilanase produksie is ook op ‘n na-transkripsionele vlak aktief gereguleer deur die autotrofiese transformante in reaksie tot die vlak van aminosuur beskikbaarheid. Die biosintetiese kapasiteit vir vreemde proteïen-produksie van beide rekombinante S. cerevisiae en P. stipitis is ook in reaksie tot die fisiologiese toestand van die gis en die beskikbaarheid van voedingstowwe gereguleer. Die teenwoordigheid van hierdie regulatoriese meganismes het die willekeurige manipulasie van sellulêre proteïen-biosintese bemoeilik.

The effect of mechanical shear on brewing yeast was investigated with a focus on losses incurred through cell rupture and viability loss. The influence of various environmental conditions was studied with regards to the influence on Saccharomyces cerevisiae's ability to resist mechanical shear. Further investigation was performed in order to locate a structure within the yeast cell that contributes to mechanical shear resistance. / It was found that yeast cells grown anaerobically in limited glucose media were more prone to losses in cell viability than cells grown aerobically in the same media, when subjected to mechanical shear. Cells grown anaerobically in high glucose concentrations and allowed to ferment the media to exhaustion were slightly more resistant to mechanical shear compared to cells grown anaerobically without fermentation in minimal glucose media. Higher ethanol concentrations lead to marginally decreased resistance to mechanical shear. / Cell walls of S. cerevisiae were partially digested or extracted using enzymatic treatment or chemical attack. It was found that while the outer mannoprotein layer does not contribute significantly, the inner beta-(1 → 3)-glucan structure plays a significant role in resistance to mechanical shear.

No description available.

Yarrowia lipolytica é uma levedura estritamente aeróbia e oleaginosa, pertencente ao filo Ascomycota. Atualmente é uma das espécies de levedura não convencional mais estudadas para aplicações biotecnológicas. Se comparada a outras leveduras, como Saccharomyces cerevisiae, observam-se notáveis distinções relacionadas a sua fisiologia, genética e filogenia. É conhecida sua capacidade de excretar altas quantidades de ácidos orgânicos (ácidos cítrico, isocítrico, cetoglutárico e pirúvico) e de secretar diferentes enzimas (lipases, proteases etc.), permitindo a degradação de diferentes substratos, incluindo os de caráter hidrofóbico. O presente trabalho teve como objetivo implementar técnicas de cultivo em meio sólido ou líquido para a levedura Yarrowia lipolytica IMUFRJ 50682, que permitissem avaliar sua capacidade em utilizar diferentes fontes de carbono, além de calcular os principais parâmetros da fisiologia celular, como velocidades específicas e fatores de conversão. Nos cultivos em meio sólido, verificou-se que esta levedura apresenta fraco ou nenhum crescimento nas fontes de carbono sacarose, xilose, citrato e acetato. Além disto, observou-se que a tiamina é fator de crescimento essencial ao desenvolvimento desta levedura quando cultivada em glicose e glicerol. Verificou-se também que esta linhagem apresenta maior tolerância ao NaCl, se comparada à linhagem Y. lipolytica W29 (tradicionalmente empregada nos estudos acadêmicos). Nos cultivos em meio líquido, foi estabelecida uma composição de meio de cultivo totalmente definido, que permitiu o crescimento desta levedura com velocidade específica máxima de 0,35 h-1 em glicose e 0,46 h-1 em glicerol, como únicas fontes de carbono. Nestes cultivos, os fatores de conversão de substrato a células, durante a fase exponencial de crescimento, foram 0,32 g de massa seca de células/g glicose e 0,48 g de massa seca de células/g glicerol. Durante os cultivos em meio líquido, que foram realizados a 28 oC e 200 rotações por minuto em incubador rotativo, empregando frascos do tipo erlenmeyer com deflectores, fechados com algodão e um quinto do volume ocupado com meio líquido e 2,5 g.l-1 iniciais da fonte de carbono, não foi observada a formação de nenhum metabólito extracelular, o que é indicativo de um metabolismo energético puramente respiratório. Nestas condições, usando-se ureia como fonte de nitrogênio, o pH permaneceu estável do início ao final dos cultivos. Observou-se também que a levedura estudada não é capaz de crescer em pH menor que 2,0. / Yarrowia lipolytica is a dimorphic, strictly aerobic and oleaginous yeast belonging to the phylum Ascomycota. It is currently one of the non-conventional yeast species most studied for biotechnological applications. If compared to other yeasts, such as S. cerevisiae, notable distinctions are observable related to its physiology, genetics and phylogeny. It is well known for its ability to secrete high quantities of organic acids (citric acid, isocitric acid, ketoglutaric acid and pyruvic acid) and innate ability to secrete various enzymes (lipases, proteases etc.), which allows the species to degrade different substrates, including hydrophobic ones. The present work aimed at the implementation of cultivation techniques in solid or liquid media for the yeast Yarrowia lipolytica IMUFRJ 50682, enabling to assess its ability to assimilate different carbon sources, and to calculates the main parameters of cellular physiology, such as specific growth rate and specific yields. In solid media assays, it was found that Yarrowia lipolytica IMUFRJ 50682 presents a weak to almost null growth when sucrose, xylose, citrate and acetate are used as sole carbon sources. Furthermore, it was observed that thiamine is an essential growth factor for the proper assimilation of the carbon sources glucose and glycerol. Response towards osmotic stress was assessed in complex solid media, in which the strain Yarrowia lipolytica IMUFRJ 50682 exhibited higher halotolerance, up to 1,0 M NaCl, if compared to the other strain Yarrowia lipolytica W29 (traditionally employed in academic studies). A defined liquid medium composition was adapted, enabling the assessment of the specific growth rates for both strains, of which Yarrowia lipolytica IMUFRJ 50682 exhibited specific growth rates of 0.35 h-1 on glucose and 0.46 h-1 on glycerol, when used individually as sole carbon sources. On those assays, the substrate to biomass yields, during the exponential growth phase, was 0.32 g cell dry weight/g glucose and 0.48 g cell dry weight/g glycerol. During the cultivations in liquid medium, which were carried out in thermostatted orbital shaker incubator at 200 rpm and 28 oC, employing baffled cotton capped erlenmeyer flasks with a fifth of its volume occupied with the growth medium and 2.5 g.l-1 of the carbon source of choice, it was not observed any extracellular metabolic formation, indicating a pure respiratory metabolism. The addition of urea as nitrogen source was crucial at maintaining a stable pH during the whole cultivations. It was also observed that the studied yeast strains are not capable of growing at a pH lower than 2.0.

Yarrowia lipolytica é uma levedura estritamente aeróbia e oleaginosa, pertencente ao filo Ascomycota. Atualmente é uma das espécies de levedura não convencional mais estudadas para aplicações biotecnológicas. Se comparada a outras leveduras, como Saccharomyces cerevisiae, observam-se notáveis distinções relacionadas a sua fisiologia, genética e filogenia. É conhecida sua capacidade de excretar altas quantidades de ácidos orgânicos (ácidos cítrico, isocítrico, cetoglutárico e pirúvico) e de secretar diferentes enzimas (lipases, proteases etc.), permitindo a degradação de diferentes substratos, incluindo os de caráter hidrofóbico. O presente trabalho teve como objetivo implementar técnicas de cultivo em meio sólido ou líquido para a levedura Yarrowia lipolytica IMUFRJ 50682, que permitissem avaliar sua capacidade em utilizar diferentes fontes de carbono, além de calcular os principais parâmetros da fisiologia celular, como velocidades específicas e fatores de conversão. Nos cultivos em meio sólido, verificou-se que esta levedura apresenta fraco ou nenhum crescimento nas fontes de carbono sacarose, xilose, citrato e acetato. Além disto, observou-se que a tiamina é fator de crescimento essencial ao desenvolvimento desta levedura quando cultivada em glicose e glicerol. Verificou-se também que esta linhagem apresenta maior tolerância ao NaCl, se comparada à linhagem Y. lipolytica W29 (tradicionalmente empregada nos estudos acadêmicos). Nos cultivos em meio líquido, foi estabelecida uma composição de meio de cultivo totalmente definido, que permitiu o crescimento desta levedura com velocidade específica máxima de 0,35 h-1 em glicose e 0,46 h-1 em glicerol, como únicas fontes de carbono. Nestes cultivos, os fatores de conversão de substrato a células, durante a fase exponencial de crescimento, foram 0,32 g de massa seca de células/g glicose e 0,48 g de massa seca de células/g glicerol. Durante os cultivos em meio líquido, que foram realizados a 28 oC e 200 rotações por minuto em incubador rotativo, empregando frascos do tipo erlenmeyer com deflectores, fechados com algodão e um quinto do volume ocupado com meio líquido e 2,5 g.l-1 iniciais da fonte de carbono, não foi observada a formação de nenhum metabólito extracelular, o que é indicativo de um metabolismo energético puramente respiratório. Nestas condições, usando-se ureia como fonte de nitrogênio, o pH permaneceu estável do início ao final dos cultivos. Observou-se também que a levedura estudada não é capaz de crescer em pH menor que 2,0. / Yarrowia lipolytica is a dimorphic, strictly aerobic and oleaginous yeast belonging to the phylum Ascomycota. It is currently one of the non-conventional yeast species most studied for biotechnological applications. If compared to other yeasts, such as S. cerevisiae, notable distinctions are observable related to its physiology, genetics and phylogeny. It is well known for its ability to secrete high quantities of organic acids (citric acid, isocitric acid, ketoglutaric acid and pyruvic acid) and innate ability to secrete various enzymes (lipases, proteases etc.), which allows the species to degrade different substrates, including hydrophobic ones. The present work aimed at the implementation of cultivation techniques in solid or liquid media for the yeast Yarrowia lipolytica IMUFRJ 50682, enabling to assess its ability to assimilate different carbon sources, and to calculates the main parameters of cellular physiology, such as specific growth rate and specific yields. In solid media assays, it was found that Yarrowia lipolytica IMUFRJ 50682 presents a weak to almost null growth when sucrose, xylose, citrate and acetate are used as sole carbon sources. Furthermore, it was observed that thiamine is an essential growth factor for the proper assimilation of the carbon sources glucose and glycerol. Response towards osmotic stress was assessed in complex solid media, in which the strain Yarrowia lipolytica IMUFRJ 50682 exhibited higher halotolerance, up to 1,0 M NaCl, if compared to the other strain Yarrowia lipolytica W29 (traditionally employed in academic studies). A defined liquid medium composition was adapted, enabling the assessment of the specific growth rates for both strains, of which Yarrowia lipolytica IMUFRJ 50682 exhibited specific growth rates of 0.35 h-1 on glucose and 0.46 h-1 on glycerol, when used individually as sole carbon sources. On those assays, the substrate to biomass yields, during the exponential growth phase, was 0.32 g cell dry weight/g glucose and 0.48 g cell dry weight/g glycerol. During the cultivations in liquid medium, which were carried out in thermostatted orbital shaker incubator at 200 rpm and 28 oC, employing baffled cotton capped erlenmeyer flasks with a fifth of its volume occupied with the growth medium and 2.5 g.l-1 of the carbon source of choice, it was not observed any extracellular metabolic formation, indicating a pure respiratory metabolism. The addition of urea as nitrogen source was crucial at maintaining a stable pH during the whole cultivations. It was also observed that the studied yeast strains are not capable of growing at a pH lower than 2.0.

L’expression aromatique fruitée des vins rouges a été le sujet de nombreuses études qui démontrent qu’au moins une composante de cette expression est le reflet d’interactions perceptives impliquant des esters. La synthèse de ces esters peut être affectée par la levure réalisant la fermentation alcoolique mais aussi par d’autres paramètres, plus technologiques, comme le niveau de maturité de la vendange. De plus, peu d’études ont été réalisé afin de valider le rôle physiologique tenu par les esters pour la levure.Dans le but de mettre en évidence plus précisément le rôle de ces facteurs dans la synthèse des esters, des souches de levures délétés des majeures estérases ont été construites, et testées en fermentation en milieu œnologique, dans des moûts de merlot et de tempranillo récoltés à deux maturités différentes. Ainsi, la concentration en chaque esters linéaires et substitués a pu être déterminée dans chaque modalité de vinification.L’étude des différents mutants de délétion, a permis, pour la première fois, de valider le rôle des principales estérases dans la synthèse des esters linéaires chez la levure mais aussi, la mise en évidence de deux gènes, non étudiés jusqu’alors en condition œnologique, sur la synthèse des esters substitués. L’analyse de l’expression génétique de la délétion des estérases chez la levure a permis aussi de valider que ces gènes permettent une véritable stabilité physiologique de la levure en conditions stressante.L’impact du degré de maturité de la vendange a été étudié à la fois chez les vins fermentés avec une levure standard commerciale mais aussi fermentés avec une levure délétée des 4 principales estérases. Une maturité avancée, des raisins de merlot seulement, entraine une baisse de 50% de la teneur en esters linéaire avec la levure standard, ce qui n’est plus observé avec la levure mutante. Cette diminution de la concentration en esters linéaire dans ces vins de merlot de maturité avancée, est bien corrélée à une diminution de leur perception fruitée. De plus, des reconstitutions aromatiques faites dans ces matrices, ont permis de valider l’implication totale des esters dans la perception de l’arôme fruité des vins rouges réalisés avec des matrices de maturité normale. En revanche en ce qui concerne les vins de merlot de maturité avancée, il existerait d’autres composés en plus des esters qui pourraient expliquer les différences sensorielles observées dans ces vins.Enfin, une approche de transcriptomique est mise en œuvre pour tâcher d’éclairer les facteurs à l’origine des modifications de production d’esters en fonction du niveau de maturité des raisins de merlot. Il en est sorti que l’effet maturité n’existe pas seul mais est bien combiné avec l’avancée de la fermentation alcoolique. L’effet global de la matrice qui peut expliquer les différences observées entre les vins de merlot. / A lot of studies highlighted the perceptual role of esters in fruity aromatic expression of red wines, demonstrating that at least partially it was due to perceptive interactions. The synthesis of these esters can be affected by the yeast making the alcoholic fermentation but also by other parameters, more technological, such as the level of maturity of the harvest. In addition, few studies have been conducted to validate the physiological role of esters for yeast.In order to more precisely highlight the role of these factors in the synthesis of esters, deleted yeast strains of the major esterases have been constructed, and tested in fermentation in an oenological environment, in merlot and tempranillo musts harvested at two different maturities. Thus, the concentration of each linear and substituted ester could be determined in each vinification modality.The study of the different deletion mutants allowed, for the first time, to validate the role of the main esterases in the synthesis of linear esters in yeast but also the demonstration of two genes, not yet studied in oenological conditions, on the synthesis of substituted esters. The analysis of the genetic expression of the esterase deletion in yeast has also validated that these genes allow a true physiological stability of the yeast under stressful conditions.The impact of the degree of maturity of the harvest has been studied both in wines fermented with a standard commercial yeast but also fermented with a yeast deleted from the 4 main esterases. Advanced maturity, merlot grapes only, results in a 50% drop in linear ester content with standard yeast, which is no longer observed with the mutant yeast. This decrease in the concentration of linear esters in these mature Merlot wines is well correlated with a decrease in their fruity perception.In addition, aromatic reconstitutions made in these matrices made it possible to validate the total involvement of esters in the perception of the fruity aroma of red wines made with matrices of normal maturity. On the other hand, for mature merlot wines, there are other compounds besides esters that could explain the sensory differences observed in these wines.Finally, a transcriptomic approach is implemented to try to shed light on the factors that cause ester production changes as a function of the maturity level of merlot grapes. It has emerged that the maturity effect does not exist alone but is well combined with the progress of alcoholic fermentation. The overall effect of the matrix that can explain the differences observed between merlot wines.

Indiana University-Purdue University Indianapolis (IUPUI) / An investigation of the lipid metabolism pathway in the yeast Yarrowia lipolytica was conducted. Yarrowia is an oleaginous ascomycete that is capable of growing on many different substrates, which derives its name from its high efficiency of growth on lipids. Once the exogenous lipids are converted into free fatty acids and internalized by the yeast, the primary mode of degradation is through β-oxidation mediated by the peroxisomal oxidases, or POX genes. These enzymes catalyze the formation of a trans double bond, producing the trans-2-enoyl product. Our study looked at the comparison of the Y. lipolytica prototrophic strain against a knockout of the Pox2 gene on the uptake, incorporation, and degradation of relevant fatty acids. To construct this gene knockout, a novel gene deletion method using a combination of Cre recombinase and the AHAS* gene was synthesized, developed, and tested successfully. This knockout system allows for serial deletion of genes with the use of only one resistance marker, with excision of the marker after selection.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA polymerase II (RNAPII) is regulated by multiple modifications to the C-terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification.