Single step chromatographic purification of plasmid DNA from neutralised lysate aiming its application in gene therapy and vaccination

Orientadores: Everson Alves Miranda, Adriano Rodrigues AzzoniDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: O número de estudos em terapia gênica com vetores plasmídiais (pDNA) têm aumentado nestes últimos anos. Como resultado, a demanda para preparações de pDNA em conformidade com as recomendações das agências reguladoras (EMEA, FDA) também aumentou. O DNA plasmidial é frequentemente obtido através da fermentação de Escherichia coli transformada e purificada por uma série de operações unitárias, incluindo a cromatografia. Este trabalho teve como objetivo o desenvolvimento de um processo cromatográfico para a recuperação e purificação do pDNA superenovelado (sc pDNA) a partir do lisado neutralizado. Os ligantes fenil (hidrofóbico) e mercaptopirimidina (tiofílico) foram imobilizados em matrizes de agarose e celulose. A seletividade destes ligantes para com o sc pDNA foi determinada através de estudos de adsorção utilizando citrato de sódio 1,5 mol/L e fosfato de potássio 2,0 mol/L como tampões de adsorção. A cromatografia com o adsorvente fenil-agarose e o citrato de sódio 1,5 mol/L permitiu recuperar 58% do pDNA sem contaminação por gDNA, proteínas e endotoxinas, sendo uma alternativa potencial para a recuperação primária do sc pDNA. O resultado mais promissor foi obtido com a cromatografia com o adsorvente mercaptopirimidina-agarose e fosfato de potássio 2,0 mol/L como tampão de adsorção. Este sistema tampão de adsorção/adsorvente permitiu a obtenção de pDNA com 100% de pureza e dentro das recomendações das agências reguladoras no tocante à contaminação por RNA e endotoxinas. Assim, este trabalho lançou as bases para o desenvolvimento de dois métodos cromatográficos para a recuperação primária ou purificação de pDNA diretamente do lisado neutralizado, ambos potencialmente aplicáveis em larga escalaAbstract: The number of studies in gene therapy with plasmid vectors (pDNA) has witnessed an increase in the recent years. As result the demand for preparations of pDNA in compliance with recommendations of the regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is oftenly obtained through fermentation of transformed Escherichia coli and purified by a series of unit operations, including chromatography. This work aimed the development of a chromatographic process for the recovery and purification of supercolied pDNA (sc pDNA) directly from neutralized cell lysate. Phenyl (hydrophobic) and mercaptopyrimidine (thiophilic) molecules immobilized in agarose and cellulose matrices were the ligands used to capture the pDNA. Their selectivity towards sc pDNA was evaluated through adsorption studies using sodium citrate 1.5 mol/L and potassium phosphate 2.0 mol/L as the adsorption buffers. The chromatography with the adsorbent phenyl-agarose and sodium citrate 1.5 mol/L was able to recover 58% of sc pDNA without gDNA, proteins and endotoxins contamination, being an potential alternative for the primary recovery of sc pDNA. The most promising result was obtained with the chromatography with mercaptopyrimidine-agarose and potassium phosphate 2.0 mol/L adsorpition buffer. With the latter buffer/adsorbent system it was possible to obtain in a single step pDNA with 100% purity and within the recommendations of regulatory agencies with regard to contamination by RNA and endotoxins. Thus, this work laid the basis for the development of two chromatographic process for the recovery or purification of pDNA directly from the neutralized lysate, both potentially applicable in larger scaleMestradoDesenvolvimento de Processos BiotecnologicosMestre em Engenharia Químic

Single Cell Oil Producing Yeasts Lipomyces Starkeyi And Rhodosporidium Toruloides: Selection Of Extraction Strategies And Biodiesel Property Prediction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.8650405052Science without Borders Program (Brazil)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Bio4energy (Sweden)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

High concentrations of dried sorghum stalks as a biomass feedstock for single cell oil production by Rhodosporidium toruloides

Environmental crisis and concerns for energy security have made the research for renewable fuels that will substitute the usage of fossil fuels an important priority. Biodiesel is a potential substitute for petroleum, but its feasibility is hindered by the utilization of edible vegetable oil as raw material, which is responsible for a large fraction of the production cost and fosters the food versus fuel competition. Microbial oils are an interesting alternative as they do not compete with food production, and low cost renewable materials could serve as raw materials during cultivation of microorganisms. Sweet sorghum is an excellent candidate as substrate for microbial oil production, as it possesses high photosynthetic activity yielding high amounts of soluble and insoluble carbohydrates, and does not require high fertilization and irrigation rates8FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

C/N ratio and carbon source-dependent lipid production profiling in Rhodotorula toruloides

Microbial oils are lipids produced by oleaginous microorganisms, which can be used as a potential feedstock for oleochemical production. The oleaginous yeast Rhodotorula toruloides can co-produce microbial oils and high-value compounds from low-cost substrates, such as xylose and acetic acid (from hemicellulosic hydrolysates) and raw glycerol (a byproduct of biodiesel production). One step towards economic viability is identifying the best conditions for lipid production, primarily the most suitable carbon-to-nitrogen ratio (C/N). Here, we aimed to identify the best conditions and cultivation mode for lipid production by R. toruloides using various low-cost substrates and a range of C/N ratios (60, 80, 100, and 120). Turbidostat mode was used to achieve a steady state at the maximal specific growth rate and to avoid continuously changing environmental conditions (i.e., C/N ratio) that inherently occur in batch mode. Regardless of the carbon source, higher C/N ratios increased lipid yields (up to 60% on xylose at a C/N of 120) but decreased the specific growth rate. Growth on glycerol resulted in the highest specific growth and lipid production (0.085\ua0g lipids/gDW*h) rates at C/Ns between 60 and 100. We went on to study lipid production using glycerol in both batch and fed-batch modes, which resulted in lower specific lipid production rates compared with turbisdostat, however, fed batch is superior in terms of biomass production and lipid titers. By combining the data we obtained in these experiments with a genome-scale metabolic model of R. toruloides, we identified targets for improvements in lipid production that could be carried out either by metabolic engineering or process optimization

Lipidomic Profile of Rhodotorula toruloides by GC/MS and Antioxidant Capacity of the Oil by DPPH and TLC-Plate Methods

This work was undertaken to evaluate the antioxidant capacity of Rhodotorula toruloides lipid extract in TLC plate, using the (DPPH) (1,1-diphenyl-2-picril-  hydrazine) method as an innovative way to visualise lipid groups that comprise this activity. Similarly, carotenoids and crude oil were analysed for  antioxidant capacity by the DPPH and β-carotene/linoleic acid methods. The lipidomic profile extract analysis was performed by GC/MS and HPLC/DAD.  The sample preparation for the GC/MS analysis was made by ultrasound-assisted transesterification. Free compounds were silylated with BSTFA (N,O-Bis  (trimethylsilyl) trifluoracetamide) + 1% TMCS (Trimethylchlorosilane). The analysis of the lipid extract showed that in the saponifiable fraction saturated  fatty acids (SFA) and monounsaturated fatty acids (MUFA) were present; and in the unsaponifiable fraction were steroids and carotenoids. The antioxidant  capacity was expressed as IC50 reaching 6.4 mg/L that means relative efficiency. The oil profile, using TLC, shows the chemical groups:  carotenoids, acylglycerols, free fatty acids and steroids. Similarly, the GC/ MS analysis shows the fatty acids and steroids. The HPLC analysis describes the  carotenoids profile, highlighting b-carotene as the majority and the presence of β-carotene-5,8-epoxide, zeaxanthin and b-cryptoxanthin, characterising  the lipidomic study of this yeast

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi‑quantitative and quantitative RT‑PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α‑galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish‑green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.O objetivo deste trabalho foi identificar genes que possam ser utilizados como marcadores moleculares para reconhecimento de fases fenológicas, durante o desenvolvimento de frutos do cafeeiro (Coffea arabica). Quatro cultivares foram avaliadas quanto à expressão diferencial de genes associados a processos de desenvolvimento e maturação de frutos. A caracterização da expressão gênica foi realizada pelas técnicas de RT‑PCR semi‑quantitativa e quantitativa, em frutos coletados em sete estádios de desenvolvimento, durante três safras. Não foi observado nenhum polimorfismo de tamanho ou expressão diferencial entre as cultivares, para os genes avaliados; porém, perfis de expressão distintos durante o desenvolvimento dos frutos foram determinados para cada gene. Quatro entre os 28 genes avaliados apresentaram perfil de expressão constante, em todas as cultivares e safras e, portanto, foram validados como genes candidatos a marcadores fenológicos de frutos de cafeeiro. O gene α‑galactosidase pode ser usado como marcador do estágio de fruto verde, o gene de cafeína sintase como marcador do estádio de transição entre fruto verde e fruto verde‑cana, e o isocitrato liase e o etileno‑receptor 3 como marcadores das fases finais de maturação

Perfil da expressão gênica durante o desenvolvimento de frutos de café \ud e identificação de genes marcadores para fases fenológicas

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene a-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.O objetivo deste trabalho foi identificar genes que possam ser utilizados como marcadores \ud moleculares para reconhecimento de fases fenológicas, durante o desenvolvimento de frutos do cafeeiro (Coffea \ud arabica). Quatro cultivares foram avaliadas quanto à expressão diferencial de genes associados a processos de \ud desenvolvimento e maturação de frutos. A caracterização da expressão gênica foi realizada pelas técnicas de \ud RT‑PCR semi‑quantitativa e quantitativa, em frutos coletados em sete estádios de desenvolvimento, durante \ud três safras. Não foi observado nenhum polimorfismo de tamanho ou expressão diferencial entre as cultivares, \ud para os genes avaliados; porém, perfis de expressão distintos durante o desenvolvimento dos frutos foram \ud determinados para cada gene. Quatro entre os 28 genes avaliados apresentaram perfil de expressão constante, \ud em todas as cultivares e safras e, portanto, foram validados como genes candidatos a marcadores fenológicos \ud de frutos de cafeeiro. O gene α‑galactosidase pode ser usado como marcador do estágio de fruto verde, o gene \ud de cafeína sintase como marcador do estádio de transição entre fruto verde e fruto verde‑cana, e o isocitrato \ud liase e o etileno‑receptor 3 como marcadores das fases finais de maturação.Consorcio Brasileiro de Pesquisa e Desenvolvimento do CafeConsorcio Brasileiro de Pesquisa e Desenvolvimento do Caf

Microbial oil production by adapted strains of oleaginous yeasts from hemicellulosic hydrolysates aiming at their application in biorefineries

Orientador: Everson Alves MirandaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuímicaResumo: Hidrolisados hemicelulósicos como os provenientes de bagaço de cana-de-açúcar (HBCA) e de cavacos de bétula (HEHB) são um substrato de baixo custo para a produção de óleos microbianos (SCO). Porém, estes hidrolisados têm como desvantagens a presença de inibidores do crescimento microbiano ou baixas razões carbono/nitrogênio. A adaptação da levedura, a destoxificação deste material e o cultivo em batelada alimentada são estratégias para se suplantar a presença de inibidores. Quanto à questão da baixa razão C/N, pode-se efetuar a concentração dos hidrolisados ou a adição de alguma fonte de carbono para aumentar essa razão. Diante disso, o objetivo deste trabalho foi adaptar as leveduras oleaginosas Rhodosporidium toruloides e Lipomyces starkeyi e utilizá-las na produção de SCO em hidrolisados hemicelulósicos não destoxificados. Foram feitas as adaptações das leveduras R. toruloides e L. starkeyi em HBCA e HEHB, respectivamente. O cultivo da cepa adaptada de L. starkeyi em HEHB resultou em uma baixa concentração de lipídeos (1,8 g/L) e por isso os estudos continuaram apenas com a cepa adaptada de R. toruloides. Esta cepa de R. toruloides foi capaz de produzir 225% mais lipídeos em meios com mistura de xilose e glicose e apresentou uma maior expressão de genes ligados à via das pentoses fosfato e ao acúmulo de lipídeos quando comparada a cepa parental. Foram realizados diferentes estudos de cultivo da cepa adaptada em HBCA, concentrado ou não e com ou sem a adição de glicerol. A concentração e a adição de glicerol ao HBCA foram responsáveis por aumentarem os conteúdos, as concentrações e as produtividades de lipídeos em no mínimo 108%, 175% e 118%, respectivamente, quando comparados ao cultivo utilizando apenas o HBCA. Em biorreator, o conteúdo lipídico e a produtividade aumentaram em 33% e 54%, respectivamente. Concluiu-se que a adaptação resultou em uma cepa com produção de lipídeos superior à cepa parental e, esta, superexpressa genes-chave para o acúmulo de lipídeos. A concentração do hidrolisado ou a adição de glicerol são estratégias simples de serem aplicadas e que melhoram consideravelmente a produção de SCO em hidrolisados com baixa razão C/N. Além disso, foi possível a produção de lipídeos em hidrolisado concentrado não destoxificado pela levedura R. toruloides, fato inédito na literaturaAbstract: Hemicellulosic hydrolysates such as those from sugarcane bagasse (HBCA) and birch woodchips (HEHB) are low cost substrates for single cell oil (SCO) production. However, these hydrolysates have low carbon/nitrogen (C/N) ratios and contain microbial growth inhibitors. Yeast adaptation, detoxification or fed-batch cultivation are strategies for overcoming the presence of these inhibitors and concentration or the addition of crude glycerol (a residue of biodiesel production) are alternatives to solve the low C/N ratio limitation. This work aimed at the adaptation of oleaginous yeasts Rhodosporidium toruloides and Lipomyces starkeyi for their use in SCO production using hemicellulose hydrolysates overcoming the low C/N ratio and inhibitors effects. R. toruloides and L. starkeyi adaptions were done in HBCA and in HEHB, respectively. The adapted strain of L. starkeyi was cultured in HEHB but low lipid concentration (1.8 g/L) was obtained and the studies were continued only with adapted R. toruloides. This strain of R. toruloides produced 225% more lipids in a mixture of xylose and glucose than the parental one. Also, the adapted strain overexpressed some genes of the pentose phosphate pathway and some genes specifically related to lipid accumulation. Concentration of the HBCA or the addition of glycerol were responsible for increasing the lipid content, concentration, and productivity in at least 108%, 175%, and 118%, respectively, compared to fermentation using only HBCA. Further experiments on bioreactors increased lipid content and productivity by 33% and 54%, respectively. From the data aforementioned, it can be concluded that adaptation resulted in a R. toruloides strain with superior trait for lipid production in hydrolysates that overexpress key genes related to lipid accumulation. Hydrolysate concentration or glycerol addition are simple and easy strategies to be applied that can increase considerably SCO production in hydrolysates. The production of lipids in non-detoxified concentrated hydrolysate by the yeast R. toruloides is not yet reported in the literatureDoutoradoEngenharia QuímicaDoutora em Engenharia Quimica2013/03103-5FAPES