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Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniera en Biotecnología Molecular. / La enzima L-asparaginasa II, que hidroliza L-asparagina en ácido aspártico y amonio, es utilizada actualmente como agente quimioterapéutico, en el tratamiento de leucemia linfoblástica aguda infantil, leucemia mieloblastica aguda (LLA), linfoma no Hodgkin, melanosarcoma y carcinoma hepatocelular entre otros, dado su efecto antineoplásico sobre ciertos tipos de células tumorales que son incapaces de producir L-asparagina y dependen de la circulante para su proliferación. A pesar de su rol exitoso en el tratamiento de estas enfermedades, su uso es constantemente reevaluado ya que genera efectos secundarios, asociados a la actividad inespecífica de la enzima sobre un segundo sustrato estructuralmente similar a la Lasparagina, la glutamina, generando pancreatitis, disfunción renal, trombosis, hemorragia y reacciones de hipersensibilidad. Además, otro factor que afecta su uso en tratamientos a largo plazo es la generación de anticuerpos en los tejidos posterior a su aplicación, neutralizando su efecto sobre células tumorales. La presencia de esta enzima se ha descrito en diversos organismos, siendo las actualmente utilizadas las producidas por microorganismos. Entre ellos, las bacterias marinas del orden actinobacteria son de gran interés, ya que se han descrito Lasparaginasas II sin actividad glutaminasa. Dado el interés por encontrar nuevas asparaginasas con baja o nula actividad glutaminasa es que se estudió una nueva Lasparaginasa II descrita en el genoma de la actinobacteria Salinispora tropica CNB 440 que hasta el momento no ha sido caracterizada. Para ello el gen que codifica la enzima L-asparaginasa II fue amplificado a partir del genoma de S. tropica CNB 440 y clonado en el vector de expresión pET22b(+), con el cual se quimiotransformó cepas de E. coli BL21(DE3) quimiocompetentes. Dada la presencia de inducción basal, las cepas fueron co-transformadas con el vector pLysS, obteniendo colonias doble transformantes de E. coli BL21(DE3) ASPII/pLysS, solucionando este problema. Posteriormente se indujo la expresión del constructo en células co-transformadas con IPTG, obteniendo la expresión de la proteína recombinante de forma soluble para los cultivos inducidos a 25ºC con 0.1 y 0.2 mM de IPTG. Luego se evaluó la actividad a 37ºC de la enzima L-asparaginasa II a partir de extractos crudos de los cultivos anteriores, mediante Nesslerización para los sustratos L-asparagina y L-glutamina, obteniendo exclusivamente actividad asparaginasa por parte de la enzima. Luego se realizó purificación de la proteína recombinante mediante cromatografía en columna Ni-NTA, obteniendo muchas proteínas contaminantes, por lo que se utilizó la técnica de cromatografía Ni-NTA en batch, para aumentar la especificidad por la proteína recombinante; sin embargo, su correcta purificación no fue posible. Finalmente se realizó una estimación de la actividad enzimática a partir de la muestra de extracto crudo inducido a 25ºC con 0.1 mM de IPTG, obteniéndose una actividad específica de la Lasparaginasa II de 117,81 U/mg y no actividad sobre L-glutamina. / The enzyme L-asparaginase II, which hydrolyzes L-asparagine to aspartic acid and ammonium, is currently used as a chemotherapeutic agent in the treatment of acute lymphoblastic leukemia, acute myeloblastic leukemia, non-Hodgkin lymphoma, melanosarcoma and hepatocellular carcinoma, among others, due to its antineoplastic effect on certain types of tumor cells that are incapable of producing L-asparagine and depend on the circulating L-asparagine for their proliferation. Despite its successful role in the treatment of these diseases, its use is constantly reevaluated since it generates secondary effects, associated with the specificity of the enzyme to a second substrate due to its structural similarity, glutamine. These effects include pancreatitis, renal dysfunction, thrombosis, hemorrhage and hypersensitivity reactions. In addition, another factor that affects its use in long-term treatments is the generation of antibodies in the tissues after its application, neutralizing its effect on tumor cells. Asparaginases are distributed among living organisms, and those produced by microorganisms are currently used. Among them, marine bacteria of the actinobacteria order are of great interest, since L-asparaginases II without glutaminase activity has been reported. Given the interest in finding new asparaginases with low or no glutaminase activity, a new L-asparaginase II described in the genome of the actinomycete Salinispora tropica CNB 440 that has not been characterized yet, was studied. To accomplish this, the gene coding for the enzyme L-asparaginase II was amplified from the genome of S. tropica CNB 440 and cloned into the expression vector pET22b(+), which was used to chemotransform chemocompetent E. coli BL21 (DE3) strains. Given the presence of basal induction, the strains were co-transformed with the vector pLysS, obtaining double transformant colonies named E. coli BL21(DE3) ASPII/pLysS, solving this problem. Subsequently, the expression of the construct was induced with IPTG in cotransformed cells, obtaining the expression of the recombinant protein in a soluble form for the cultures induced at 25 ° C with 0.1 and 0.2 mM of IPTG. The activity at 37ºC of the enzyme L-asparaginase II was evaluated from crude extracts of the cultures, by Nesslerization for the substrates L-asparagine and L-glutamine, obtaining only asparaginase as enzymatic activity. Purification of the recombinant protein was initially performed by Ni-NTA column chromatography, obtaining a low-purity recombinant protein, so the technique was varied to Ni-NTA chromatography in batch, to increase the affinity for the asparaginase. However, its correct purification was not possible. Finally, an estimation of the enzymatic activity was made from crude extract induced at 25°C with 0.1 mM of IPTG, obtaining a specific activity of L-asparaginase II of 117.81 U/mg, and no activity on L-glutamine.

Tesis para optar al grado de Doctora en Ciencias de la Ingeniería Mención Ingeniería Química y Biotecnología / Salinosporamide A is a cytotoxic that has been proven to combat various types of cancer and malaria. It is currently in phases II and III of approval as an anticarcinogen. The advantages that poses over other cytotoxics are greater activity at low concentrations and highly specific. It acts on the proteasome-ubiquitin system, responsible of apoptosis in cells. This secondary metabolite is naturally ocurred in the actinomycetes bacterium strictly marine, Salinispora tropica that needs a specific ionic force in the medium to grow. Due to his nature it is a promisory source of secondary metabolites for pharmaceutical use hereby is constantly studied. The CNB440 strain is the representative strain of the species and posses a Genome-Scale Metabolic Model (GSM). The goal of this work was to implement diverse metabolic and genetic strategies that allow improve the production of Salinosporamide A. Chapter 4 of this thesis details the proves to establish the protocols for growth and determination of Salinosporamide A, Define the sensitivity of bacteria to kanamycin (100 ug/ml), thiostreptone (12 µg/ml) and apramycin (12 µg/ml). The growth curves in several minimum mediums, and stablish the methodology for the determination of Salinosporamide A. Chapter 5 describes the genetic strategy used to modify the bacterium and generate a higher concentration of Salinosporamide A. The strategy followed was by recombination homologous with the temperature sensitive vector (pGM1190) and transferred to S. tropica by conjugation with the strain E. coli ET12567/pUZ8002, to delete specific sites on the chromosome of S. tropica. These molecular tools have been successfully used in the transformation of various Streptomyces, but had not been tested in Salinispora. The sites suggested to be deletedto increase the production of the secondary metabolite were 3 clusters of genes sporolides, lymphostine and salinilactam. But due to various complications in the development of the present work only the deletion of the sporolide gene cluster was evaluated and this resulted in an increase of 20% in metabolite production. Chapter 6 details the use of genome-scale metabolic model iCC908 for increase the production of Salinosporamide A. The first stage consisted in establishing the working environment of the model, to increase the accuracy of the model, integrated growth data, metabolites in medium production and determination of Salinosporamide A, with this was also able define in silico the supplementation of medium production, to obtain more Salinosporamide A. . The second stage consisted of applying different algorithms OptKnock, OptGene, OptOrf, GDLS, FSEOF, which browse reactions or genes within the genome-scale model that could be, deleted, blocked or overexpressed to increase the production of the secondary metabolite. We found several candidates that were evaluated in silico and we proposed to evaluate the deletion of two genes. As the last stage, were evaluated the metabolic pathways that increase production by gene overexpression. The evaluation of this metabolic pathways consist in add diverse substrates that increase the flow in the pathway of the gene to be overexpresed, tyrosine at a concentration of 5mM increase the production of the secondary metabolite Salinosporamide A by 180%, enhancing the presence of phenylalanine in the medium. With these results it was possible to obtain a medium production that increased in 2.8 times Salinosporamide A by fermentation based on the use of the genome-scale metabolic model. And also was possible transform the strain S. tropica with genetic tools previously proved in Streptomyces.