L'increment de la dosi gènica com a estratègia de millora de la producció de proteïnes recombinades al llevat Pichia Pastoris: Revertint les limitacions genètiques del sistema biològic

Student thesis: Doctoral thesis


Recombinant protein production is becoming the preferred option to synthesized biopharmaceutical and industrial enzymes. As a host system, yeast factories provide numerous advantages over other microorganisms, such as their capacity to perform post-translational protein modifications, the wide genetic available tools, and the facility to growth at high densities, among others. In this context, the suitability of methylotrophic yeast Pichia pastoris for recombinant protein production has stand out over the classical yeast Saccharomyces cerevisiae, presenting an increasing relevance for the production of commercial recombinant products. Among several factors, the presence of the strong and tightly regulated AOX1 promoter (PAOX1) has contributed to the popularity of this expression system. To further increase the recombinant protein production, several strategies have been attempted, being the increase of the heterologous gene dosage one of the most widely reported. Although a heterologous productivity improvement can be achieved with this methodology, high gene dosages normally entail a metabolic burden in the cell that results in lower productivities. In the course of this thesis, it has been used the lipase of Rhizopus oryzae (Rol) as a protein model expressed under the control of the PAOX1. Firstly, we constructed a series of P. pastoris strains harbouring an increase gene dosage of ROL, and we reported for the first time the use of Droplet Digital PCR (ddPCR) for an accurate copy number determination in P. pastoris, allowing a higher precision than using conventional quantitative real-time PCR (qPCR). Secondly, a transcriptomic study of a set of multicopy strains was carried out using microarrays, to better understand the impact of ROL gene dosage on the metabolic pathways. Concretely, transcriptomic data revealed a high downregulation of methanol-related pathways (among others), in agreement with the physiological data that had been showed that C-source uptake rate and growth yields were gene dosage dependents. Based on these observations, a strain carrying 4 copies of ROL was remodified to express a deregulated variant of the Mxr1 transcription factor, the main regulator of methanol metabolism. Physiological data and transcriptional levels of the main genes involved in methanol metabolism confirmed the improvement of Rol production and the increasing expression levels of methanol-related genes and ROL. Finally, it was performed a comparative transcriptomic analysis of the response of P. pastoris to the expression of three different recombinant proteins, to study the impact of protein complexity and the use of constitutive or inducible expression systems. Overall, in the present study we describe the first systems-level approach to determine the impact of increasing gene dosage in the yeast P. pastoris, hence allowing the design of novel cell engineering strategies to generate high-producing strains by means of the synthetic biology and metabolic engineering.
Date of Award4 Nov 2016
Original languageCatalan
SupervisorJoan Albiol Sala (Director) & Pau Ferrer Alegre (Tutor)

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