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Bioprocess Efficiency As a Roadmap to Establish Pichia Pastoris As a Reliable Cell Factory

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Bioprocess Efficiency As a Roadmap to Establish Pichia Pastoris As a Reliable Cell Factory ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en ESCOLA D’ENGINYERIA Departament d’Enginyeria Química, Biològica i Ambiental Bioprocess efficiency as a roadmap to establish Pichia pastoris as a reliable cell factory Memòria per obtenir el grau de Doctor per la Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia Directors: Francisco Valero i José Luis Montesinos Xavier Ponte Font Bellaterra, 2017 Abstract 0. Bioprocess efficiency as a roadmap to establish Pichia pastoris as a reliable cell factory ABSTRACT The present work is focused, with a bioprocess engineering point of view, in the production of the heterologous Rhizopus oryzae lipase expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol-induced alcohol oxidase 1 promoter; and, specially, the improvement of the bioprocess efficiency by the use of proper operational strategies in fed-batch mode for bioreactors of different scale. With that, P. pastoris is stablished as a versatile, robust and competent platform for the production of recombinant proteins. In a first part of the dissertation, the emphasis is put on reaching optimal levels of the main performance indexes of industrial interest at lab scale in methanol feeding strategies as the only carbon source and inducer. Prior to this final goal, though, preliminary studies and operational improvements were carried out to outcome system limitations and to give more versatility and robustness to the system. They included upgrades in system control and supervising in LabVIEW, installation of new hardware for instrumentation and a new feeding strategy for the nitrogen source added to the cultures. Later, a systematic study on the effect of the oxygen tension was conducted. It was found that, in normoxic conditions, dissolved oxygen can limit the process in both excess and lack. An optimal value of 25 % air saturation was found for all specific rates and results were validated by a consistency check and further data reconciling. From that point, the optimization of the bioprocess in bench-top bioreactor by the mathematical design of methanol feeding profiles was fulfilled. From modelling data, and by the use of a simple, versatile optimization tool, the simulation of the optimal methanol concentration profiles were carried out to maximize product titer and volumetric productivity, and then compared to previous strategies and then successfully applied experimentally. On the other hand, the next part of this document focuses on studies of scale down and up from the bench-top scale. The first change is downwards to a microbioreactor system of a 1000 µL scale. Cultures from that scale were successfully reproduced succesfully at lab scale to verify the system as suitable for clone screening and design of simple fed- batch strategies. Finally, the bioprocess was brought up to a 50 L scale. Unfortunately, the attempts did not succeed on reproduce the bioprocess efficiency, but it was possible to discard some of the hypothesis that were brought up, and to settle some other ones that can be further investigated in the future, like oxygen mass transfer limitations. I Abstract 0. Bioprocess efficiency as a roadmap to establish Pichia pastoris as a reliable cell factory RESUM El present document està centrat en un punt de vista d’enginyeria de bioprocés, de cara a la producció heteròloga de la lipasa de Rhizopus oryzae, expressada en el llevat metilotròfic Pichia pastoris sota el control del promotor de l’alcohol oxidasa 1, induïble amb metanol. Especialment, l’objectiu es focalitza a la millora de l’eficiència del bioprocés mitjançant l’ús d’estratègies d’operació en discontinu alimentat en bioreactors de diferent escala. Amb això, es pretén establir P. pastoris com una plataforma versàtil, robusta i competent per a la producció de proteïnes recombinants. A la primera part de la tesi, l’èmfasi es troba en l’assoliment de nivells òptims dels principals índexs de rendiment d’interès industrial a escala de laboratori, utilitzant estratègies d’operació amb metanol com a font única de carboni i inductor del promotor. Prèviament a aquest objectiu, però, es van realitzar estudis preliminars i millores operacionals per tal de superar certes limitacions del sistema i per aportar-hi versatilitat i robustesa. Entre ells, es va actualitzar i incorporar millores al programa de supervisió i control del procés en LabVIEW, s’hi instal·là nou hardware d’instrumentació i es va implementar una nova estratègia d’alimentació de la font de nitrogen. Posteriorment, un estudi sistem àtic de l’efecte de la disponibilitat d’oxigen es va dur a terme. Es va obtenir que, en condicions de normòxia, l’oxigen dissolt pot limitar el procés tant per un excés com per nivells escassos. El 25 % de saturació d’aire es va establir com a valor òptim de cara a les velocitats específiques, i els resultats van ser validats per un test de consistència i reconciliació de dades. A partir d’aquest punt, s’assolí l’optimització del bioprocés a escala de laboratori mitjançant el disseny de perfils d’alimentació de metanol amb eines matemàtiques d’optimització. A partir de dades de modelització i amb una eina d’optimització simple i versàtil, es van dur a terme les simulacions de perfils òptims de concentració de metanol per maximitzar la concentració de producte i la productivitat volumètrica, es van comparar amb estratègies anteriors i es van aplicar experimentalment amb èxit. Per altra banda, la següent part del document se centra en estudis de canvi d’escala. El primer que es realitza és a escala de microbioreactor. Es van reproduir uns cultius realitzats a aquella escala al laboratori per verificar aquest sistema com a vàlid per realitzar-hi assajos d’expressió i selecció de soques i per dissenyar-hi estratègies simples d’operació en discontinu alimentat. Finalment, el bioprocés va ser portat a escala pilot de 50 L. Malauradament, els intents realitzats no van assolir reproduir l’eficiència del bioprocés, tot i que va ser possible descartar-ne algunes hipòtesis plantejades i proposar- ne d’altres que hauran de ser investigades en un futur, com ara limitacions en la transferència d’oxigen. II TABLE OF CONTENTS 1. Introduction: heterologous protein production by Pichia pastoris. A bioprocess engineering comprehensive design ....................................................................................1 2. Objectives ................................................................................................................................ 43 3. Preliminar results: Pichia pastoris producing Rhizopus oryzae lipase under PAOX1 control. first steps to the optimization of bioprocess efficiency ................................ 45 4. Bioprocess efficiency of Rhizopus oryzae production under the control of PAOX1 is oxygen tension dependent. ................................................................................................. 61 5. Towards optimal substrate feeding for heterologous protein production in Pichia pastoris fed batch process under PAOX1 control: a modelling aided approach. ..... 91 6. The microbioreactor as a tool to evaluate fed-batch strategies and high- throughput clone screening. ..............................................................................................123 7. Upgrading to the first step up to industry. pilot plant scale production of ROL by Pichia pastoris under PAOX1 control ...................................................................................153 8. General conclusions ............................................................................................................ 185 III IV 1. Introduction Bioprocess efficiency as a roadmap to establish Pichia pastoris as a reliable cell factory 1 INTRODUCTION: HETEROLOGOUS PROTEIN PRODUCTION BY Pichia pastoris. A BIOPROCESS ENGINEERING COMPREHENSIVE DESIGN 1.1 Bioprocess engineering “Bioprocess engineering is the discipline that puts biotechnology to work”. This forceful statement was published by the National Research Council in 1995 [1]. Since the quite recent inception of Biotechnology as an industrial applied science during this last century, bioprocess engineering has been stablished and promoted itself as an alternative to classic industrial processes and for the synthesis of new products. Biotechnology can be divided into several color-classes, white biotechnology (industrial processes, fine chemical products, enzymes, biofuels), red biotechnology (pharma research), green biotechnology (agriculture), blue biotechnology (sea resources) and grey biotechnology (environmental bioengineering). Biotechnology and bioprocesses are, then, present in a wide heterogeneous range of fields. Some notorious examples are the production of biopharmaceuticals like recombinant second generation insulin or growth factors [2]; wastewater treatment plants or countless applications in the food industry, from brewery [3] to enzyme production for hydrolysis in
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