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Suspension Bioreactor Strategies for Itaconic Acid Production Using Engineered Microorganisms

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Suspension Bioreactor Strategies for Itaconic Acid Production Using Engineered Microorganisms Suspension bioreactor strategies for itaconic acid production using engineered microorganisms Nuno Miguel Sousa Marques Thesis to obtain the Master of Science Degree in Biotechnology Supervisors: Prof. Dr. Frederico Castelo Alves Ferreira Dr. Nuno Ricardo Torres Faria Examination committee Chairperson: Professor Helena Maria Rodrigues Vasconcelos Pinheiro Supervisor: Professor Frederico Castelo Alves Ferreira Member of the commitee: Professor Nuno Gonçalo Pereira Mira November 2017 Acknowledgments It is acknowledged funding from Portuguese Foundation for Science and Technology (FCT) through iBB-Institute for Bioengineering and Biosciences (FCT reference: UID/BIO/04565/2013 and POL2020 reference 007317, including iBB grant ITACYEAST), project CRUISEPTDC/AAG- TEC/0696/2014 and scholarship SFRH/BPD/108560/2015. During this journey, I would like to thank to my supervisors for all the collaboration and guidance during this process. Also Prof. Dr. Nuno Mira for all the collaboration and availability to help me. To all my work colleagues that during these months helped me, with special references for André Costa, António Maduro, Carlos Rodrigues, Flávio Ferreira, João Santos, Margarida Silva, Marisa Santos, Ricardo Pereira and Dona Rosa. To my colleagues from Biotechnology and for my brothers of another mother, Carlos Fernandes, Miguel Chapado, Miguel Nascimento (Los Abadia), Diogo Mendonça, João Lopes, João Nunes, Miguel Antunes, Pedro Reis, Silvestre Leite, Tiago Nuno Jorge, and all the others I am not mentioning, thank you for all the moments together. For all my Sport Lisboa e Benfica colleagues that during this time help me doing one of the things I like more. To Bara, Pavla and Martina. To Katka for all our time together and all the things we already did. Learn Portuguese to read the Portuguese paragraph. My Slovak is far away better than your English! Last but not the least, to my family, my brother, my mom, my dad, and Benny, thank you for all. Agradeço ao financiamento da Portuguese Foundation for Science and Technology (FCT) through iBB-Institute for Bioengineering and Biosciences (FCT reference: UID/BIO/04565/2013 and POL2020 reference 007317, including iBB grant ITACYEAST), project CRUISEPTDC/AAG- TEC/0696/2014 and scholarship SFRH/BPD/108560/2015. Durante esta aventura, tenho de agradecer aos meus supervisores por toda a colaboração, ajuda e conhecimento ao longo deste processo de aprendizagem. Ao Prof. Dr. Nuno Mira por toda a colaboração e disponibilidade para me ajudar. A todos os meus colegas de laboratório que me ajudaram, alguns tornando-se até amigos, com especial referência a André Costa, António Maduro, Carlos Rodrigues, Flávio Ferreira, João Santos, Margarida Silva, Marisa Santos, Ricardo Pereira e claro, à Dona Rosa. A todos os meus colegas de Biotecnologia e para os meus irmãos de outra mãe, Carlos Fernandes, Miguel Chapado, Miguel Nascimento (Los Abadia), Diogo Mendonça, João Lopes, João Nunes, Miguel Antunes, Pedro Reis, Silvestre Leite, Tiago Nuno Jorge (por todo o meu conhecimento de NBA que te vai fazer rico deves-me (mais) uma imperial). A todos os não mencionados, um obrigado por todos os momentos juntos. A todos os meus colegas do Sport Lisboa e Benfica com quem durante este tempo partilhei muitas horas da minha vida, o meu obrigado. Só vos menciono em português porque o Mota só fala chinês agora malta. Por isso obrigado Diogo, Mota, Marc, Bernardo e Andrés e Nuno Azevedo por termos passado ii tantas horas juntos que nunca foram desperdiçadas. À Bára, Pavla e Martina. À Katka por tudo o que vivemos juntos. Vê lá se aprendes português, eu já falo eslovaco fluententemente… E claro, aos meus pais, o maior motivo para escrever em português aqui (tenho de vos pagar o curso de inglês?). Ser o primeiro licenciado cá de casa só me mostrou que os cursos valem muito pouco comparado com aquilo que sabemos fazer. E não definem e nunca vão definir quem somos ou quem seremos. Usar o que sabemos fazer no que queremos fazer. E se não sabemos, aprendemos. E ao meu irmão querido, que iniciou agora a sua vida académica, espero que não te percas para o álcool e afins. iii Abstract The present work aims to study the potential conditions for production of itaconic acid (ITAC) using engineered microorganisms (Saccharomyces cerevisiae and Issatchenkia orientalis) in suspension bioreactor. Firstly, it was assessed that I. orientalis is the best ITAC producer between the two species and so, further work was developed with this microorganism. Daily carbon source addition strategies led to higher ITAC titres, with the consumption of all the sugar and the reducing of the main by-product concentration, citric acid. Carbon source feeds have influence in citric acid concentration during the fermentation time. Cheese whey was used as a possible carbon-source and rich-medium supplement. The best ITAC titre was obtained in shake-flask with 50% v/v of synthetic minimal fermentation medium (MMF) and 50% (v/v) of clarified (protein-free) cheese whey. A titre in ITAC of 9.83 g/l was obtained after eight days of fermentation. With MMF in shake- flask and with fed-batch, 2.1 g/l of ITAC was obtained after five days of fermentation. Scale-up of the process from shake-flasks to liter bioreactors was done. pH control reveals to be fundamental regarding improvement of ITAC titres in bioreactor. Control of pH 4 and daily feeding of 20 g of glucose per liter of fermentation, resulted in 2.79 g/l of ITAC at day six of the fermentation. Bioreactor with 50% v/v cheese whey and 50% synthetic MMF medium led to 2.66 g/l of ITAC but a higher concentration of citric acid when compared to control (MMF only, no cheese whey). Keywords: ITAC, suspension bioreactor, I.orientalis, protein-free cheese whey, pH control iv v Resumo O trabalho desenvolvido tem como objetivo encontrar as melhores condições para a produção de ácido itacónico (ITAC) usando microorganismos engenheirados (S. cerevisiae e I. orientalis) em bioreactores em suspensão. Foi testado o melhor produtor de ITAC entre as duas espécies, que se revelou ser I. orientalis, e o trabalho futuro foi desenvolvido com esta levedura não convencional. Adições diárias de fonte de carbono foram usadas como estratégia para aumentar a concentração de ITAC produzido, levando a um aumento na concentração de ITAC no meio, com o consumo de toda a fonte de carbono disponível e redução da concentração do subproduto, ácido cítrico. A concentração de ácido cítrico durante o tempo de fermentação é influenciada pela adição de fonte de carbono. Soro de leite clarificado (sem proteína) foi testado como uma possível fonte de carbono e meio rico de fermentação. A melhor concentração de ITAC foi obtida em shake-flask com 50% v/v de meio sintético (MMF) e 50% v/v de soro de leite clarificado, com 9.83 g/l obtidos após oito dias de fermentação. Com MMF em shake-flask e um sistema de fed- batch, 2.1 g/l de ITAC foram obtidos em cinco dias de fermentação. A pH 4 e com adições diárias de fonte de carbono, 2.79 g/l de ITAC foram obtidos no sexto dia de fermentação. Um bioreactor com 50% v/v soro de leite e 50% MMF levou à produção de 2.66 g/l de ITAC mas também a uma maior concentração de ácido cítrico no meio (apenas MMF, sem soro de leite). Palavras-chave: ITAC, bioreactor em suspensão, I. orientalis, soro de leite desproteinizado, controlo de pH vi Index Acknowledgments .......................................................................................................................... ii Abstract ......................................................................................................................................... iv Resumo ......................................................................................................................................... vi List of Tables ................................................................................................................................. ix List of figures ................................................................................................................................. xi Abbreviations ............................................................................................................................... xiii 1. Introduction ............................................................................................................................ 1 1.1 Motivation ............................................................................................................................ 1 1.2 Objectives and challenges .................................................................................................. 2 1.3 Research Questions ............................................................................................................ 3 1.4 Research strategy ............................................................................................................... 3 2. Literature Review ...................................................................................................................... 4 2.1 Chemical synthesis and biological production of ITAC with microorganisms ..................... 4 2.2 Pathways and regulation ..................................................................................................... 5 2.3 From modifications in natural ITAC producers to biological engineered ITAC factories..... 7 2.4 I. orientalis as a possible ITAC producer ............................................................................ 9 2.5 Fermentation Conditions ..................................................................................................
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