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David Manuel Nogueira Ribas Timization Using Indus Universidade do Minho Escola de Ciências trial microbes David Manuel Nogueira Ribas timization using indus Structural-functional studies of carboxylic vel tools in production op s: no acid transporters: novel tools in production ter optimization using industrial microbes lic acid transpor xy tudies of carbo Structural-functional s a Ribas vid Manuel Nogueir Da 3 1 UMinho|20 October, 2013 Universidade do Minho Escola de Ciências David Manuel Nogueira Ribas Structural-functional studies of carboxylic acid transporters: novel tools in production optimization using industrial microbes Master Thesis Master in Molecular Genetics Work done under supervision of Professora Doutora Margarida Casal Professora Doutora Sandra Paiva October, 2013 DECLARAÇÃO Nome: David Manuel Nogueira Ribas Endereço eletrónico: [email protected] Número do bilhete de identidade: 13254792 Título da tese: Structural-functional studies of carboxylic acid transporters: novel tools in production optimization using industrial microbes Orientadores: Prof. Doutora Margarida Casal Prof. Doutora Sandra Paiva Ano de conclusão: 2013 Designação do mestrado: Genética Molecular DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE/TRABALHO Universidade do Minho:__/__/__ Assinatura:________________________________ iii ACKNOWLEDGMENTS/AGRADECIMENTOS À Professora Doutora Margarida Casal agradeço toda a confiança depositada em mim e a fantástica oportunidade de iniciar um projeto de investigação com um potencial enorme na minha área de formação, acrescendo o facto de ser gerido em parceria internacional com um instituto de investigação de conhecida reputação. Agradeço também a aposta na minha formação e no meu currículo e da qual me orgulho muito, assim como toda a ajuda e compreensão prestada ao longo de todo o mestrado. Eternamente grato. À Professora Doutora Sandra Paiva agradeço toda a ajuda e acompanhamento que me prestou, em particular a aposta na minha formação. Agradeço também a ajuda preciosa na parte final da dissertação da tese. Obrigado por tudo À doutora Joana Sá-Pessoa a forma como me recebeu e acompanhou ao longo de todo o mestrado, a confiança que depositou em mim, todos os conhecimentos que me transmitiu e toda a ajuda e apoio que me prestou. Agradeço também a aposta no meu currículo como investigador. A dedicação e perseverança na forma como faz ciência são uma referência para mim. Obrigado por tudo. Aos colegas do Laboratório de Biotecnologia Molecular da Universidade do Minho agradeço a forma como me integraram no grupo e ajudaram ao longo de todo o projeto de investigação. É um orgulho ter pertencido a este grupo de pessoas e cientistas. Obrigado por tudo. Ao Centro de Biotecnologia Molecular e Ambiental e ao “VTT Research Institute” agradeço todos os recursos disponibilizados. iv ABSTRACT Structural-functional studies of carboxylic acid transporters: novel tools in production optimization using industrial microbes The rising global energy demands in last century drive to worrying depletion levels of fossil fuels, leading to a growing interest in microbial biofuel synthesis, particularly in model organisms like Saccharomyces cerevisiae. The microbial conversion of sugars to biofuels is a promising technology, thus envisaging for more efficient metabolic pathways has been attempted. Indeed byproducts of biomass pretreatment processes and the biofuels themselves are often toxic at industrially-relevant levels., therefore efforts to improve production yield by engineering efflux systems to overcome toxicity problems and secret inhibitory chemicals from the cell has been revealed as a crucial alternative. In this scope, the aim of the present work was to screen a wide range of promising membrane transporters for the transport of carboxylic acids, particularly for xylonic acid, mucic acid, saccharic acid, gluconic acid and xylaric acid in the yeast S. cerevisiae. During this study a considerable number of membrane proteins, known as carboxylic acid transporters from different yeasts species were screened and functionally characterized in regard to the transport of mono- and dicarboxylic acids with biotechnological application, such as xylonic and gluconic acids, and saccharic, mucic and xylaric acids, respectively. Among the transporters tested, we have found evidences for the transport of four carboxylic acids, mucic, xylaric, gluconic and saccharic acids with the following specificities: gluconic acid (Ki of 13.2 mM and 40.6 mM), xylaric acid (Ki of 27.6 mM), mucic acid (Ki of 32.9 mM) and saccharic acid (Ki of 24.1 mM and 24.7 mM). These results revealed to be very promising for future work aiming at engineering proper microbial strains with increased ability to export biofuel acids to external medium. v RESUMO Estudos funcionais/estruturais de transportadores de ácidos carboxílicos: novas ferramentas para otimização da produção em microrganismos industriais. A crescente procura de combustíveis fósseis a que se assistiu no último século levou a que se atingissem limites de exploração preocupantes, redirecionando a atenção para a síntese microbiana de biocombustíveis, em particular através organismo modelo Saccharomices cerevisiae. A conversão microbiana de açúcares em biocombustíveis tem- se revelado uma tecnologia promissora, pelo que novas tentativas de melhorar a eficiência das vias metabólicas envolvidas no processo de síntese têm sido testadas. Tanto os produtos resultantes do processamento de biomassa como os próprios biocombustíveis exercem regularmente um efeito tóxico na produção à escala industrial. Nesse sentido, importantes esforços têm sido levados a cabo para desenvolver novos sistemas de efluxo com o intuito de reduzir problemas de toxicidade e conduzir para o exterior da célula diferentes compostos químicos. Neste contexto, o objetivo do presente trabalho é desvendar o potencial de diferentes transportadores de membrana para o transporte de ácidos carboxílicos em S. cerevisiae. Ao longo deste estudo, várias proteínas de membrana, conhecidas como transportadores de ácidos carboxílicos provenientes de diferentes espécies de leveduras foram testadas e caracterizadas funcionalmente no que respeita ao transporte de ácidos mono e dicarboxílicos com interesse biotecnológico, tais como ácidos xilónico e glucónico, e ácidos sacárico, mucico e xilárico, respetivamente. De entre os transportadores testados foram encontradas evidências para o transporte de quatro ácidos carboxílicos, a saber os ácidos mucico, xilárico, glucónico e sacárico com as respetivas especificidades: ácido glucónico (Ki de 13,2 mM e 40,6 mM), ácido xilárico (Ki de 27,6 mM), ácido mucico (Ki de 32,9 mM) e ácido sacárico (de 24,1 mM e 24,7 mM). Estes resultados revelam-se bastante promissores para futuros estudos que envolvam a obtenção de estirpes microbianas com capacidade desenvolvida para excretar biocombustíveis. vi TABLE OF CONTENTS Acknowledgements/Agradecimentos…………………………………………..……..iv Abstract……………………………………………………………………………..…..v Resumo………………………………………………………………………………....vi Abbreviations and symbols………………………………………………………..….IX List of publications……………………………………………………………………XI 1. Introduction……………………………………………………………….……….…1 1.1 Carboxylic acids: general properties and nomenclature………………………..1 1.2 Carboxylic acids in industry………………………………………………………2 1.2.1 Carboxylic acids production in Escherichia coli………………………….….6 1.2.2 Carboxylic acids production in Saccharomyces cerevisiae…………………..8 1.3 Sugar acids bio-production……………………………………………………...10 1.3.1 Gluconic acid microbial production…………………………………………10 1.3.2 Xylonic acid microbial production…………………………………………..11 1.3.3 Saccharic acid microbial production………………………………………..12 1.3.4 Mucic acid microbial production……………………………………………13 1.3.5 Xylaric acid microbial production…………………………………………...14 1.4 Plasma membrane carboxylic acid transporters in S. cerevisiae……………..14 1.4.1 ScJen1 transporter and homologues………………………………………..17 1.4.2 Ady2 transporter and homologues………………………………………….22 1.4.3 Carboxylic acids toxicity in S. cerevisiae…………………………………….24 1.4.4 Mechanisms to avoid a futile cycle of diffusional entry and active extrusion of organic acids………………………………………………………………………...25 1.5 Outline of the thesis……………………………………………………………...25 2. Materials and methods……………………………………………………………..27 2.1 Biological material……………………………………………….………………27 2.2 Yeast cells culture conditions……………………………………………………29 2.3 Transport assays………………………………………………………..…….….30 3. Results…………………………………………………………………………….…32 3.1 Seeking for novel production pathways of sugar acids in S. cerevisiae………32 3.1.1 Screening a ScJen1p mutant collection for potential export properties of xylonic, mucic and saccharic acids…………………………………………………...32 vii 3.1.2 Screening Jen1p homologous proteins in different yeast strains for potential export properties of carboxylic acids……………………………………...37 3.1.3 Accessing carboxylic acids export properties in S. cerevisiae strains expressing heterologously CaJen1, CaJen2, KlJen1 and KlJen2…………………..41 3.2 Comparative analysis of inhibition constant (Ki) between the carboxylic acid transporters……………………………………………………………………………47 4. Discussion…………………………………………………………………………..48 4.1 Final remarks………………………………………………………………….....48 4.2 Future Perspectives……………………………………………………………...51 5. Bibliography…………………………………………………………………..……53 Appendix…………………….………………………………………………………...59 viii ABBREVIATIONS AND ACRONYMS AMPK - Adenosine Monophosphate-activated Protein Kinase ald – aldehyde dehydrogenase COOH – Carboxyl group dhaB – glycerol dehydratase
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