Miguel Antunes

Class 2018

PhD Grant: PD/BD/142944/2018

PhD thesis

Mechanisms of Yeast Adaptation and Tolerance to Acetic Acid Stress: physiological genomic approaches and genotype-phenotype associations

Supervisors

• Isabel Sá-Correia,Professor @ IST/ULisboa, PT

Collaborators

• Pedro T. Monteiro, Professor @ IST/ULisboa, PT
• Jean-Luc Legras (SPO – ADEL  team, INRAE, Montpellier, France)
• Hana Sychrova (Institute of Physiology, Czech Academy of Sciences, Prague , Czech Republic)
• Joseph Schacherer (Laboratory for Molecular Genetics, Genomics and Microbiology, CNRS, University of Strasbourg)

PhD Degree complete: Discussion on May 10, 2024

THESIS ABSTRACT

This thesis work explores the mechanisms of yeast adaptation and tolerance to stresses of biotechnological relevance. The understanding of these mechanisms is crucial for enhancing yeast robustness towards a sustainable circular bio-based economy. Yeast cells employ several strategies to respond and adapt to acetic acid-imposed stress, including the regulation and maintenance of ion homeostasis. The role of the Saccharomyces cerevisiae protein kinase Hrk1, a determinant of tolerance to acetic acid and a member of the NPR/Hal family of kinases, was found to contribute to Pma1 activation during cultivation of S. cerevisiae under acetic acid stress, thereby counteracting the decrease of intracellular pH and the dissipation of plasma membrane electrochemical potential. Additionally, potassium was demonstrated to be a key factor in adaptation to acetic acid stress, and the H+/K+, Na+ antiporter Nha1, a proposed phosphorylation target of Hrk1, was suggested to be involved. This thesis also explores non-Saccharomyces yeast species such as Rhodotorula toruloides, addressing the challenges posed by stresses of biotechnological relevance, particularly in lignocellulosic-based bioprocesses. This thesis work also contributed to YEASTRACT+ portal update and upgrade to facilitate cross-species transcription regulatory analysis and prediction of regulatory networks in less-characterized yeasts. Based on a multi-stress tolerant evolved R. toruloides strain, previously obtained in our laboratory by Adaptive Laboratory Evolution (ALE), the genome and transcriptome were compared with those of the original strain. Results revealed several mutations, copy number variants and increased transcript levels from genes related to cell envelope biogenesis and remodeling, DNA repair, and stress-responsive pathways. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid -induced stresses. The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance, underscoring the potential of the ALE approach for enhancing the robustness of industrial yeast strains.


RESUMO DA TESE

Foram explorados mecanismos de adaptação e tolerância de leveduras a stresses de relevância em Biotecnologia. A compreensão destes mecanismos é crucial para aumentar a robustez de leveduras visando uma bioeconomia circular sustentável. As leveduras utilizam várias estratégias para responderem e se adaptarem a stress por ácido acético, incluindo a regulação e manutenção da homeostase iónica. A contribuição da proteína cinase Hrk1, uma determinante de tolerância a ácido acético e membro da família de cinases NPR/Hal, na ativação da Pma1 em Saccharomyces cerevisiae durante o cultivo sob stress por ácido acético, foi demonstrada. Tal permite contrariar a diminuição do pH intracelular e a dissipação do potencial eletroquímico da membrana plasmática. Foi ainda demonstrado que a concentração de potássio constitui um fator essencial nessa adaptação e tolerância, tendo o envolvimento do transportador H+/K+,Na+ Nha1 sido proposto enquanto alvo de fosforilação por Hrk1. Esta tese também explorou espécies de leveduras não-Saccharomyces, como Rhodotorula toruloides, importantes para superar os stresses impostos em bioprocessos baseados em hidrolisados de biomassa lenhocelulósica. Este trabalho contribuiu ainda para a atualização e expansão do portal YEASTRACT+ para facilitar a análise da regulação transcricional em diferentes espécies de leveduras e a previsão de redes regulatórias em espécies menos caracterizadas. Utilizando uma estirpe de R. toruloides evoluída, tolerante a múltiplos stresses e previamente obtida no nosso laboratório por Evolução Laboratorial Adaptativa (ALE), realizou-se uma análise do seu genoma e transcritoma comparados com os da estirpe original. Foram reveladas várias mutações, variação no número de cópias e aumento do nível de transcritos de genes relacionados com a biogénese e remodelação do invólucro celular, reparação de DNA e vias de sinalização a stress. Baseado nessas respostas, o fenótipo de tolerância a múltiplos stresses foi estendido a outros stresses: osmótico, salino, oxidativo, genotóxico e induzidos por etanol e ácidos gordos de cadeia média. A análise global desta estirpe evoluída proporcionou a oportunidade de obter conhecimento mecanístico subjacente à aquisição de tolerância a múltiplos stresses, evidenciando o potencial da abordagem ALE no aumento da robustez de estirpes de leveduras industriais.

PUBLICATIONS

Papers

Antunes, M., Mota, M. N., Sá-Correia, I. (2024). Cell envelope and stress-responsive pathways underlie an evolved oleaginous Rhodotorula toruloides strain multi-stress tolerance. Biotechnology for Biofuels and Bioproducts. 17(1):71. DOI: 10.1186/s13068-024-02518-0.

Antunes, M., & Sá-Correia, I. (2024). The role of ion homeostasis in adaptation and tolerance to acetic acid stress in yeasts. FEMS Yeast Research, foae016. DOI: 10.1093/femsyr/foae016

Antunes, M., Kale, D., Sychrová, H., & Sá-Correia, I. (2023). The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis. Microbial Cell, 10(12), 261.

Teixeira, M. C., Viana, R., Palma, M., Oliveira, J., Galocha, M., Mota, M. N., … & Monteiro, P. T. (2023). YEASTRACT+: a portal for the exploitation of global transcription regulation and metabolic model data in yeast biotechnology and pathogenesis. Nucleic Acids Research, 51(D1), D785-D791.

Antunes, M., & Sá-Correia, I. (2022). The NPR/Hal family of protein kinases in yeasts: biological role, phylogeny and regulation under environmental challenges. Computational and Structural Biotechnology Journal.Oliveira, J., Antunes, M., Godinho, C. P., Teixeira, M. C., Sá-Correia, I., & Monteiro, P. T. (2021). From a genome assembly to full regulatory network prediction: the case study of Rhodotorula toruloides putative Haa1-regulon. BMC Bioinformatics, 22(1), 1-17.

Godinho, C. P., Palma, M., Oliveira, J., Mota, M. N., Antunes, M., Teixeira, M. C., Monteiro, P. T., & Sá-Correia, I. (2021). The NC Yeastract and CommunityYeastract databases to study gene and genomic transcription regulation in non-conventional yeasts. FEMS Yeast Research, 21(6), foab045.

Monteiro, P. T., Oliveira, J., Pais, P., Antunes, M., Palma, M., Cavalheiro, M., Galocha, M., Godinho, C. P., Martins., L. C., Bourbon, N., Mota, M. N., Ribeiro, R. A., Viana, R., Sá-Correia, I., Teixeira, M. C., YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts. Nucleic Acids Research, 48(D1), D642-D649, 2020.

Antunes, M., Palma, M., & Sá-Correia, I., Transcriptional profiling of Zygosaccharomyces bailii early response to acetic acid or copper stress mediated by ZbHaa1. Scientific Reports, 8(1), 1-14, 2018.

Teixeira, M. C., Monteiro, P. T., Palma, M., Costa, C., Godinho, C. P., Pais, P., Carvalheiro, M., Antunes, M., Lemos, A., Pedreira, T., & Sá-Correia, I. YEASTRACT: an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae. Nucleic acids research, 46(D1), D348-D353, 2018.

Oral Communications

Antunes, M., Kale, D., Sychrová, H., Sá-Correia, I. The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis. COST YEAST4BIO WG Meeting October 17-18, 2023, Lisbon, Portugal.

Poster Communications

Antunes, M., Kale, D., Sychrová, H., Sá-Correia, I. The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis. III iBB Workshop November 13, 2023, Lisbon, Portugal.

Antunes, M., Tsouris, A., Schacherer, J., Sá-correia, I. Identification of Saccharomyces cerevisiae genetic variants associated with tolerance to industrially relevant stress conditions based on GWAS. 8th Conference on physiology of yeasts and filamentous fungi (PYFF8) June 5-8, 2023, Cork, Ireland.

Antunes, M., Palma, M., & Sá-Correia, I., “The Protein Kinase Hrk1 Enhances Glucose Uptake Through the Hexose Transporter Hxt1 in Saccharomyces cerevisiae.” 29th International Conference on Yeast Genetics and Molecular Biology (ICYGMB) August 18-22, 2019, Gothenburg, Sweden.

Antunes, M., Oliveira, J., Pais, P., Palma, M., Cavalheiro, M., Galocha, M., Godinho, C. P., Martins., L. C., Bourbon, N., Mota, M. N., Ribeiro, R. A., Viana, R., Monteiro, P. T., Sá-Correia, I., Teixeira, M. C., “YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts.” Microbiotec’19 December 5-7, 2019, Coimbra, Portugal.

DOCTORAL PROGRAM (36 ECTS)

• General Doctoral Training (6 ECTS)

• Advanced Experimental Techniques (6 ECTS)

• Bioentrepreneurship (6 ECTS)

• Genomics Proteomics and Bioinformatics (6 ECTS)

• Outreach and Teaching Skills (6 ECTS)

• Molecular and Cell Biophysics (6 ECTS)

Advanced courses:

• BioData.pt Crash Course: Microbiome Visualization with Biome-Shiny
• Ready For BioData Management? – Introduction to Data Management Plans
• Elixir PT/BioData.pt – Data Carpentry Workshop – Genomics Data
• Specialization course: Radiological Protection and Safety (Level III) – Industrial Area (Utilization of sealed and unsealed sources) (1 ECTS)

Current position
05/2024-08/2024: Researcher (2022.01501.PTDC WeakAc_R) @ Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal