Industrial biotechnology is a segment of the bioeconomy in which microorganisms are considered core assets, playing a central role as cell factories in fermentation processes. Despite numerous proof-of-concept studies performed in industrial models, many commercial bioprocesses are based on non-conventional microorganisms with optimized innate metabolic potential. In fact, the choice between engineering microbial models or improving the innate metabolism of non-conventional microorganisms has been a major question in the field. Ashbya gossypii is a filamentous fungus that stands in the intersection between industrial models and non-conventional microorganisms. It is considered a paradigm of the industrial biotechnology owing to the success of the bioprocess implemented almost 30 years ago that makes use of its overwhelming capacity to overproduce riboflavin (vitamin B2). Until the beginning of this decade, its biotechnological relevance remained restricted to this application. Since then, interesting potentialities of its natural metabolism have been unravelled. Therefore, with the objective of developing A. gossypii as an alternative cell factory and of identifying novel biotechnological products, this thesis focused on the systematic exploration of this fungus natural metabolism through its physiological and molecular characterization. The overproduction of riboflavin is the most distinctive trait of this hemiascomycete. It has been proposed to act as an ecological defence, as it is triggered by environmental stress. Envisioning a better understanding of this trait, a presumed genotoxic effect associated with the overproduction of riboflavin was investigated. For assessing that, the Ashbya Comet Assay was developed, which was able to reproducibly measure DNA damage. Using this protocol, exposure to sun-mimicking light during growth was found to significantly increase DNA damage in riboflavin-overproducing cells, but not in non-overproducing ones. In overproducing cells, light induced the intracellular accumulation of reactive oxygen species and increased the production of riboflavin 1.5-fold. These results draw attention for the importance of controlling the exposure to light of riboflavin biotechnological processes. Proceeding with the influence of riboflavin overproduction on the global metabolism of A. gossypii, the molecular basis for the unusual sensitivity to uracil displayed by the A. gossypii Agura3 pyrimidine auxotroph was identified. Uracil phosphoribosyltransferase (UPRTp) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRTp, produced and purified from Escherichia coli, revealed that uracil concentrations above 1 mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Decreased UPRT activity ultimately favours the preservation of PRPP, which may be directed to promote riboflavin production. Thus, this UPRTp modulation reveals a putative means of saving precursors essential for riboflavin overproduction. The A. gossypii Agura3 mutant, which displays a riboflavin-overproducing phenotype, was further identified as a suitable host for the production of a novel high-value compound, orotic acid. This strain was found to accumulate and excrete orotic acid to culture supernatants. Evidences are discussed indicating that the metabolic feature crucial for this phenotype is a putative mitochondrial dihydroorotate dehydrogenase encoded by AgURA9. Further engineering of this strain with the overexpression of a gene involved in glycerol metabolism (AgGUP1) improved its glycerol consumption profile, as well as its glycerol-dependent hyperosmotic tolerance. Thus, a flexible and robust strain for the production of high-value chemicals was constructed, which is able to sustain their production from a medium completely based on industrial by-products. Finally, an alternative production process for ?-lactones is presented. The biotechnological exploration of these valuable fragrances relied solely in biotransformation processes, which limited their production from more sustainable substrates. Here, the innate ability of A. gossypii for de novo biosynthesis of ?-lactones from glucose was uncovered and improved. Characterization of nine strains of this fungus revealed the presence of seven chemically different ?-lactones. To understand this biosynthetic process, metabolic engineering strategies were applied to the fatty acid biosynthesis and the ?-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid into linoleic acid, and deletion of AgELO624, which encodes an elongase that catalyzes the formation of longer fatty acids increased the production of ?-lactones up to 6.4-fold. Further substitution of AgPOX1 by a codon-optimized POX2 gene from Yarrowia lipolytica fine-tuned the biosynthesis of ?-decalactone. By means of metabolic engineering, this study elucidated key steps in the biosynthesis of these compounds, and demonstrated the potential of A. gossypii as a model and future platform for the de novo biosynthesis of ?-lactones. In sum, this thesis contributed to the goal of establishing A. gossypii as an alternative cell factory by disclosing two novel biotechnological applications that emerged from the exploration and understanding of this fungus natural metabolism.
DP_AEM External Thesis Comitee – Miguel Teixeira (IST)
More details in:
Papers in international peer-reviewed journals (during DP_AEM )
· Díaz-Fernández D, Aguiar TQ, Martín VI, Romaní A, Silva R, Domingues L, Revuelta JL, Jiménez A, 2019. Microbial lipids from industrial wastes using xylose-utilizing Ashbya gossypii strains. Bioresource Technology 293:122054. https://doi.org/10.1016/j.biortech.2019.122054
· Silva R, Aguiar TQ, Coelho E, Jiménez A, Revuelta JL, Domingues L, 2019. Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of ?-lactones. Microbial Cell Factories 18:62. https://doi.org/10.1186/s12934-019-1113-1
· Silva R*, Aguiar TQ*, Oliveira C, Domingues L, 2019. Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii. New Biotechnology 50:1-8. https://doi.org/10.1016/j.nbt.2018.12.004
· Silva R, Aguiar TQ, Oliveira R, Domingues L, 2019. Light exposure during growth increases riboflavin production, ROS accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains. FEMS Yeast Research 19:foy114. https://doi.org/10.1093/femsyr/foy114
· Aguiar TQ, Silva R, Domingues L, 2017. New biotechnological applications for Ashbya gossypii: Challenges and perspectives. Bioengineered 8:309–15. https://doi.org/10.1080/21655979.2016.1234543
Other papers in international peer-reviewed journals
· Aguiar TQ*, Silva R*, Domingues L, 2015. Ashbya gossypii beyond industrial riboflavin production: a historical perspective and emerging biotechnological applications. Biotechnology Advances 33:1774–1786. . https://doi.org/10.1016/j.biotechadv.2015.10.001
· Silva R*, Aguiar TQ*, Domingues L, 2015. Blockage of the pyrimidine biosynthetic pathway affects riboflavin production in Ashbya gossypii. Journal of Biotechnology 193:37–40. https://doi.org/10.1016/j.jbiotec.2014.11.009
More details in:
· Silva R, Aguiar TQ, Domingues L, 2019. “Towards the valorization of crude glycerol to high-value chemicals by engineered Ashbya gossypii” 7thConference on Physiology of Yeasts and Filamentous Fungi (PYFF7) – Book of Abstracts, 67. Milan, Italy, June 24-27. http://hdl.handle.net/1822/60772
· Silva R, Aguiar TQ, Oliveira R, Domingues L, 2017. “Ashbya gossypii riboflavin overproducing strains are highly susceptible to light-induced oxidative DNA damage” MICROBIOTEC’17 - Book of Abstracts, 61. Porto, Portugal, December 7-9. http://hdl.handle.net/1822/48498
· Silva R, Aguiar TQ, Domingues L, 2016. “Engineering osmotic stress tolerance in the riboflavin producer Ashbya gossypii” BIOIBEROAMÉRICA 2016 - Book of Abstracts, 494. Salamanca, Spain, June 5-8. http://hdl.handle.net/1822/42088
Oral communications by invitation
· Silva R, 2020. “Diversifying beyond Saccharomyces cerevisiae: the engineering potential of the filamentous fungus Ashbya gossypii for biomass-based products and energy” 1st Workshop of BBRI – Biomass and Bioenergy Research Infrastructure. Lisbon, Portugal, February 19.
· Silva R, 2020. “The filamentous fungus Ashbya gossypii as an alternative cell factory” 8th Workshop of the Doctoral Program in Applied and Environmental Microbiology. Braga, Portugal, February 11.
· Silva R, Coelho E, Aguiar TQ, Jiménez A, Revuelta JL, Domingues L, 2018. “Volatile compounds’ profiling and engineering of Ashbya gossypiistrains for de novo biosynthesis of lactones” Metabolic Engineering 12 – Book of Abstracts, 85. Munich, Germany, June 24-28. http://hdl.handle.net/1822/58854
· Aguiar TQ, Silva R, Oliveira C, Domingues L, 2017. “Substrate inhibition of uracil phosphoribosyltransferase (UPRT) in distinct microbial species and its implications in the phenotype of uracil auxotrophs” MICROBIOTEC’17 - Book of Abstracts, 437. Porto, Portugal, December 7-9. http://hdl.handle.net/1822/48497
· Silva R, Oliveira R, Aguiar TQ, Domingues L, 2017. “The Ashbya gossypii Comet Assay: measuring oxidative and non-oxidative damage in the DNA” 30 years Molecular Biology of Ashbya gossypii. Basel, Switzerland, April 28. http://hdl.handle.net/1822/56493
· Silva R, Aguiar TQ, Domingues L, 2017. “Deciphering the uracil growth sensitivity of Ashbya gossypii pyrimidine auxotrophs” 30 years Molecular Biology of Ashbya gossypii. Basel, Switzerland, April 28. http://hdl.handle.net/1822/56492
· Silva R, Aguiar TQ, Domingues L, 2016. “Why does uridine supplementation properly rescues the growth of Ashbya gossypii uracil auxotrophs, but not uracil?” XIX National Congress of Biochemistry. Guimarães, Portugal, December 8-10. http://hdl.handle.net/1822/43666
· Silva R, Aguiar TQ, Domingues L, 2016. “Analysis of the sensitivity of different fungal species to uracil” PYFF6 - 6th Conference on Physiology of Yeast and Filamentous Fungi (Programme & Abstract Book). No. P96, 173. Lisbon, Portugal, July 11-14. http://hdl.handle.net/1822/42683
Departamento de Microbiología y Genética, University of Salamanca – Salamanca, Portugal (May 2017 - July 2017)
Scientific short-term mission during the PhD: Construction of engineered strains for the de novo biosynthesis of lactones.
Communication Skills DP_AEM/Ana Salgado/AScience (course level 1), 2016 Braga, Portugal, September 30.
Communication Skills DP_AEM/Ana Salgado/AScience (course level 2), 2017. Braga, Portugal, February – July.
PhD Thesis concluded in 16/09/2019 - Approved, with the final grade of Very Good (http://hdl.handle.net/1822/66077)
Rui Silva holds a PhD degree in Chemical and Biological Engineering (University of Minho - 2019). He worked several years as Scientist and has R&D expertise in Biotechnology, Microbiology and Molecular Biology as well as in laboratory management (Associate Director at https://twitter.com/LBMlab). His research focused on the development of microbial cell factories using metabolic and genetic engineering approaches for the production of high-value chemicals by fermentation. In this role, Rui was author of 8 scientific articles in international peer-reviewed journals, dozens of oral and written communications in scientific conferences and directly contributed to raise ~250k € for research projects.
Currently, Rui is an Innovation Consultant at Innovayt A/S helping companies and public organisations to develop and fund their research and innovation projects.
More details in: