Strategies to overcome industrial stress factors in engineered Saccharomyces Cerevisiae and impact on bioreactor fermentation

Student thesis: Doctoral Thesis

Abstract

Saccharomyces cerevisiae fermentation is a valuable process to produce industrially relevant compounds sustainably and cost-effectively. However, biomolecule production by fermentation is usually impaired by harsh environmental conditions that may lead to yeast stress resulting in stuck or sluggish fermentations. Thus, enhance yeast tolerance to environmental stresses through genetic engineering or culture media supplementation may improve biomolecule productivity. Accordingly, this thesis focuses on the study of solutions to overcome the impact of oxidative and ethanol stresses on β-farnesene producer yeast in bioreactor fermentation. Culture medium supplementation with antioxidant compounds is a strategy commonly applied to overcome yeast oxidative stress. Therefore, the impact of an antioxidant peptide extract (APE), obtained from industrial spent yeast, was evaluated in the fermentation of S. cerevisiae in the presence and absence of the oxidative stress inducer hydrogen peroxide (H2O2). Under 2 mM of H2O2 exposure, the addition of 0.7 g/L APE led to the reduction of intracellular reactive oxygen species (ROS) levels up to 3-fold in shake-flasks and 1.7-fold in batch bioreactor fermentations. As a result, cell density and biomolecule concentration were enhanced up to 2-fold and 2.8-fold in shake-flasks, while, in bioreactors were increased up to 1.5-fold and up to 1.6-fold, respectively. Culture medium supplementation with APE showed to be a promising strategy to overcome yeast oxidative stress and improve βfarnesene production, while allowing for the valorisation of biomass waste as a sustainable and eco-friendly solution for the biotechnology industry. Yeast stress tolerance is also often improved using genetic engineering. Since yeast membrane is the first target of ethanol toxicity, improving the synthesis of mono-unsaturated fatty acids can be a good strategy to overcome ethanol stress. The effect of overexpressing OLE1 and TniNPVE genes, coding homologous and insect delta-9 fatty acids desaturases, respectively, on oleic acid production was evaluated in strains modified with these genes under a range of different pGAL promoter strengths (pGAL3 << pGAL2 < pGAL10 < pGAL1). OLE1 overexpression with pGAL10 (pGAL10-OLE1) increased oleic acid content between 30.9% and 77.3% compared to control, while TniNPVE overexpression with pGAL2 (pGAL2-TniNPVE) enhanced oleic acid between 18.4 and 34.7% in shake-flasks fermentations. Additionally, pGAL10-OLE1 and pGAL2-TniNPVE grew faster than control strain with a max 6 to 7% higher and produced more 55% of -farnesene. Besides, pGAL10-OLE1 and pGAL2-TniNPVE were exposed to 12.5% and 15% ethanol at different stages of fermentation and demonstrated to be more sensitive to ethanol than the control strain. Therefore, the higher oleic acid levels obtained by genetic modification improved yeast growth and productivity but not its resistance to ethanol. To clarify if high levels of mono-unsaturated fatty acids content can positively affect yeast growth and productivity at a larger scale, fed-batch fermentations with pGAL10-OLE1 were conducted for 13 days in 2-L bioreactors. pGAL10-OLE1 presented increments up to 59.5% in oleic acid and 49.1% in palmitoleic acid relative to control. These changes in monounsaturated fatty acids content resulted in β-farnesene productivity improvements between 5.5% (day 13) and 21.2% (day 2). In conclusion, improve yeast tolerance to oxidative stress, by reducing ROS accumulation with APE medium supplementation, benefited yeast growth and productivity. In contrast, OLE1 overexpression did not improve yeast tolerance to ethanol. However, the increase in mono-unsaturated fatty acids content led to higher farnesene productivity, which can benefit the industrial production of the biomolecule.
Date of Award22 Mar 2024
Original languageEnglish
Awarding Institution
  • Universidade Católica Portuguesa
SupervisorCarla Oliveira (Supervisor), João Azevedo Silva (Co-Supervisor) & Erdem Carsanba (Co-Supervisor)

Keywords

  • Saccharomyces cerevisiae
  • Oxidative stress
  • Ethanol stress
  • Bioreactor fermentation
  • Lipids

Designation

  • Doutoramento em Biotecnologia

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