1^st Speaker: Mengistu Fentahun Mekureyaw (Postdoc, Agro-Biotechnology Science Group)

Title: Laccase catalyzed defluorination of Per- and polyfluoroalkyl substances (PFAS)

Abstract: Per- and polyfluoroalkyl substances (PFAS) are heterogenous and synthetic organic compounds with multiple chemically inert and almost unassailable C-F bonds. They have wide applications with extreme persistence in the environment with strong resistance to common biological and chemical degradation. Natural enzyme-catalyzed oxidative humification reactions (ECOHR) are known to degrade persistent pollutants, including PAH and pesticides; however, due to their remarkable stability, PFAS takes a very long time for an effective degradation. Laccases, multi-copper oxidases which use molecular oxygen (O₂) for their activity showed potential signs of PFAS degradation. We tested a few commercially available enzymes and cloned enzymes based on reported genes for PFAS degradation. From our preliminary reactions laccase from Agaricus bisporus showed decreasing of PFOA concentration in mass spectrometry and F^‒ signal along with reaction time. Also, laccase from Aspergillus sp. showed a clear substrate specificity, which is more active for longer chain PFAS than the shorter ones. But all these reactions were relatively slower because different reasons, low activity of native laccases, low redox potential, narrow substrate scope towards short-chain PFAS constrains their capacity to complete defluorination. Now we are planning and working on engineering of two representative laccases from Agaricus bisporus and Aspergillus laccases for improving their expression, activity, redox potential, and catalytic activity.

2^nd Speaker: Viswanada Reddy Bysani Kondagari (Postdoc, Agro-Biotechnology Science Group)

Title: Engineering C1-fixation in yeast - aiming to achieve the established C₁-Cn biopath

Abstract: Using captured CO₂ and C₁-feedstocks like formate and methanol derived from electrochemical activation of CO₂ are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO₂-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO₂ assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO₂. ALE improved the RGP-dependent growth by 8-fold. ¹³C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we re-established formate and CO₂-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more ¹³C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO₂ assimilation. Here at Aarhus University, we aim to construct yeast strains capable of operating CO₂ fixation/assimilation via CBB, rTCA, and CETCH cycles while deriving the energy from another methanol to meet the energy demand. Further, the C1-fixing strain will used as a platform strain for the alkane/alkenes (Cn) compounds.