Spheroplasts for membrane-bound catalysts Francesca Paradisi University in Bern, Switzerland
Squalene-hopene cyclases (SHCs) are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale [1, 2]. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability [3-5]. Here we present an unexplored strategy for biocatalysis: the application of SHC spheroplasts [6]. By removing the outer cell membrane, we produced stable and substrate-accessible biocatalysts. SHC spheroplasts exhibited up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability was also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a new concept for the carrier-free immobilization of spheroplasts via crosslinking, CLS (crosslinked spheroplasts). The CLS maintained the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the SHCs, but also to make biocatalytic processes even greener and more cost-efficient. References 1. Eichenberger, M. et al. Asymmetric Cation-Olefin Monocyclization by Engineered Squalene–Hopene Cyclases. Angew. Chemie - Int. Ed.60, 26080–26086 (2021). 2. Schneider, A., Jegl, P. & Hauer, B. Stereoselective Directed Cationic Cascades Enabled by Molecular Anchoring in Terpene Cyclases. Angew. Chemie - Int. Ed. 60, 13251–13256 (2021). 3. Eichhorn, E. et al. Biocatalytic Process for (-)-Ambrox Production Using Squalene Hopene Cyclase. Adv. Synth. Catal. 350, 2339–2351 (2018). 4. Julsing, M. K. et al. Outer membrane protein alkL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli. Appl. Environ. Microbiol. 78, 5724–5733 (2012). 5. Gu, Y. et al. Directed Evolution of Artificial Metalloenzymes in Whole Cells. Angew. Chemie - Int. Ed. 61, (2022). 6. Benitez-Mateos, A. I. et al., Spheroplasts preparation boosts the catalytic potential of a squalene-hopene cyclase, ChemRxiv (2022). doi:10.26434/chemrxiv-2022-w08d0 preprint.
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© The Author(s), 2022
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