Rapid enzyme stabilization by computationally designed libraries of HMF oxidase
Caterina Martin1, Hein Weijma1,2, Marco W. Fraaije1,3*
1University of Groningen, Groningen, The Netherlands
HMF oxidase (HMFO) from Methylovorus sp. is a recently characterized flavoprotein oxidase 1. HMFO is able to oxidize 5-(hydroxymethyl)furfural (HMF) into 2,5-furandicarboxylic acid (FDCA). Because HMF can be formed from fructose or other sugars and FDCA is a polymer building block, the oxidase has attracted attention as industrially relevant biocatalyst. The dicarboxylic acid FDCA can be polymerized with ethylene glycol to produce polyethylene furanoate (PEF). This renewable and bio-based polyester can be a valid alternative to the petroleum-based polyethylene terephthalate (PET) thanks to its similar characteristics.
The first step to the development of an HMFO with improved catalytic properties is the engineering of the enzyme to enhance its thermostability using the recently developed FRESCO method 2.
FRESCO (Framework for Rapid Enzyme Stabilization by Computational libraries) is a computational approach to determine thermostabilizing point mutations in a protein structure. FRESCO has the potential to become a more valid alternative to random approaches like direct evolution when the protein structure is known. The first steps consist of in silico screening of single variants. After this screening the selected mutants are subject to experimental verification for improved TM and preserved catalytic activity. Finally, the combination of stabilizing mutations are combined with a novel Golden Gate based technique to lead to highly stabilized variants.
I will present the results obtained by using the FRESCO method combined with the new gene shuffling technique developed: the stability and activity profiles of the generated HMFO mutants 3'4.
References
(1) Dijkman, W. P., and Fraaije, M. W. (2014) Discovery and characterization of a 5-hydroxymethylfurfural oxidase from Methylovorus sp. strain MP688. Appl. Environ. Microbiol. 80, 1082–1090.
(2) Wijma, H. J., Floor, R. J., Jekel, P. A., Baker, D., Marrink, S. J., and Janssen, D. B. (2014) Computationally designed libraries for rapid enzyme stabilization. Protein Eng. Des. Sel. 27, 49–58.
(3) Martin, C., Maqueo, A. O., Wijma, H. J., and Fraaije, M. W. (2018) Biotechnology for Biofuels Creating a more robust 5 ‑ hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design 1–9.
(4) Fürst, M. J. L. J., Martin, C., Lončar, N., and Fraaije, M. W. (2018) Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins. Methods Enzymol. 608, 151–187.
