Creating a more robust 5-hydroxymethylfurfural oxidase by combining computational predictions with a novel effective library design

Abstract

BackgroundHMF oxidase (HMFO) from Methylovorus sp. is a recently characterized flavoprotein oxidase. HMFO is a remarkable enzyme as it is able to oxidize 5-hydroxymethylfurfural (HMF) into 2,5-furandicarboxylic acid (FDCA): a catalytic cascade of three oxidation steps. Because HMF can be formed from fructose or other sugars and FDCA is a polymer building block, this enzyme has gained interest as an industrially relevant biocatalyst.ResultsTo increase the robustness of HMFO, a requirement for biotechnological applications, we decided to enhance its thermostability using the recently developed FRESCO method: a computational approach to identify thermostabilizing mutations in a protein structure. To make this approach even more effective, we now developed a new and facile gene shuffling approach to rapidly combine stabilizing mutations in a one-pot reaction. This allowed the identification of the optimal combination of seven beneficial mutations. The created thermostable HMFO mutant was further studied as a biocatalyst for the production of FDCA from HMF and was shown to perform significantly better than the original HMFO.ConclusionsThe described new gene shuffling approach quickly discriminates stable and active multi-site variants. This makes it a very useful addition to FRESCO. The resulting thermostable HMFO variant tolerates the presence of cosolvents and also remained thermotolerant after introduction of additional mutations aimed at improving the catalytic activity. Due to its stability and catalytic efficiency, the final HMFO variant appears to be a promising candidate for industrial scale production of FDCA from HMF.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1051-x) contains supplementary material, which is available to authorized users.

Stabilization of cyclohexanone monooxygenase by computational and experimental library design

“…We previously created a combinatorial library of wild‐type and mutant residues by golden gate shuffling using synthetic genes (Martin et al, ). To explore a method that avoids DNA synthesis, we now applied a PCR‐ and Gibson assembly‐based method.…”

“…After an in silico screening step supported by molecular dynamics simulations, selected mutants are expressed and tested for improved stability, and successful hits are combined to a final stabilized mutant. So far, we applied the FRESCO procedure to five enzymes, and achieved considerable improvements, with T m increases up to 35°C (Arabnejad et al, ; Floor et al, ; Martin, Ovalle Maqueo, Wijma, & Fraaije, ; Wijma et al, ; Wu et al, ).…”

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

“…(EC 1.1.3.13), glucose oxidase form Aspergillus niger (EC 1.1.3.4), and choline oxidase wild-type and an engineered choline oxidase variant (AcCO6) from Arthrobacter chlorophenolicus. [16,17,[22][23][24] Except for (AcCO6), all these oxidases did not convert 4 same reaction conditions as with AOX*. Furthermore, AcCO6 was found to suffer from severe substrate inhibition.…”

“…This FAD-containing biocatalyst can oxidize 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid in three consecutive oxidations. [16,17] Another recent demonstration of the biocatalytic potential of a flavoprotein alcohol oxidase was recently reported by Turner and co-workers. They showed that choline oxidase can be engineered towards a wider substrate acceptance, for the selective oxidation of primary alcohols to aldehydes.…”

An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols

“…Recently, however, alcohol oxidases have begun to attract more attention. A fungal alcohol oxidase was engineered towards activity with glycerol, and 5‐hydroxymethylfurfural oxidase (HMF oxidase) has been engineered for enhanced catalytic activity, improved overoxidation to the acid, thermostability and activity (albeit low) towards a secondary alcohol …”