Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences.

Shiran Barber-Zucker,Jonathan J Weinstein,Miguel Alcalde,Sarel J Fleishman,Eva Garcia-Ruiz,Vladimir Mindel

doi:10.1021/jacs.1c12433

Abstract

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

Highlights

The need for developing economical and environmentally friendly energy sources is undeniable
We visually compared the models with the VP from Pleurotus eryngii (VPL) experimental structure finding that the models retained the intricate arrangement of amino acids in the hemebinding pocket and the ion-binding sites (Figure S1)
The DNA encoding each protein was codon-optimized for yeast expression, ordered as synthetic gene fragments that were incorporated into the pJRoC30

Summary

Introduction

The need for developing economical and environmentally friendly energy sources is undeniable. Lignocellulose, into biofuels is a promising route for sustainable and renewable energy production.[1] The amorphous and highly cross-linked structure of lignin, obstructs the accessibility of chemicals and enzymes to cellulose and impedes their conversion into biofuels and other high-value chemicals.[2−4] lignin itself comprises potentially valuable chemicals that could be valorized. The most efficient natural system for lignin depolymerization is observed in white-rot basidiomycetes These fungi secrete a repertoire of high-redox potential oxidoreductases (laccases and peroxidases) that degrade lignin synergistically.[6−8] Of these, versatile peroxidases (VPs; EC 1.11.1.16) are of particular interest for biotechnological use due to their broad substrate scope ranging from low- to high-redox potential substrates. Some fungal species secrete several VP paralogs, suggesting that VPs may act synergistically.[10,11] VPs are especially challenging for heterologous production, limiting their use in research, let alone as an enzyme repertoire or in industrial applications

Methods

Results

Conclusion