Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences

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.

Designed high-redox potential laccases exhibit high functional diversity

“…Furthermore, these methods usually implement only very few mutations in the active site and hence are limited in the number of variants they can generate with versatile reactivity profiles 18,41 . We recently used PROSS to generate several stable and functionally diverse VPs 25 . Here, we used a different approach for diversification, demonstrating that evolution-guided atomistic design methods 48 can be used to construct a repertoire of stable and diverse HRPLs using a two-step design protocol, again with a very low experimental effort.…”

“…Thus, PROSS and FuncLib are compatible and complementary methods that rationalize and dramatically accelerate many of the iterative steps that are necessary for enzyme optimization. We recently used PROSS to enable the functional expression of enzymes as complex as versatile peroxidases (VPs) in yeast 25 . This encouraged us to extend these methods to other ligninolytic oxidoreductases, with the ultimate aim of constructing an artificial yeast secretome for efficient lignin depolymerization 5 .…”

“…Computational design methodologies can be used as effective alternatives to iterative engineering approaches. The PROSS 20 and FuncLib 21 algorithms previously developed in our lab have been used to improve diverse proteins in one-shot design; that is, without iterating modeling, mutagenesis and screening [20][21][22][23][24][25] . Both algorithms use Rosetta atomistic design calculations and phylogenetic data to restrict design to structurally tolerated substitutions that are likely to occur in natural evolution.…”

Designed High-Redox Potential Laccases Exhibit High Functional Diversity

“…Computational design strategies can be used as effective alternatives to iterative engineering approaches or in combination with them. The PROSS and FuncLib algorithms previously developed in our lab have been used to improve diverse proteins in one-shot design; that is, without iterating modeling, mutagenesis, and screening. − Both algorithms use Rosetta atomistic design calculations and phylogenetic data to restrict design to structurally tolerated substitutions that are likely to occur in natural evolution. PROSS designs mutants that exhibit higher stability and expressibility; hence, design is allowed only outside the active sites. , By contrast, FuncLib aims to improve the catalytic activity or alter the substrate scope, and accordingly, the design process is limited to residues in the active site .…”

“…Thus, PROSS and FuncLib are compatible and complementary methods that rationalize and dramatically accelerate many of the iterative steps that are necessary for enzyme optimization. We recently used PROSS to enable the functional expression of enzymes as complex as versatile peroxidases (VPs) in yeast . This encouraged us to extend these methods to other ligninolytic oxidoreductases, with the ultimate aim of constructing an artificial yeast secretome for efficient lignin depolymerization …”