Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity.

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants.

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.