Biodegradation of Polyhydroxybutyrate by Bacterial Strains, Native Extracellular PHB Depolymerases, and Structural Variants

Abstract

Thanks to its biodegradability and biocompatibility, among other properties, the biodegradable polymer polyhydroxybutyrate (PHB) has the potential to become an extensively used plastic in the production of a variety of products, from polymer films and everyday objects to specialized devices like biosensors. Extracellular PHB depolymerases (PhaZs) — enzymes responsible for the biodegradation of this polymer — play a crucial role in understanding the life cycle of PHB and developing new PHB-based applications. Even though these enzymes have been investigated since the 1960s, new discoveries in this field continue to improve knowledge of PhaZs, their mechanisms and their properties. In addition, the tools provided by molecular and synthetic biology open the door for new applications and new enzymatic features. This dissertation presents advances in the understanding of PhaZs and their degradation of PHB that can lead to the development of PHB and PhaZs-based applications.Rigorous comparisons of PHB degradation by nine bacterial strains with either demonstrated or predicted PhaZ activity were performed through bioinformatics and experimental approaches. This enabled direct assessments, which were previously not possible from literature as correlations between different studies conducted under different conditions are, at the very least, very difficult; and demonstrated that at least one predicted strain could perform PHB degradation. The observations obtained can provide a starting point for applications such as PHB recycling.Next, a rationalized method to produce recombinant PhaZs was presented. This approach was demonstrated with five purified PhaZs (and four PhaZ variants in a subsequent study). Through a simple and rapid PHB plate-based method, the activity of five recombinant PhaZs was rapidly compared at different temperatures and pH values. These methods can be used to produce and test PhaZs of native or synthetic origin. The activity of a PhaZ from the marine strain Marinobacter algicola DG893 (PhaZMal) was then experimentally validated and characterized, adding a new entry to the PhaZs found in literature. This enzyme is of interest as it is one of the few known PhaZs from marine bacteria, and because marine environments are specifically under threat due to the plastic accumulation crisis. In addition, comparisons were established with the well characterized PhaZ from the soil originating bacterium Comamonas testosteroni 31A (PhaZCte).PhaZCte was the focus of the final study presented in this dissertation due to its high activity and other attractive features. The structure of PhaZCte was modified through the removal or inclusion of domains and the fusion with protein sequences showing no affinity to PHB. This allowed the determination of the contribution of the domains to degradation, and of the robustness of this enzyme and its variants under several storage conditions. In addition, the demonstration that activity was retained when the enzyme is fused to the small outer capsid protein (Soc) from bacteriophage T4, shows potential for the integration of this enzyme in a bacteriophage expression system and in enzyme-responsive biosensors.