Bioplastic-eating animals: Polyhydroxyalkanoate degrading enzymes in a chemosymbiotic worm

Abstract

Bacteria and halophilic archaea synthesize the bioplastic polyhydroxyalkanoate (PHA). PHA represents an important carbon and energy storage compound build up under nutrient limitation. PHA can make up to 90% of the microorganism’s dry weight. When growth conditions are lifted, the microorganisms degrade PHA into their monomers, dimers or a mix of oligomers. Microorganism will metabolize the resulting degradation products to yield CO2, CH4, H2O and energy. To degrade PHA, microorganisms, including bacteria, archaea, fungi and a few protist species, use a PHA depolymerase enzyme (PHAD). Until now, it was largely thought that animals were unable to produce the PHAD enzyme to breakdown PHA. In Chapter I of my dissertation, I identified the first animal PHAD in the gutless worm Olavius algarvensis. I characterized the enzyme structure, function and expression pattern. The host PHAD degrades extracellular PHA and expresses all genes needed to generate energy from PHA degradation, which likely indicates that the worm benefits from the PHA degradation. Surprisingly, I discovered that additional 67 metazoan species from nine distinct animal phyla encode for at least one PHAD. All of the animal species that encode for a PHAD access PHA through their diet. My in-depth analysis of the earthworm PHAD in Chapter III contradicted my hypothesis that animals encode for a PHAD to meet their nutritional requirements. Using immunohistochemistry assays, I found that the Lumbricus rubellus PHAD was localized in the epidermis. One possible explanation for my findings is that the PHAD degrades PHA of invading bacteria. Alternatively, earthworms might excrete their PHADs to their habitat to target extracellular PHA in the soil. Therefore, I predict that animal PHADs might have multiple benefits for metazoans. PHADs degrade PHA either intracellularly or extracellularly. Intracellular PHADs function on native PHA inside the cell. In contrast, extracellular PHADs function on denatured PHA that occurs outside the cell. The affinity of the PHAD for either intracellular or extracellular PHA is reflected in the enzymes structure. PHADs are typically classified based on sequence homology of the predicted protein. In Chapter II of my dissertation, I showed that the classification of PHADs from the gammaproteobacterial group Chromatiales is often misleading. One challenge is that there is less known about the protein structure of intracellular PHADs, which makes predicting the type of PHAD by sequence homology alone uncertain. Using AlphaFold2 to generate and compare PHAD models from multiple Chromatiales PHAD enzymes, I showed that true intracellular PHADs lack a signal peptide and have an altered substrate binding site. Enzyme assays on selected Chromatiales species, including Thiocapsa rosea, confirmed the initial hypothesis. Experimental evidence is thus crucial to reveal the true functions of PHADs. It is important to correctly classify the type of PHAD because it alters our interpretation of PHA degradation in natural ecosystems and consequently our understanding of carbon cycling. In conclusion, my dissertation adds new data to the field of PHA degradation. I revealed that experimental evidence is needed to classify PHADs. Additionally, I identified a novel group of PHADs that likely function after lysis of microbial species. Significantly, I showed that PHA degradation is not limited to bacteria, fungi, archaea, protists but that the ability to degrade PHA is widespread in animals.