Functional, structural characterizations and biotechnological application of lytic polysaccharide monooxygenases from the lower termite Coptotermes gestroi

Abstract

Termites are social insects that live in colonies dividing the labor between castes. Generally, these insects are decomposers and their diet is mainly based on lignocellulosic compounds. The worker caste is responsible for the degradation of cellulose and hemicellulose, and this process can reach about 75-90% of efficiency. Moreover, the lignin is partially degraded or modified during the digestion. The lignocellulose breakdown occurs in the termite gut throughout the secretion of Carbohydrate-Active Enzymes (CAZymes), which includes the glycoside hydrolases, such as cellulases and hemicellulases. These enzymes are endogenously secreted by the termite in the foregut and midgut, and by the symbionts in the hindgut, such as protist and bacteria. Based on recent literature, along with studies of our group, the catalytic diversity of termite and symbiotic glycoside hydrolases is not sufficient to explain the high efficiency in the degradation of recalcitrant lignocelluloses by termites, which is the main urban pest in São Paulo state. In this context, the literature and results from our research group suggested that oxidative mechanisms might be involved in termite gut, adding hydrolytic enzymes for the degradation of the lignocelluloses. Unpublished data from our group revealed the presence of two genes in the genome of the lower termite Coptotermes gestroi encoding two proteins, described as hypothetical, which contains a chitin binding domain (PFAM 03067), recently re-classified as "lytic polysaccharide monooxygenase from family 10" or LPMO_10. This protein domain encodes oxidative metalloenzymes know as Lytic Polysaccharides Monooxygenases (LPMOs), which cleave cellulose/hemicellulose by oxidation rather than hydrolysis. This class of enzyme has recently become the focus of academic and industrial research, due to their biotechnological importance. However, functional characterizations were carried out only with enzymes of bacterial origin, and anything is known for LMPOs from animal origin. According to the Pfam database, there are over 200 orthologous putative LMPOs distributed through 44 organisms in Metazoa kingdom, especially from the phylum Arthropoda and Insecta class. Thus, this proposal aims to investigate the protein domain LPMO_10 in termites and in Metazoa kingdom, especially in the phylum Arthropoda, emphasizing the role of these enzymes play in the digestive physiology of C. gestroi. To achieve these objetives, the genes of C. gestroi CGAA10-1 and CGAA10-2, and 6 more orthologous genes in Metazoa kingdom will be subjected to codon optimization and in-vitro synthesis for heterologous expression in E. coli. The purified proteins will be subjected to functional characterization seeking to correlate its enzymatic activity with the evolution of specificity for substrates in LPMOs. In addition, comparative structural studies will be carried out with between CgAA10-1 or CgAA10-2 with AA10 from bacteria available in the PDB database. Thus, it is expected that this study present a new class of enzyme, provide the basis to open a new group of LPMOs from Metazoa origin. Moreover, the potential biotechnological applications for these LPMOs will be tested, including supplementation of commercial cellulolytic cocktails, since LPMOs are described by increasing the saccharification of lignocellulosic material in about 40%. (AU)