Identification and characterisation of lignin degrading bacteria and enzymes fromlarval guts of the African Palm Weevil (Rhynchophorus phoenicis)

Abstract

As developing economies continue to grow, the world’s demand for energy which currently stands at 84MB (million barrels oil) per day is projected to rise to 116 MB per day by the year 2030. The need to meet this continuous rise in demand for energy while lowering the emission of CO2 and other greenhouse gases has necessitated a shift in focus from the exploitation of fossil fuels which are limited, to more renewable and environmentally safe biological resources such as lignocellulosic biomass, the main structural components of plant cell walls. Lignocellulose, however, is resistant to degradation, thus there is a high cost and energy requirement associated with its pretreatment in order to access the lignin bound polysaccharides for subsequent hydrolysis, fermentation and conversion to biofuels and biomaterials.Xylophagous (wood-feeding) insects such as the African palm weevil (Rhynchophorus phoenicis) have developed the ability to effectively utilize lignocellulosic substrates as an energy source due to the synergistic association with their gut microbes. This makesthem viable resources to explore for novel lignocellulose degrading enzymes.Metagenomics allows access to the entire microbiome present in a particular environment and has been adopted in recent studies, rather than culture-based methods, thereby allowing for discovery of novel genes and enzymes from both culturable and non-culturable microbes.In this study, we carried out taxonomic profiling of the bacteria in the gut of the African palm weevil’s larvae using 16S rRNA gene amplicon sequencing with particular interest in identifying lignin degrading bacteria. We also performed functional metagenomicsanalysis from whole metagenome sequencing data derived from whole gut metagenomic DNA of APW larvae to identify genes and by extension, enzymes that can deconstruct lignocellulose and degrade its lignin component. The predominant bacterial genera found across all gut segments were Enterococcus, Lactococcus,Shimwellia, Lelliotia, Klebsiella and Enterobacter, with the foregut having the mostdiverse and abundant lignin degraders mostly from the Proteobacteria phylum. Onethousand, one hundred and forty-one (1,141) annotated genes identified from the R. phoenicis larval gut bacterial metagenome aligned with genes encoding CAZymes and 249 of these belonged to the “Auxiliary Activities” class which harbours the suite of genes implicated to play different roles in lignin deconstruction. Three genes of putative lignin degraders were successfully amplified by PCR, one of the three amplified genes B-38773 (encoding a putative deferrochelatase/ peroxidase of approximately 46kDa insize that has a conserved domain match to the dye decolourising peroxidase superfamily) was produced by heterologous expression and was found to exhibit activity on the peroxidase substrate ABTS, and the anthraquinone dye RB19 but no activity was observed with kraft lignin. Specific activity of 12.9Umg-1 at optimum temperature of 40°C and pH of 4 were recorded when B-38773 enzyme was assayed against ABTS as a substrate. Kinetic parameters: Vmax, Km, Kcat, and catalytic efficiency were determined to be 3.68 μMol/min, 1.089mM, 540.9S-1 and 4.96 X 105 M-1S-1respectively.This study elucidates the lignocellulose degrading potential of the gut community associated with the African palm weevil (APW) by robustly defining the bacterial community structure of the APW gut. Also, massive data from the metagenomic library generated will serve as a storehouse from where genes with various potential functions identified by the inhabitant gut bacteria can be harvested to contribute to areas of biotechnological relevance for industrial applications