Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium

Abstract

Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial challenging economic factors. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge enrichment strategies can be applied to develop microbial communities with an efficient CAZymes arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of ERAC disclosed 41 metagenome-assembled genomes (MAGs), harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The majority of the CAZymes predicted (60 % of the total) shared less than 90 % amino acid identity compared to sequences found in public databases. Additionally, a clostridial MAG identified in this study produces proteins during consortia development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism. IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described herein represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharides degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied towards lignocellulose-based biorefineries.

Comparative genomic analysis ofPlanctomycetotapotential towards complex polysaccharide degradation identifies phylogenetically distinct groups of biotechnologically relevant microbes

“…At present, there is a viable interest in understanding how microorganisms employ different strategies for complex polysaccharide degradation to reinforce the current solutions or develop new industrial technologies [52, 60, 98]. For example, CAZyme gene clusters allow for the coordinated production of multiple, often synergistically acting enzymes [51, 99].…”

An insight into cellulolytic capacity of the Trichoderma harzianum P49P11 revealed by omics approaches

“…For carbohydrate-active enzymes (CAZyme) prediction, all genomic proteins in the T. harzianum P49P11 genome were screened for homologs in the dbCAN version 4 database (YIN, et al, 2012) using the HMMER 3.1b package [44] as previously described [45]. To reduce a single proteins sequence with multiple annotations, we manually analyzed the putative CAZymes using PRIAM [46] and Pfam databases, and then best blast hits were used as possible enzymatic activities.…”

A Review on Bacterial Contribution to Lignocellulose Breakdown into Useful Bio-Products

“…Microbial communities generally consist of organisms that produce various enzymes that specifically breakdown certain substrates. This pack of enzymes are useful in lignocellulose driven bio-refineries [ 48 ]. Biological enzymes are an economical, green, more efficient option which uses little energy in the production process.…”

“…These arise from a bio-refinery which is a platform that employs the use of biomass to produces bioenergy [ 45 ]. The saccharification of lignocellulose biomass leads to a release of sugars which can be used for downstream production of useful products [ 48 ]. Biogas, bioethanol and biodiesel are the major products from lignocellulose biomass after various reactions have occurred.…”