Destructuring plant biomass: Focus on fungal and extremophilic cell wall hydrolases

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown

Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform “community-level” metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes. Here, we investigated the microbial degradation of wheat straw in liquid cultures that had been inoculated with wheat straw compost. Samples taken at selected time points were subjected to multi-omics analysis with the aim of identifying new microbial mechanisms for lignocellulose degradation that could be applied in industrial pre-treatment of feedstocks. Phylogenetic composition of the community, based on sequenced bacterial and eukaryotic ribosomal genes, showed a gradual decrease in complexity and diversity over time due to microbial enrichment. Taxonomic affiliation of bacterial species showed dominance of Bacteroidetes and Proteobacteria and high relative abundance of genera Asticcacaulis, Leadbetterella and Truepera. The eukaryotic members of the community were enriched in peritrich ciliates from genus Telotrochidium that thrived in the liquid cultures compared to fungal species that were present in low abundance. A targeted metasecretome approach combined with metatranscriptomics analysis, identified 1127 proteins and showed the presence of numerous carbohydrate-active enzymes extracted from the biomass-bound fractions and from the culture supernatant. This revealed a wide array of hydrolytic cellulases, hemicellulases and carbohydrate-binding modules involved in lignocellulose degradation. The expression of these activities correlated to the changes in the biomass composition observed by FTIR and ssNMR measurements. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics has enabled the identification of a large number of lignocellulose degrading enzymes that can now be further explored for the development of improved enzyme cocktails for the treatment of plant-based feedstocks. In addition to the expected carbohydrate-active enzymes, our studies reveal a large number of unknown proteins, some of which may play a crucial role in community-based lignocellulose degradation.

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Defining functional diversity for lignocellulose degradation in a microbial community using multi-omics studies.

Biocatalysts for biomass deconstruction from environmental genomics.

The stability of gut microbiota is essential for the host health. Parabacteroides spp., core members of the human gut microbiota, have average abundance of 1.27% in the human of 12 populations. Parabacteroides has been recently reported to have a close relationship with host health (E.g., metabolic syndrome, inflammatory bowel disease and obesity). Parabacteroides have the physiological characteristics of carbohydrate metabolism and secreting SCFAs. However, antimicrobial resistance of Parabacteroides to antibiotic (such as clindamycin, moxifloxacin and cefoxitin) should not be ignored. In this review, we primarily focused on Parabacteroides distasoniss, Parabacteroides goldsteinii, Parabacteroides johnsonii and Parabacteroides merdae and discussed their relationships with host disease, diet and the prevention or induction of diseases. P. distasonis and P. goldsteinii may be viewed as the potential next generation probiotics (NGP) candidate due to their protective effects on inflammation and obesity in mice. We also discussed the potential therapeutic application of Parabacteroides spp. in maintaining host-intestine homeostasis.

Roles of intestinal Parabacteroides in human health and diseases.

Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this “genomic dark matter.” Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as “genomic dark matter” and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. Biotechnol. Bioeng. 2014;111: 1550–1565. © 2014 Wiley Periodicals, Inc.

Changbin Du

Papers

Identification of novel biomass‐degrading enzymes from genomic dark matter: Populating genomic sequence space with functional annotation