Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities

Abstract

Producing cellulosic biofuels from plant material has recently emerged as a key US Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using Kristen M. DeAngelis and John M. Gladden contributed equally to this manuscript. Bioenerg. Res. (2010) 3:146-158 DOI 10.1007 SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest. Electronic supplementary material

Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass

“…Adaptive cultivation on other feedstocks, such as lignin, cellulose, and CMC, has also generated simplified communities and, along with this study, indicates that this method is a useful tool for developing simplified biomass-degrading consortia tailored to deconstruct a designated feedstock under defined conditions, such as temperature or pH (6,22,26).…”

“…DNA was isolated from the pellets generated during isolation of culture supernatants described above. DNA isolation and sequencing were performed as previously described (6). Sequencing tags were quality trimmed and analyzed using the pyroclust version of the software tool PyroTagger (http://pyrotagger.jgi-psf.org) with a 220-bp sequence length threshold and an accuracy of 10% for low-quality bases (9,19).…”

“…), which is often based on homology to known enzymes (28). The complexity of microbial communities in environmental samples also hampers bioinformatic enzyme discovery efforts, because it often prevents assembly of full-length genes from metagenomic databases (1,6). Enrichment cultures on lignocellulosic biomass provide a method to reduce the complexity of the microbial communities and provide more tractable samples for detailed investigations.…”

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts

“…Thus, engineered culture conditions are used to enrich for desirable traits and lead to the discovery of functional microbial consortia or specific microorganisms. For example, starting with microbial communities collected from compost and soil environments, a selection of simplified communities have been found that can deconstruct lignocellulosic biomass in extreme conditions, such as high temperature, high-solid loading, and low moisture (DeAngelis et al 2010;Jimenez et al 2014;Reddy et al 2013;Simmons et al 2014;Yu et al 2015). The enrichment process generates less complex lignocellulolytic microbial communities that can facilitate targeted discovery of potential enzymes and microorganisms for biomass deconstruction .…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…Attempts to address the various limitations of functional metagenomics include selective enrichment of the uncultured microbial communities prior to construction of metagenomic [59,109,[127][128][129][130][131] to improve functional expression of heterologous genes. Recently, metagenomic libraries from thermophilic habitats were successfully screened for novel esterases using the bacterium Thermus thermophilus as host [132].…”