UC Davis UC Davis Previously Published Works Title Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities Permalink https://escholarship.org/uc/item/5bh2q8rd Journal BioEnergy Research, 3(2) ISSN 1939-1242 Authors DeAngelis, Kristen M. Gladden, John M. Allgaier, Martin et al. Publication Date 2010-06-01 DOI 10.1007/s12155-010-9089-z Peer reviewed eScholarship.org Powered by the California Digital Library University of California Bioenerg. Res. (2010) 3:146–158 DOI 10.1007/s12155-010-9089-z Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities Kristen M. DeAngelis & John M. Gladden & Martin Allgaier & Patrik D’haeseleer & Julian L. Fortney & Amitha Reddy & Philip Hugenholtz & Steven W. Singer & Jean S. Vander Gheynst & Whendee L. Silver & Blake A. Simmons & Terry C. Hazen Published online: 30 March 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Producing cellulosic biofuels from plant material are often too complex for enzyme discovery using current has recently emerged as a key US Department of Energy metagenomic sequencing technologies. One potential strat- goal. For this technology to be commercially viable on a egy to overcome this problem is to selectively cultivate the large scale, it is critical to make production cost efficient by microbial communities from these complex ecosystems on streamlining both the deconstruction of lignocellulosic biomass under defined conditions, generating less complex biomass and fuel production. Many natural ecosystems biomass-degrading microbial populations. To test this efficiently degrade lignocellulosic biomass and harbor premise, we cultivated microbes from Puerto Rican soil or enzymes that, when identified, could be used to increase green waste compost under precisely defined conditions in the the efficiency of commercial biomass deconstruction. presence dried ground switchgrass (Panicum virgatum L.) or However, ecosystems most likely to yield relevant lignin, respectively, as the sole carbon source. Phylogenetic enzymes, such as tropical rain forest soil in Puerto Rico, profiling of the two feedstock-adapted communities using Kristen M. DeAngelis and John M. Gladden contributed equally to this manuscript. Electronic supplementary material The online version of this article (doi:10.1007/s12155-010-9089-z) contains supplementary material, which is available to authorized users. : K. M. DeAngelis : J. M. Gladden : M. Allgaier : P. D’haeseleer : A. Reddy J. S. Vander Gheynst J. L. Fortney : A. Reddy : P. Hugenholtz : S. W. Singer : Department of Biological and Agricultural Engineering, J. S. Vander Gheynst : B. A. Simmons : T. C. Hazen University of California, Microbial Communities Group, Deconstruction Division, Davis, CA, USA Joint BioEnergy Institute, Emeryville, CA, USA W. L. Silver Ecosystem Sciences, Policy and Management, K. M. DeAngelis : J. L. Fortney : S. W. Singer : W. L. Silver University of California, Earth Sciences Division, Berkeley, CA, USA Lawrence Berkeley National Lab, Berkeley, CA, USA B. A. Simmons Biomass Science and Conversion Technology Department, J. M. Gladden : P. D’haeseleer Sandia National Laboratory, Livermore, CA, USA Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, T. C. Hazen (*) Livermore, CA, USA Ecology Department, Earth Sciences Division, Lawrence Berkeley National Lab, M. Allgaier : P. Hugenholtz One Cyclotron Road MS 70A-3317, Joint Genome Institute, Berkeley, CA 94720, USA Walnut Creek, CA, USA e-mail: [email protected] Bioenerg. Res. (2010) 3:146–158 147 SSU rRNA gene amplicon pyrosequencing or phylogenetic Division, US DOE Joint BioEnergy Institute (JBEI), microarray analysis revealed that the adapted communities focuses on two natural biomass-degrading ecosystems: the were significantly simplified compared to the natural tropical forest soils of Puerto Rico and municipal green communities from which they were derived. Several mem- waste compost. Wet tropical forest soils are some of the bers of the lignin-adapted and switchgrass-adapted consortia most productive and diverse terrestrial ecosystems on earth. are related to organisms previously characterized as biomass A recent study identified tropical forest soils as the fastest degraders, while others were from less well-characterized decomposing soils of plant material compared to all other phyla. The decrease in complexity of these communities biomes globally [39]. Green waste compost is another make them good candidates for metagenomic sequencing ecosystem where microorganisms rapidly break down and will likely enable the reconstruction of a greater number lignocellulosic biomass into carbon dioxide, water, and of full-length genes, leading to the discovery of novel humus. This degradation is so fast, in fact, that the compost lignocellulose-degrading enzymes adapted to feedstocks heap can heat to 60–70°C, due to the metabolic activity of and conditions of interest. the microbial community. We are using metagenomics, proteomics, and transcriptomics to investigate these com- Keywords Lignocellulolytic . Enzymes . Metagenome . munities, both in their native state and after cultivation on Community. Rain forest . Compost . PhyloChip . Pyrotag candidate bioenergy feedstocks (Fig. 1). Identifying specific genes from these ecosystems, which have a high degree of microbial diversity, is challenging. Introduction Fortunately, next-generation sequencing technologies such as 454 pyrosequencing can facilitate the discovery of The US Department of Energy has recently made alterna- relevant genes [30]. Recent metagenome studies have tive liquid fuel production from lignocellulosic biomass a demonstrated that it is possible to assign functional primary goal. Establishing such renewable, low-carbon annotations to partial gene sequences from shotgun se- liquid fuel alternatives is a critical short- and long-term quence reads with a reasonable degree of accuracy, based solution to the environmental problems and national on BLASTX hits against reference databases [38, 45]. Such security risks associated with petroleum consumption. annotation can provide a useful functional profile of a Cellulosic biofuels are one such alternative that are community and help identify gene categories of interest. receiving unprecedented international attention, owing to However, this study and others [1] indicate that shotgun the large, underutilized reservoir of renewable energy in metagenome sequence data from highly complex natural plant biomass [10, 30]. Currently, one of the major barriers microbial communities is of limited use for targeted to the large-scale production of inexpensive cellulosic enzyme discovery, because of the lack of contiguous biofuels is the ability to efficiently deconstruct biomass sequences (contigs) large enough to contain complete open into fermentable carbon sources, such as glucose and reading frames (ORFs); for cellulases, this is at least 1 kb xylose. Enzymatic saccharification of the plant cell poly- [33]. For example, Allgaier et al. [1] found only 25 mers cellulose and hemicellulose is an efficient method to potentially full-length lignocellulose-degrading enzymes obtain these sugars from biomass, but this process is costly from a switchgrass compost microbial community. To using present-day fungal commercial enzyme cocktails. discover and extract a greater number of novel functional Discovery of more efficient and robust biomass-degrading enzymes for bioenergy applications, lower complexity enzymes will drive down costs and increase the economic metagenomic data sets that are more amenable to assembly viability of this technology. are required. One possible method for generating less Many natural ecosystems, such as soils and compost, complex microbial communities is to adapt environmental almost completely mineralize plant biomass. The indige- communities to feedstocks (e.g., switchgrass, or lignin) nous microbes in these ecosystems may provide a rich under fixed conditions, such as temperature, pH, etc. that reservoir of genes relevant to the development of cellulosic are more directly relevant to the feedstocks and process biofuels. Target genes include glycosyl hydrolases, conditions expected for large-scale industrial biomass enzymes that convert simple sugar intermediates into deconstruction, with the expectation that the resulting biofuels [30], and lignolytic enzymes that can either release communities will be enriched for microbes expressing cellulose from the plant polymer lignin to increase sugar desired enzymes. yields from biomass, or facilitate lignin transformation to In this manuscript, we present the results of a shotgun biobased products. Lignin is of special interest, since sequencing by 454 Titanium technology of tropical forest currently it is a waste stream in cellulosic biofuels soils, which were sufficiently complex to resist assembly of production that is burned to recover heat [24]. Our research, full-length genes. While we present analysis of known within the Microbial Communities Group, Deconstruction lignocellulolytic enzymes and the prospects for enzyme 148 Bioenerg. Res. (2010) 3:146–158 Fig. 1 Schematic structure of our approach to metabolic anal- Novel Deconstruction ysis of complex microbial com- Enzymes & Pathways munities. Starting with natural Whole Genome samples at the bottom, a series Sequencing of analytical, biochemical,