*Manuscript Click here to download Manuscript: JBEI_BioEnergyResearch_11march2010.doc 1 2 3 4 1 5 6 7 2 Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme 8 9 10 3 Discovery in Lignocellulolytic Microbial Communities 11 12 4 13 14 1,2,* 1,3,* 1,4 1,3 15 5 Kristen M. DeAngelis , John M.Gladden , Martin Allgaier , Patrik D’haeseleer , Julian 16 17 6 L. Fortney1,2, Amitha Reddy1,5, Philip Hugenholtz1,4, Steven W. Singer1,2, Jean S. Vander 18 19 1,5 2,6 1,7 1,2,6,+ 20 7 Gheynst , Whendee L. Silver , Blake A. Simmons , and Terry C. Hazen 21 22 8 23 24 1 25 9 Affiliations: Microbial Communities Group, Deconstruction Division, Joint BioEnergy Institute, 26 27 10 Emeryville CA; 2Earth Sciences Division, Lawrence Berkeley National Lab; 3Physical and Life 28 29 11 Sciences Directorate, Lawrence Livermore National Laboratory; 4Joint Genome Institute, Walnut 30 31 5 32 12 Creek, CA; Department of Biological and Agricultural Engineering, University of California, 33 34 13 Davis; 6Ecosystem Sciences, Policy and Management, University of California, Berkeley; 35 36 7 37 14 Biomass Science and Conversion Technology Department, Sandia National Laboratory, 38 39 15 Livermore, CA 40 41 * 42 16 These authors contributed equally to this manuscript. 43 44 17 +Corresponding author: Ecology Department, Earth Sciences Division, Lawrence Berkeley 45 46 47 18 National Lab, One Cyclotron Road MS 70A-3317, Berkeley CA 94720. Tel. 510 486 6223; fax 48 49 19 510 486 7152; email [email protected] 50 51 20 52 53 54 55 22 56 57 58 59 23 60 61 62 Enrichment Cultures for Enzyme Discovery, page 1 of 31 63 64 65 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California. Ernest Orlando Lawrence Berkeley National Laboratory is an equal opportunity employer. 1 2 3 4 24 Abstract 5 6 7 25 8 9 26 Producing cellulosic biofuels from plant material has recently emerged as a key U.S. Department 10 11 12 27 of Energy goal. For this technology to be commercially viable on a large scale, it is critical to 13 14 28 make production cost efficient by streamlining both the deconstruction of lignocellulosic 15 16 29 biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic 17 18 19 30 biomass and harbor enzymes that, when identified, could be used to increase the efficiency of 20 21 31 commercial biomass deconstruction. However, ecosystems most likely to yield relevant 22 23 24 32 enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme 25 26 33 discovery using current metagenomic sequencing technologies. One potential strategy to 27 28 29 34 overcome this problem is to selectively cultivate the microbial communities from these complex 30 31 35 ecosystems on biomass under defined conditions, generating less complex biomass-degrading 32 33 34 36 microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or 35 36 37 green waste compost under precisely defined conditions in the presence dried ground switchgrass 37 38 38 (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling 39 40 41 39 of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or 42 43 40 phylogenetic microarray analysis revealed that the adapted communities were significantly 44 45 46 41 simplified compared to the natural communities from which they were derived. Several 47 48 42 members of the lignin-adapted and switchgrass-adapted consortia are related to organisms 49 50 51 43 previously characterized as biomass degraders, while others were from less well-characterized 52 53 44 phyla. The decrease in complexity of these communities make them good candidates for 54 55 45 metagenomic sequencing and will likely enable the reconstruction of a greater number of full- 56 57 58 59 60 61 62 Enrichment Cultures for Enzyme Discovery, page 2 of 31 63 64 65 1 2 3 4 46 length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to 5 6 7 47 feedstocks and conditions of interest. 8 9 48 10 11 12 49 Keywords Lignocellulolytic; enzymes; metagenome; community; rain forest; compost; 13 14 50 PhyloChip; pyrotag 15 16 51 17 18 19 52 20 21 53 Introduction 22 23 24 54 25 26 55 The US Department of Energy has recently made alternative liquid fuel production from 27 28 29 56 lignocellulosic biomass a primary goal. Establishing such renewable, low-carbon liquid fuel 30 31 57 alternatives is a critical short- and long-term solution to the environmental problems and national 32 33 34 58 security risks associated with petroleum consumption. Cellulosic biofuels are one such 35 36 59 alternative that are receiving unprecedented international attention, owing to the large, 37 38 60 underutilized reservoir of renewable energy in plant biomass [30, 10]. Currently, one of the 39 40 41 61 major barriers to the large-scale production of inexpensive cellulosic biofuels is the ability to 42 43 62 efficiently deconstruct biomass into fermentable carbon sources, such as glucose and xylose. 44 45 46 63 Enzymatic saccharification of the plant cell polymers cellulose and hemicellulose is an efficient 47 48 64 method to obtain these sugars from biomass, but this process is costly using present-day fungal 49 50 51 65 commercial enzyme cocktails. Discovery of more efficient and robust biomass-degrading 52 53 66 enzymes will drive down costs and increase the economic viability of this technology. 54 55 67 Many natural ecosystems, such as soils and compost, almost completely mineralize plant 56 57 58 68 biomass. The indigenous microbes in these ecosystems may provide a rich reservoir of genes 59 60 61 62 Enrichment Cultures for Enzyme Discovery, page 3 of 31 63 64 65 1 2 3 4 69 relevant to the development of cellulosic biofuels. Target genes include glycosyl hydrolases, 5 6 7 70 enzymes that convert simple sugar intermediates into biofuels [30], and lignolytic enzymes that 8 9 71 can either release cellulose from the plant polymer lignin to increase sugar yields from biomass, 10 11 12 72 or facilitate lignin transformation to biobased products. Lignin is of special interest, since 13 14 73 currently it is a waste stream in cellulosic biofuels production that is burned to recover heat [24]. 15 16 74 Our research, within the Microbial Communities Group, Deconstruction Division, U.S. DOE 17 18 19 75 Joint BioEnergy Institute (JBEI), focuses on two natural biomass-degrading ecosystems: the 20 21 76 tropical forest soils of Puerto Rico and municipal green waste compost. Wet tropical forest soils 22 23 24 77 are some of the most productive and diverse terrestrial ecosystems on earth. A recent study 25 26 78 identified tropical forest soils as the fastest decomposing soils of plant material compared to all 27 28 29 79 other biomes globally [39]. Green waste compost is another ecosystem where microorganisms 30 31 80 rapidly break down lignocellulosic biomass into carbon dioxide, water, and humus. This 32 33 34 81 degradation is so fast, in fact, that the compost heap can heat to 60–70°C, due to the metabolic 35 36 82 activity of the microbial community. We are using metagenomics, proteomics, and 37 38 83 transcriptomics to investigate these communities, both in their native state and after cultivation 39 40 41 84 on candidate bioenergy feedstocks (Fig. 1). 42 43 85 Identifying specific genes from these ecosystems, which have a high degree of microbial 44 45 46 86 diversity, is challenging. Fortunately, next-generation sequencing technologies such as 454 47 48 87 pyrosequencing can facilitate the discovery of relevant genes [30]. Recent metagenome studies 49 50 51 88 have demonstrated that it is possible to assign functional annotations to partial gene sequences 52 53 89 from shotgun sequence reads with a reasonable degree of accuracy, based on BLASTX hits 54 55 90 against reference databases [38, 45]. Such annotation can provide a useful functional profile of a 56 57 58 91 community and help identify gene categories of interest. However, this study and others [1] 59 60 61 62 Enrichment Cultures for Enzyme Discovery, page 4 of 31 63 64 65 1 2 3 4 92 indicate that shotgun metagenome sequence data from highly complex natural microbial 5 6 7 93 communities is of limited use for targeted enzyme discovery, because of the lack of contiguous 8 9 94 sequences (contigs) large enough to contain a complete open reading frames (ORFs); for 10 11 12 95 cellulases, this is at least 1 kb [33]. For example, Allgaier et al. [1] found only 25 potentially 13 14 96 full-length lignocellulose degrading enzymes from a switchgrass-compost microbial community.