Biocatalysts for biomass deconstruction from environmental genomics

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ScienceDirect Biocatalysts for biomass deconstruction from environmental genomics Zachary Armstrong1, Keith Mewis1, Cameron Strachan2 and Steven J Hallam1,2,3 Plant biomass offers a sustainable alternative to the energy and materials produced from fossil fuels. The industrial scale production or biorefining of fermentable sugars and aromatics from plant biomass is currently limited by the lack of cost effective and efficient biocatalysts. One potential solution to this problem is the discovery of biomass deconstructing biocatalysts from uncultivated microbial communities. Here we review recent progress in recovering such biological devices from environmental genomes and consider how this information can be used to build better biorefining ecosystems. Addresses 1 Genome Science and Technology Program, University of British Columbia, Vancouver, Canada 2 MetaMixis Biologics Inc., Vancouver, BC, Canada 3 Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada Corresponding author: Hallam, Steven J ([email protected])

Current Opinion in Chemical Biology 2015, 29:18–25 This review comes from a themed issue on Energy

[3,4]. It is an aggregate of several polymers, including: cellulose, matrix polysaccharides (pectin and hemicellulose) and lignin. To harvest the fermentable sugars and aromatics provided in plant biomass, mechanical, chemical and biological processes have been developed [5,6] (Figure 1). The discovery and design of cost effective and efficient biocatalysts enabling generation of a ‘sugar platform’ for biomass deconstruction is integral to scalable production in modern biorefining ecosystems [7,8]. For over 3.5 billion years, cooperative microbial communties have been driving energy and material transfomations that create and sustain planetary living conditions. As a result, although the vast majority of microbes in nature remain uncultivated, they represent a deep reservoir of genetic information and metabolic potential [9,10]. Bioinformatic and functional screens of environmental DNA can access the hidden metabolic powers of microbial communities to recover biological devices from environmental genomes, that is, metagenomes for deconstructing plant biomass into energy and materials through processes that are more in sync with the natural world (Figure 2).

Edited by Timothy DH Bugg and Michael Resch

Sequence guided discovery http://dx.doi.org/10.1016/j.cbpa.2015.06.032 1367-5931/# Elsevier Ltd. All rights reserved.

Introduction The modern world depends on refining oil into everything from energy (e.g. gasoline) to materials (e.g. plastics, pesticides and pharmaceuticals). But this progress also has serious environmental costs from the production of climate changing greenhouse gases to other pollutants that degrade the quality of life. The truth is that we cannot continue to rely on a nonrenewable resource like oil without undermining our future success and the health of the planet. The problem is not about peak oil, it is about finding a more sustainable way to harvest energy and materials from our surroundings that minimizes environmental cost. Plant biomass is a renewable resource that can be converted into energy and materials as an alternative to fossil fuel [1,2]. Lignocellulose, the main component of plant cell walls, is arguably the most abundant biopolymer on the planet Current Opinion in Chemical Biology 2015, 29:18–25

Increases in throughput and concomitant decline in DNA sequencing costs over the last decade have led to an exponential increase in genomic information from individual cells to microbial communities inhabiting diverse natural and engineered ecosystems. Curated databases and bioinformatic tools have been developed to guide the discovery of genes and pathways for biomass deconstruction from this expanding environmental information resource. The Carbohydrate Active enZymes (CAZy) database (http://www.cazy.org/) has emerged as an integral clearinghouse for functional annotation [11]. CAZy categorizes polysaccharide degradation genes, such as glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), carbohydrate-biding modules (CBMs) and more recently lytic polysaccharide mono-oxygenases (LPMOs) [12,13], into structure-guided families. Targeted database searches combined with gene expression studies provide one effective route toward functional validation of known and novel CAZymes from individual genomes and metagenomes. For example, Heins and colleagues [14] selected and synthesized a set of 175 diverse GH1 genes from the CAZy database and a metagenomic study of the bovine rumen for characterization under biorefining relevant conditions (70 8C, 20% (v/v) ionic liquids) [15]. www.sciencedirect.com

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From microbiomes to biofactories. Microbial communities drive energy and material transformations through distributed networks of metabolite exchange. The genetic information from these communities can be harnessed using different types of search functions to construct modular biorefining ecosystems tuned to local biomass inputs, product profiles or operating conditions. (a) Cellulose, 35–50% of dry plant matter [47], is composed of 1,4-linked b-D-glucopyranose subunits which form insoluble crystalline microfibrils [48]. These microfibrils enhance structural stability and are recalcitrant to microbial degradation [49]. The matrix polysaccharides: (b) galacturonan, a main constituent of pectin, (c) xyloglucan, a major component of hemicellulose, and (d) arabinoxylan, another component of hemicellulose. The main function of matrix polysaccharides appears to be the crosslinking of cellulose microfibrils to create a more rigid cell wall [50]. (e) Lignin, 10–30% of plant biomass [51], is a polyphenolic created through radical, oxidative coupling of monolignols. This mechanism causes multifarious structural linkages to be formed which are so diverse it has been hypothesized that no two lignin molecules are identical [52]. Lignin strengthens the cell wall by crosslinking with the polysaccharide fraction [53].

In addition to functional annotation within existing CAZy families, in silico screens based on genomic context information have enabled identification and characterization of hypothetical genes relevant to biomass deconstruction. This approach was recently used to search 5500 genomes within the IMG database resulting in the identification of 56 hypothetical genes located proximal to canonical cellulases [16]. A subset of these genes were synthesized and tested for functional activity, identifying 11 novel biomass deconstructing enzymes. This ‘guilt by association’ paradigm can be extended to polysaccharide utilization loci (PULs). PULs are minimally defined as a SusC/SusD gene pairing in close proximity to genes www.sciencedirect.com

encoding carbohydrate active enzymes [17]. Hypothetical genes predicted within a PUL provide functional clues and inform downstream expression studies. With this in mind, Terrapon and colleagues developed an automated Bacteroidetes PUL prediction pipeline and web interface using genomic context information and domain annotations based on information in the CAZy database [18].

Model organisms and ecosystems While much emphasis has been placed on shotgun sequencing of microbial communities inhabiting everything from termites [19] to tropical soils [20], more recent studies have combined isolation and enrichment strategies to Current Opinion in Chemical Biology 2015, 29:18–25

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Enzyme discovery workflow. Microbial communities can be interrogated for biological devices through bioinformatic and functional screening. Environmental DNA can be extracted directly from natural and engineered ecosystems and used to construct screening libraries. A workflow for constructing large insert fosmid libraries is depicted. Fosmid library production involves high molecular weight environmental DNA preparation, ligation into a vector backbone and ‘head-full packaging’ of ligated DNA into a phage delivery system. Host cells are then transfected, plated and arrayed in 384-well plate libraries which can be interrogated with a variety of functional screens. Environmental DNA or positive clones identified in functional screens can be sequenced and a gene or cluster of functional genes identified using in silico methods. Subsequent biochemical testing and characterization of the identified biological device determines suitability for incorporation into biofactories through strain engineering.

define core microbial communities and enzymatic interactions mediating modular biomass deconstruction. Recently, O’Connor and colleagues used isolate sequencing combined with metagenomics and metaproteomics to identify a novel digestive strategy within the shipworm Bankia setacea [21]. In this model, digestive enzymes produced by symbiotic gill bacteria are secreted and transported to the cecum where they are used by the host to deconstruct biomass for nutrition. Indeed, 41 out of 42 endosymbiont encoded proteins detected in the cecum were predicted to interact with or modify plant cell wall polysaccharides (PCWPs). While most detected proteins were homologous to existing CAZy families, several contained Current Opinion in Chemical Biology 2015, 29:18–25

unknown catalytic domains linked to CBMs for cellulose and xylan. In another study, Gladden and colleagues identified thermotolerant and ionic-liquid tolerant cellulases from a microbial community enriched on switchgrass using a combination of metagenomics and expression screening [22]. Candidates identified in silico were synthesized and expressed in vitro using an Escherichia coli cell free system and in vivo using a low copy plasmid. The capacity to isolate CAZymes from the background of cellular maintenance proteins identifies minimal sets of biomass deconstructing enzymes encoded in microbial communities adapted to specific PCWPs and environmental conditions. This information combined with functional www.sciencedirect.com

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screening paradigms described below can be used to build better biorefining ecosystems tuned to local biomass sources and operating conditions to produce defined product profiles.

Functional metagenomic screens Functional metagenomic screens involve the construction of environmental DNA libraries using a suitable vector for heterologous expression in a compatible host system, for example, E. coli (Figure 2), although isolate libraries can also be constructed in the same manner [10]. Resulting libraries are screened for activity on agar [23] or in microtiter plates [24,25], using a reporter substrate,

transgene, or other form of phenotypic selection such as growth. Screening libraries sourced from a range of environmental conditions, for example, pH, temperature, ionic liquids (ILs), enables recovery of active clones with alternative substrate specificities and tolerances [14,20,26–35]. Similarly, libraries sourced from xylotrophic or wood-feeding organisms can provide insight into modular biomass deconstruction. Recently, Ruegg and colleagues, screened an isolate fosmid library sourced from the lignocellulolytic bacterium Enterobacter lignolyticus to identify genes conferring IL tolerance under biorefining conditions in an E. coli host [36]. They recovered an active clone encoding a membrane

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Compounds used for the detection of biomass deconstruction activities. Fluorogenic and chromogenic model compounds can be used in the discovery and characterization of biomass deconstruction activities. 4-Methylumbelliferyl b-D-cellobioside (MUC) releases the fluorescent 4methylumbelliferone upon hydrolysis, and has been used to detect cellulase activity [33]. Many other glycosides containing 4-methylumbelliferyl have also been developed for detecting hydrolase activity. a-O-(b-methylumbelliferyl) acetovalinone (MUAV) is a model of the b-O-4 linkage found in lignin, which similarly releases 4-methylumbelliferone upon hydrolysis, and has been used to characterize lignin modifying activities [54,55]. Widespread screening with this substrate has, however, yet to be demonstrated. The oxidation of 2,6-dimethoxyphenol (DMP), a model of the syringyl residues found in lignin, results in 3,30 ,5,50 -tetramethoxydiphenoquinone which can be monitored spectrophotometrically. Ferrer and colleagues have used DMP to develop a functional metagenomic screen for laccases [56]. www.sciencedirect.com

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transporter and transcriptional regulator enabling a 20% increase in biofuel production in the presence of 68 mM 1-ethyl-3-methylimidazolium chloride. Similarly, Bastien and colleagues screened fosmid libraries sourced from the termite Pseudacanthotermes militaris gut and fecal combs [34]. This species cultivates a termite specific basidiomycete fungus, Termitomyces sp., which thrives upon combs made of termite feces. Functional screening recovered 101 clones acting on a range of model substrates containing arabinoxylan and xylan moieties and identified differences in biomass deconstruction potential between microbial communities inhabiting the gut and comb milieus.

more soluble than cellulose, or glycosides containing a reporter aglycone (Figure 3), which can offer exceptional signal-to-noise ratios. Emerging technologies based on capillary electrophoresis and mass spectrometry show potential for their use in rapid and precise activity screening on intact biomass. For example, a recent method to characterize enzymatically released oligosaccharides using capillary electrophoresis can rapidly quantify sugars released from the action of xylanases on wheat flour arabinoxylan down to femtomolar ranges while differentiating between the activities of GH10 and GH11 xylanases [37]. Another recent method used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect changes in the lignin and sugar content of milled red spruce after treatment with a commercial cellulase cocktail with or without xylanase addition [38]. Future

Functional screens typically employ synthetic reporter substrates, such as carboxy-methyl cellulose, which is far

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Biosensor for detecting lignin transformation. Strachan and colleagues [46] developed a biosensor that detects lignin transformation products. The transcriptional repressor EmrR binds to the emrRAB promoter region. Derepression of this promoter occurs in the presence of monoaromatic compounds released during lignin transformation. The reporter strain also contains a plasmid with green fluorescent protein (GFP) downstream of the emrRAB promoter, resulting in fluorescence in the presence of lignin modification products. When used in co-culture screens, this reporter enables rapid recovery of active clones encoding lignin transformation phenotypes. Current Opinion in Chemical Biology 2015, 29:18–25

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application of these methods in functional metagenomic screens has the potential to recover clones acting on more complex polymeric substrates used under biorefining conditions.

Lignin transforming enzymes Lignin transformation can increase the release of fermentable sugars and aromatics for energy and material production. However, compared to hemicellulose and cellulose, biorefining processes for lignin transformation are incipient [39]. Although several bacterial strains have been implicated in lignin transformation, including Enterobacter lignolyticus SCF1 and Rhodococcus jostii RHA1 [40– 42], basidiomycete fungi remain the primary sources of lignin-transforming enzymes, including laccases, manganese-dependent peroxidases, and lignin peroxidases [43]. Together with different oxidative mediators, these enzymes trigger a non-specific cascade of bond splitting reactions that has been compared to combustion [44]. Until recently, screens for recovering lignin transforming enzymes have focused on a narrow range of substrates, representing model compounds or non-specific activities that give a colorimetric or fluorescent output upon oxidation [45]. While such methods have enabled identification of fungal or bacterial lignin transforming isolates, they have proven less effectual in functional metagenomic screens. Biosensor-based detection of lignin transformation products provides an alternative screening paradigm that can be used to search across a range of substrate specificities and sensitivities. For example, Strachan and colleagues recently developed an E. coli biosensor for lignin transformation based on the emrR transcriptional regulator, emrRAB promoter and green fluorescent protein [46]. They used this biosensor in co-cultures to recover active clones sourced from coal bed fosmid libraries that confer differential lignin transformation profiles that synergize in combination (Figure 4). Complete sequencing and transposon insertion identified six functional classes including oxidoreductases, hydrogen peroxide generating, secretion, signalling, transport and chemotaxis modules necessary for lignin transformation activity. One clone in particular harboured a pseudo laccase most closely related to a copper resistance protein from Psuedomonas putida (CopA) that catalyzed the oxidation of model compounds 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and DMP, albeit at much slower rates than lignin peroxidases found in basidiomycetes. In addition to catalyzing the oxidation of model compounds CopA was able to release small molecules from lignin including vanillin in a copper-dependent manner. In addition to harbouring functional classes implicated in lignin transformation, many of the active clones contained mobile genetic elements suggesting that bacterial lignin transformation is an adaptive trait that can be used to engineer biorefining www.sciencedirect.com

ecosystems with defined product profiles based on different class combinations.

Conclusions By searching environmental sequence information sourced from natural and engineered ecosystems we can recover biological devices for biomass deconstruction. Bioinformatic screens can be used to identify genes encoding these devices based on CAZyme homology or genomic context information, and define functional modules mediating specific substrate conversion processes such as PULs. The integration of gene expression profiling, for example, metatranscriptomics and metaproteomics with plurality and single-cell genome sequence analysis validates gene prediction models and focuses attention on regulatory networks underlying biomass deconstruction in the environment. Moreover, new screening paradigms capable of accurate and sensitive activity detection using intact biomass promise to identify functional modules acting on more complex polymeric substrates such as lignin. By programming industrial strains with cooperative functional modules it will become possible to build biorefining ecosystems based on the same design principles used by uncultivated microbial communities to drive energy and material transfomations in the environment. Such biofactories will be tunable to local biomass inputs and product profiles, and more robust to process perturbations.

Conflict of interest C.R.S. and S.J.H. are cofounders of MetaMixis, Inc., a synthetic biology company that uses coculture-based biosensor screening to interrogate metagenomic libraries for the production of industrial enzymes and active pharmaceutical intermediates.

Acknowledgements This work was performed under the auspices of the Natural Sciences and Engineering Research Council (NSERC) of Canada, Genome British Columbia, Genome Alberta, Genome Canada, Canada Foundation for Innovation (CFI), the Tula Foundation funded Centre for Microbial Diversity and Evolution and the Canadian Institute for Advanced Research (CIFAR). Z.A. and K.M. were both supported by the NSERC CREATE Genome Sciences and Technology (GSAT) training program at the University of British Columbia. We would like to thank Rahul Singh, Lindsay Eltis and Steve Withers for insightful conversations regarding biomass deconstruction and the future of biorefining.

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