High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development.

Functional metagenomics is a powerful tool for both the discovery and development of biocatalysts. This study presents the high-throughput functional screening of 22 large-insert fosmid libraries containing over 300,000 clones sourced from natural and engineered ecosystems, characterization of active clones, and a demonstration of the utility of recovered genes or gene cassettes in the development of novel biocatalysts. Screening was performed in a 384-well-plate format with the fluorogenic substrate 4-methylumbelliferyl cellobioside, which releases a fluorescent molecule when cleaved by ß-glucosidases or cellulases. The resulting set of 164 active clones was subsequently interrogated for substrate preference, reaction mechanism, thermal stability, and optimal pH. The environmental DNA harbored within each active clone was sequenced, and functional annotation revealed a cornucopia of carbohydrate-degrading enzymes. Evaluation of genomic-context information revealed both synteny and polymer-targeting loci within a number of sequenced clones. The utility of these fosmids was then demonstrated by identifying clones encoding activity on an unnatural glycoside (4-methylumbelliferyl 6-azido-6-deoxy-ß-d-galactoside) and transforming one of the identified enzymes into a glycosynthase capable of forming taggable disaccharides.IMPORTANCE The generation of new biocatalysts for plant biomass degradation and glycan synthesis has typically relied on the characterization and investigation of one or a few enzymes at a time. By coupling functional metagenomic screening and high-throughput functional characterization, we can progress beyond the current scale of catalyst discovery and provide rapid annotation of catalyst function. By functionally screening environmental DNA from many diverse sources, we have generated a suite of active glycoside hydrolase-containing clones and demonstrated their reaction parameters. We then demonstrated the utility of this collection through the generation of a new catalyst for the formation of azido-modified glycans. Further interrogation of this collection of clones will expand our biocatalytic toolbox, with potential application to biomass deconstruction and synthesis of glycans.

Author Correction: A compendium of geochemical information from the Saanich Inlet water column.

In Table 3 of this Data Descriptor the units of Mean_N2O and Mean_CH4 are incorrectly stated as "Nanomolar (µM)". This should instead read "Nanomolar (nM)".

A compendium of multi-omic sequence information from the Saanich Inlet water column.

Marine oxygen minimum zones (OMZs) are widespread regions of the ocean that are currently expanding due to global warming. While inhospitable to most metazoans, OMZs are hotspots for microbial mediated biogeochemical cycling of carbon, nitrogen and sulphur, contributing disproportionately to marine nitrogen loss and climate active trace gas production. Our current understanding of microbial community responses to OMZ expansion is limited by a lack of time-resolved data sets linking multi-omic sequence information (DNA, RNA, protein) to geochemical parameters and process rates. Here, we present six years of time-resolved multi-omic observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique multi-omic framework for studying microbial community responses to ocean deoxygenation along defined geochemical gradients in OMZ waters.

A compendium of geochemical information from the Saanich Inlet water column.

Extensive and expanding oxygen minimum zones (OMZs) exist at variable depths in coastal and open ocean waters. As oxygen levels decline, nutrients and energy are increasingly diverted away from higher trophic levels into microbial community metabolism, resulting in fixed nitrogen loss and production of climate active trace gases including nitrous oxide and methane. While ocean deoxygenation has been reported on a global scale, our understanding of OMZ biology and geochemistry is limited by a lack of time-resolved data sets. Here, we present a historical dataset of oxygen concentrations spanning fifty years and nine years of monthly geochemical time series observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique geochemical framework for evaluating long-term trends in biogeochemical cycling in OMZ waters.

Integrating biogeochemistry with multiomic sequence information in a model oxygen minimum zone.

Microorganisms are the most abundant lifeform on Earth, mediating global fluxes of matter and energy. Over the past decade, high-throughput molecular techniques generating multiomic sequence information (DNA, mRNA, and protein) have transformed our perception of this microcosmos, conceptually linking microorganisms at the individual, population, and community levels to a wide range of ecosystem functions and services. Here, we develop a biogeochemical model that describes metabolic coupling along the redox gradient in Saanich Inlet-a seasonally anoxic fjord with biogeochemistry analogous to oxygen minimum zones (OMZs). The model reproduces measured biogeochemical process rates as well as DNA, mRNA, and protein concentration profiles across the redox gradient. Simulations make predictions about the role of ubiquitous OMZ microorganisms in mediating carbon, nitrogen, and sulfur cycling. For example, nitrite "leakage" during incomplete sulfide-driven denitrification by SUP05 Gammaproteobacteria is predicted to support inorganic carbon fixation and intense nitrogen loss via anaerobic ammonium oxidation. This coupling creates a metabolic niche for nitrous oxide reduction that completes denitrification by currently unidentified community members. These results quantitatively improve previous conceptual models describing microbial metabolic networks in OMZs. Beyond OMZ-specific predictions, model results indicate that geochemical fluxes are robust indicators of microbial community structure and reciprocally, that gene abundances and geochemical conditions largely determine gene expression patterns. The integration of real observational data, including geochemical profiles and process rate measurements as well as metagenomic, metatranscriptomic and metaproteomic sequence data, into a biogeochemical model, as shown here, enables holistic insight into the microbial metabolic network driving nutrient and energy flow at ecosystem scales.

Molecular tools for investigating microbial community structure and function in oxygen-deficient marine waters.

Water column oxygen (O2)-deficiency shapes food-web structure by progressively directing nutrients and energy away from higher trophic levels into microbial community metabolism resulting in fixed nitrogen loss and greenhouse gas production. Although respiratory O2 consumption during organic matter degradation is a natural outcome of a productive surface ocean, global-warming-induced stratification intensifies this process leading to oxygen minimum zone (OMZ) expansion. Here, we describe useful tools for detection and quantification of potential key microbial players and processes in OMZ community metabolism including quantitative polymerase chain reaction primers targeting Marine Group I Thaumarchaeota, SUP05, Arctic96BD-19, and SAR324 small-subunit ribosomal RNA genes and protein extraction methods from OMZ waters compatible with high-resolution mass spectrometry for profiling microbial community structure and functional dynamics.