Canadian MetaMicroBiome Library

Background Publications Researchers Samples Contact

The microbial world harbours a wealth of DNA sequence information that can be harnessed for the production of bioproducts and enzymes that mediate industrially relevant chemical reactions. In some cases, the biological processes encoded by these gene products can produce high-energy organic compounds that may begin to directly replace fossil fuel-based technologies that are harmful to the environment. For many years, access to the full richness of the genetic material in the microbial world has been hampered by the inability to cultivate most microbes present in microbial communities. Molecular techniques now provide the methods to access the DNA from this uncultivated microbial majority; it is now possible to construct gene libraries from DNA that is extracted directly from a handful of soil, and these libraries then represent the genetic material from each of the microorganisms present in that soil sample. This captured environmental DNA (the 'metagenome') is hosted in readily cultivated bacteria and the corresponding expression of captured genes can be detected and screened for the expression of targeted gene products. In this way, diverse and novel genes from uncultivated microorganisms that carryout desired functions maybe exploited, without the prerequisite of cultivation.

The Canadian MetaMicroBiome Initiative is a publicly accessible collection of libraries of environmental DNA representing Canadian soil microbial communities. Soil samples from disparate Canadian biomes will be chosen due to their unparalleled microbial diversity and corresponding metabolic richness. These DNA libraries will be characterized by powerful DNA sequencing techniques to determine the microbial community diversity of each sample. In partnership with Iogen Corp., we will show that there is DNA of interest in these environmental libraries by isolating new genes for the conversion of lignocellulosic biomass for biofuel and bioproducts applications. Detailed characterization of the enzyme activities and structures will aid in the development or improvement of processes for the production of high value-added chemicals.

Trevor C. Charles, Ph.D.

NSERC Proposal

This proposal lies firmly within the target area of Quality Foods and Novel Bioproducts, research topic Novel Bioproducts. Here we describe the development and implementation of metagenomics-based methods for the isolation of genes encoding novel enzymes from uncultivated soil microbes. Some of these enzymes will then be candidates for enzyme improvement using directed evolution methods. This research project will be carried out in close partnership with Iogen Corporation of Ottawa. Glycosyl hydrolases are enzymes of significant economic importance as environmentally friendly tools for a range of industrial applications. Currently cellulases are used in the textile industry to achieve the stone-washed look of denim garments, for polishing fabric to prevent the formation of pills during washing, and as additives in laundry detergents. Xylanases and cellulases are used in the pulp and paper industry to reduce the need for chemicals in the bleaching process and to alter fibers to create novel and improved paper properties. Food-grade enzymes, particularly pectinases, are used for clarification in the manufacture of juices, and for texture control in baking. Amylases break down starch to produce corn syrup and “corn ethanol,” and other glycosyl hydrolases are used to improve the efficiency of grain processing. Most recently, conversion of lignocellulosic biomass to sugars for subsequent fermentation to high value products (primarily fuel-grade ethanol) is a cutting-edge technology whose development has become an important global initiative with Iogen Corporation as a leading company in the field. Lignocellulose (plant biomass) is an enormously abundant potential source of valuable hydrocarbon chemicals consisting primarily of cellulose, hemicellulose, and lignin. It occurs in high natural abundance in agricultural, industrial and domestic waste. Lignocellulose can be used as a carbon source by a wide variety of cellulolytic organisms that produce glycosyl hydrolases to obtain fermentable sugars from the biomass. Identifying novel enzymes for efficient conversion of biomass to sugars, for subsequent fermentation to fuel-grade ethanol (“cellulose ethanol,” in contrast to “corn ethanol”) or other high-value chemicals is a major interest for industrial enzyme and chemical manufacturers. Underlining the timeliness and value of this research proposal, in March of 2008 Canada’s Environment Minister (John Baird) stated “thanks to our Government’s allocation of $500 million for next generation biofuels, Canada is one step closer to making our country’s first full-scale cellulosic ethanol fuel facility a reality. With technologies such as this, Canada is well-positioned to be a world leader in the renewable fuels industry.”

Modern molecular microbiology has revealed a hidden diversity of life far greater than ever predicted. However, our ability to access the biotechnological potential that resides within the DNA of that diversity is currently limited by lack of methodologies for direct isolation of biological components that could be used to develop the next generation of industrial enzymes. Recent advances in metagenomics, the genomics of uncultivated microbial communities, suggest a strong role for this new approach in providing tools and resources to utilize the full value of lignocellulosic feedstock for next generation biofuels and high-value chemicals.

Despite increasing metagenomic research activity, relatively few environments on Earth have been sampled for potential human benefit. This is especially true of Canadian environments. We propose the development of the Canadian MetaMicroBiome Library (CM²BL), a freely-available and sustainable resource of subsamples and genomic DNA from representative Canadian microbial communities. The C²2BL would facilitate searches for novel enzymes of industrial potential. Initially we will screen this metagenomic resource to discover new enzymes for biomass conversion and carbohydrate fiber modification for applications of immediate relevance to the industrial partner. Associated with this metagenomic repository will be taxonomic information afforded by novel high-throughput sequencing approaches. This information is critical for understanding the composition, diversity and uniqueness of the libraries stored in MetaMicroBiome to guide library selection for future bioprospecting interests.

Objectives

This proposal reflects long term objectives to establish and maintain a Canadian metagenomic resource for Canadian and International research communities. We foresee that availability of such a resource will spur research aimed at gaining a greater understanding of the microbial ecosystems while at the same time providing easy access to genetic material of potential commercial value.

The short term objectives are reflected in the following sub-project activities proposed for the three year duration of NSERC Strategic funding.

  1. Establish the Canadian MetaMicroBiome Library with soil community genomic DNA samples that have been phylotyped and captured in metagenomic libraries.
  2. Carry out functional screening of soil metagenomic libraries to identify carbohydrase enzymes of potential commercial value for biomass conversion and carbohydrate fiber modification.
  3. Fully characterize identified enzyme activities of industrial promise, and initiate enzyme improvement strategies with the aim of commercialization.

Background and Concept

Most biocatalysts used by industry today originated from organisms cultivated in a laboratory environment. However, undiscovered microorganisms may host myriad metabolic processes with diverse suites of enzymes, with the potential for biotechnological applications. Given that the majority of free-living microorganisms (often >99%) are not readily cultivated under current laboratory conditions, metagenomics has enabled bioprospecting of genetic diversity from uncultivated microorganisms from environmental samples. Metagenomics projects have provided genetic insight into microbial communities inhabiting the oceans, soil and acid mine drainage biofilms. But these have been conducted primarily from an ecological perspective. Smaller studies related to the discovery of commercially valuable enzymes have demonstrated the ability of metagenomics to capture a variety of novel target genes from a range of environments. A major goal of the research outlined in this proposal is to establish a focused Canadian functional metagenomics project, building on successful proof-of-principle studies from the past, to access bioproduct-related genes and enzymes from the microbial complexity hidden in this country’s diverse terrestrial biomes.

The proposed project will establish the Canadian MetaMicroBiome Library (CM²BL). This unique resource will house a collection of metagenomic (‘Meta’) libraries containing the combined genome sequences of microbial communities (‘Micro’) taken from soils spanning multiple Canadian biomes (‘Biome’) and ecosystems. The focus on soils is justified by the unparalleled microbial diversity of soils, low culturability of soil microorganisms, and high cell biomass for readily obtainable DNA. These characteristics will maximize the value of these DNA libraries for screening of industrially-relevant enzyme activities, and ensure that these libraries will serve as a significant genetic resource for discovering additional bioproducts relevant to human health and industry in the future.

We will generate geographic and physical/chemical parameters, and ribosomal sequence tag profiles for each of the soil samples by a combined serial analysis of ribosomal sequence tag methodology and Illumina sequencing approach: SARST-Illumina. A novel “pre-filter” step (DNA stable-isotope probing; SIP) will also be applied. Selected soils will be incubated with 13C-labelled substrates related to bioproduct metabolism. Gradient ultracentrifugation will separate DNA of metabolically active organisms from that of quiescent microbes. This pre-filter step will enrich for genes from relevant organisms regardless of representation in the total population; it has been shown to result in a high proportion of positive clones in library screens. Metagenomic libraries will be functionally screened for enzymatic activities of immediate utility to biofuel production and fiber-based industries, and the resulting target clones characterized by DNA sequence analysis. Access to the libraries will be made available on a cost-recovery basis to members of the Canadian academic, government and commercial communities.

The primary goal of the proposed metagenomic library screens is to demonstrate the strong utility of this methodological approach by accessing the genetic complexity of each of these libraries in order to obtain novel enzyme activities. Functional screening rather than sequence-based prospecting enables the identification of completely novel genes in addition to homologues of previously characterized genes. This functional genomics builds on research that has been developing over the past several years in the Charles laboratory, where we have demonstrated the isolation of novel genes for polyhydroxyalkanoate metabolism, fatty acid metabolism, phosphorous metabolism, and quorum sensing, among others, using phenotypic selection in diverse surrogate hosts.