Joint Genome Institute to Tackle 44 Sequencing Projects in 2009, Focused on Bioenergy and Environmental Applications

2 July 2008

Botryococcus braunii, subject of one of the sequencing projects, is a microalga that produces long-chain hydrocarbons. Source: NIES

The US Department of Energy Joint Genome Institute (DOE JGI) will  initiate 44 new DNA sequencing projects in 2009, with continued focus on the JGI’s mission areas: bioremediation, and global carbon cycling.  The 44 projects, culled from nearly 150 proposals received through the Community Sequencing Program (CSP), represent more 60 billion nucleotides of data to be generated through this biodiversity sampling campaign—roughly the equivalent of 20 human genomes.

Projects ranges from sequencing the Loblolly pine—the most commonly planted tree species in the US—to algae that produce long-chain hydrocarbons; to phytoplankton; to several metagenome complexes—complex microbial communities that are isolated directly from the environment or reside inside of a larger organism.

The Loblolly pine (Pinus taeda) accounts for about 75% of all seedlings planted in the US each year. Its ability to efficiently convert CO₂ into biomass and its widespread use as a plantation tree make it a promising, cost-effective feedstock for cellulosic biofuel production, said Eddy Rubin, DOE JGI Director.

Because of the pine’s enormous genome (21 billion bases), the project will begin with a targeted effort to understand the structure of the pine genome. The project is intended to zero in on genes that can be used for molecular breeding programs to improve Loblolly as a biomass feedstock, carbon sequestration tool, and source of renewable, high-quality raw materials for lumber and pulp fiber.

Botryococcus braunii is a colony-forming green microalga of the species Chlorophyceae that can grow in both freshwater and brackish environments. During the growth cycle of this organism, the algae synthesize long-chain liquid hydrocarbon compounds and sequester them in the extracellular matrix of the colony to afford buoyancy. (Typical hydrocarbon content of the organism is approximately 30-40% of the dry weight of the cells.)

Three phenotypically distinct isolates, or “races,” of B. braunii have been reported (races A, B, and L), which are  identified by the type of oil produced and accumulated by the organism.

Of the three, the oils produced by race B, a family of isoprenoid compounds termed botryococcenes, hold the most promise as an alternative energy source, according to the research team.  Botryococcenes have been converted to fuel suitable for internal combustion engines through caustic hydrolysis, and geochemical analysis has shown that botryococcenes, presumably from ancient B. braunii communities, also compose a portion of the hydrocarbon masses in several modern-day petroleum and coal deposits.

The JGI project will target the identification of specific metabolic pathways responsible for hydrocarbon synthesis to alleviate bottlenecks in biofuels production.

Greater duckweed (Spirodela polyrhiza) is the smallest, fastest growing, and simplest of flowering plants, producing biomass faster than any other flowering plant. Their carbohydrate content is readily converted to fermentable sugars by using commercially available enzymes developed for corn-based ethanol production, said Rubin.

Propagated on agricultural and municipal wastewater, Spirodela species efficiently extract excess nitrogen and phosphate pollutants. Duckweed growth on ponds effectively reduces algal growth (by shading), coliform bacteria counts, suspended solids, evaporation, biological oxygen demand, and mosquito larvae while maintaining pH, concentrating heavy metals, sequestering or degrading halogenated organic and phenolic compounds, and encouraging the growth of aquatic animals such as frogs and fowl.

The DOE JGI has selected several metagenomes to sequence. These leverage DOE JGI’s expertise from previous studies of acid mine drainage and the termite hindgut, where samples yielded scores of different microbes, producing hundreds of enzymes with potentially useful industrial applications.

One such metagenome lurks inside of Bankia setacea, the giant Pacific shipworm. Shipworms, wood-boring marine bivalves, have been nicknamed “termites of the sea.” These animals are capable of feeding solely on wood, utilizing a highly efficient system of symbiotic lignocellulose degradation that is biologically, functionally, and evolutionarily distinct from those found in termites, ruminants, and all other cellulose-consuming animals

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Like termites, the ability of shipworms to consume wood depends on symbiotic bacteria that provide enzymes, including cellulases and other hydrolases critical for digestion of wood by the host and potentially valuable for commercial bioconversion of lignocellulose to ethanol. Analysis of the shipworm symbiont community metagenome will provide important insights into the composition and function of this unique lignocellulose degrading bacterial community and will allow valuable comparisons to the recently sequenced termite symbiont metagenome.

Unlike termites, shipworms accomplish the complete degradation of lignocellulose with a simple intracellular consortium of just a few related types of microbes.

Other CSP 2009 projects include the following:

• One metagenome project entails a sampling of the foregut of Opisthocomus hoazin—a leaf-eating Amazonian pheasant-like stinkbird, or hoatzin. A prehistoric relic, its unique fermentative organ harbors an impressive array of novel microbes, like that of cows and other ruminants. Instead of a rumen, stinkbirds possess a crop, an enlargement of the esophagus where the fermentation takes place—and the source of the stink. The characterization of its contents will likely lead to the identification of novel microbial enzymes that degrade plant cell walls.

• Nanoflagellates, a group of marine microbes, prey on other microbes, such as bacteria and phytoplankton, for survival. These predatory protists play a critical role in marine carbon cycling. This project will investigate the genetic mechanism behind the processes of predation, digestion, and biomass incorporation by protists.

• The most abundant source of carbon is plant biomass, composed primarily of cellulose, hemicellulose, and lignin. Many microorganisms are capable of utilizing cellulose and hemicellulose as carbon and energy sources, but a much smaller group of filamentous fungi has evolved with the ability to depolymerize lignin, the most recalcitrant component of plant cell walls. Collectively known as white rot fungi, they possess the unique ability to efficiently depolymerize lignin in order to gain access to cell wall carbohydrates for carbon and energy sources. Ceriporiopsis subvermispora rapidly depolymerizes lignin with relatively little cellulose degradation. The annotated gene set of C. subvermispora and comparative analyses with the lignin degraders P. chrysosporium and Pleurotus ostreatus (both sequenced by DOE JGI) will advance the understanding of these complex oxidative mechanisms involved in lignocellulose conversions.

• The CSP selections draw from all three branches of life: eukaryotes (such as plants and fungi), bacteria, and archaea. Desulfurococcus fermentans, isolated from the Uzon Caldera on the Kamchatka Peninsula, is the only known archaeon that breaks down cellulose and, unlike most known microorganisms that carry out fermentation, it produces hydrogen in the presence of hydrogen while fermenting cellulose and starch without experiencing an inhibition of growth. A comparative genomics investigation of Desulfurococcus species will resolve the finer details that distinguish proton reduction (producing hydrogen) from sulfur reduction in fermentative archaea and help to define the evolutionary and metabolic relationships of the Desulfurococcus species with their archaeal relatives.

• Among the holy grails of biofuel production is the perfect concoction of enzymes capable of rendering complex biomass into fuel by a process known as simultaneous saccharification and fermentation (SSF). Hansenula polymorpha strain NCYC 495 leu1.1 is a yeast capable of fermenting xylose (five-carbon sugar), glucose (six-carbon sugar), and cellobiose (a unit of two condensed glucose molecules) to ethanol at high temperatures (45–50° C), thus holding promise for the SSF process. Commercially feasible SSF technology has not yet been developed because of the absence of a robust organism capable of fermentation at high temperatures. Sequencing of H. polymorpha will enable the identification of the limiting steps in the fermentation pathway from xylose to ethanol.

• Another key barrier to economical cellulosic biofuel production is the cost of enzymes for the degradation of cellulosic biomass. Currently, the cellulases used in pilot cellulosic ethanol plants are produced by fungi, in many cases Trichoderma reesei strain Qm6a (whose genome sequence analysis was published in Nature Biotechnology by DOE JGI and collaborators). The widespread use of T. reesei in cellulase production underscores the importance of this organism and the need for understanding the mechanisms behind enzyme secretion. This project will sequence five T. reesei strains with varying levels of cellulase production and derived from strain Qm6a with the purpose of characterizing the cellular machinery behind enzyme secretion.

• The use of microbes to directly generate electricity from the biodegradation of waste organic matter in microbial fuel cells is a technology that shows promise. A 2009 project will sequence the electricity-generating photosynthetic bacterium Rhodopseudomonas palustris strain DX-1 to help highlight the mechanistic basis for this unusual biological property. This project will add to the growing literature describing the complexity of this genus by complementing the six other strains of I that have been sequenced to date by DOE JGI.

Established in 2005, the Community Sequencing Program (CSP) provides the scientific community at large with access to high-throughput sequencing at DOE JGI for projects of relevance to DOE missions. Sequencing projects are chosen based on scientific merit—judged through independent peer review—and relevance to issues in bioenergy, global carbon cycling, and bioremediation.

The US Department of Energy Joint Genome Institute, supported by the DOE Office of Science, unites the expertise of five national laboratories—Lawrence Berkeley, Lawrence Livermore, Los Alamos, Oak Ridge, and Pacific Northwest—along with the Stanford Human Genome Center to advance genomics in support of the DOE missions related to clean energy generation and environmental characterization and cleanup. DOE JGI’s Walnut Creek, CA, Production Genomics Facility provides integrated high-throughput sequencing and computational analysis.

Resources

• Sequencing projects for 2009