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Genomics of cellulosic biofuels

The development of alternatives to fossil fuels as an energy source is an urgent global priority. Cellulosic biomass has the potential to contribute to meeting the demand for liquid fuel, but land-use requirements and process inefficiencies represent hurdles for large-scale deployment of biomass-to-biofuel technologies. Genomic information gathered from across the biosphere, including potential energy crops and microorganisms able to break down biomass, will be vital for improving the prospects of significant cellulosic biofuel production.

 

The capture of solar energy through photosynthesis is a process that enables the storage of energy in the form of cell wall polymers (that is, cellulose, hemicellulose and lignin). The energy stored in these polymers can be accessed in a variety of ways, ranging from simple burning to complex bioconversion processes. The high energy content and portability of biologically derived fuels, and their significant compatibility with existing petroleum-based transportation infrastructure, helps to explain their attractiveness as a fuel source. Despite the increasing use of biofuels such as biodiesel and sugar- or starch-based ethanol, evidence suggests that transportation fuels based on lignocellulosic biomass represent the most scalable alternative fuel  ource.                      

 

Lignocellulosic biomass in the form of plant materials (for example, grasses, wood and crop residues) offers the possibility of a renewable, geographically distributed and relatively greenhouse-gas-favourable source of sugars that can be converted to ethanol and other liquid fuels. Calculations of the productivity of lignocellulosic feedstocks, in part based on their ability to grow on marginal agricultural land, indicates that they can probably have a large impact on transportation needs without significantly compromising the land needed for food crop production.

Lignocellulosic biofuel production involves collection of biomass, deconstruction of cell wall polymers into component sugars (pretreatment and saccharification), and conversion of the sugars to biofuels (fermentation) (Fig. 1). Partially because of the historically low demand for biologically based transportation fuels, each step in this process is in the early stages of optimization for efficiency and throughput. The crops from which biomass is currently derived have not been domesticated for this particular purpose and the present methods for saccharification and fermentation are inefficient and expensive. However, the recent and pressing desire to develop alternatives to fossil fuels has made the rapid improvement of biofuel production a high priority, in which biologically derived energy (‘'bioenergy’')-relevant genomic insights and resources will have an important role

Biomass

From the perspective of transportation fuels, plants can be viewed as solar energy collectors and thermochemical energy storage systems. It is the storage of energy in a form that can later be accessed via thermochemical or enzymatic conversion that distinguishes biomass from other renewable energy sources. Cellulosic biomass, sometimes referred to as lignocellulosic biomass, is an abundant renewable resource that can be used for the production of alternative transportation fuels. The three main components of lignocellulose are cellulose, hemicellulose and lignin, with the relative proportions of the three dependent on the material source4. Cellulose, the main structural component of plant cell walls, is a long chain of glucose molecules, linked to one another primarily by glycosidic bonds. Hemicellulose, the second most abundant constituent of lignocellulosic biomass, is not a chemically well defined compound but rather a family of polysaccharides, composed of different 5- and 6-carbon monosaccharide units, that links cellulose fibres into microfibrils and cross-links with lignin, creating a complex network of bonds that provide structural strength5. Finally lignin, a threedimensional polymer of phenylpropanoid units, can be considered as the cellular glue providing the plant tissue and the individual fibres with compressive strength and the cell wall with stiffness, in addition to providing resistance to insects and pathogens.

  1

As we can retrospectively view the features that made certain wild plants desirable for domestication thousands of years ago to become today’'s food crops, we are now prospectively defining criteria to choose plants with potential to serve as dedicated bioenergy crops in the future. These include cell wall composition, growth rate, suitability for growth in different geographical regions, and resource-use efficiencies. With these features in mind, a list of potential bioenergy crops is being developed and targeted for different growing conditions. Most plants assimilate their CO2 first into a C3 compound, whereas a smaller subset use a C4 compound. Plants using C4 photosynthesis tend to be among the most productive, having higher maximum efficiencies of light, nitrogen and water use in assimilating carbon. The C4 group of potential energy crops includes various perennial grasses such as switchgrass and Miscanthus. These grasses have the advantages of not requiring replanting after a yearly harvest, rapid growth, high biomass density per unit area, and low nutrient and water needs, enabling growth on marginal agricultural land. Disadvantages are that C4 plants are rare in cold climates and unable to grow at temperatures below 10 uC. In these environments, trees, which exclusively depend on C3 photosynthesis, provide the only candidate species. The C3 group of potential energy crops includes trees, such as poplar and eucalyptus, which have relatively rapid growth potential in difficult-to-plough environments. It is highly likely that multiple different energy feedstocks will be deployed depending on latitude, geography, water availability and landowner acceptance.

 

Until recently, minimal effort has been directed towards optimizing potential energy crops for the generation of transportation fuels. This is in stark contrast to the agronomic development of food crops, which have been domesticated for thousands of years to maximize productivity. Teosinte, the wild precursor to modern maize, was first recognized by Native Americans more than 5,000 years ago as a potential food crop. The domestication of teosinte resulted in its conversion from a wild plant, the characteristics of which had been orchestrated by natural selection maximizing survival and reproduction, into a plant whose morphology and physiology had been extensively altered by artificial selection to increase its nutritional yield and ease of harvest8. More recently, selective breeding as well as agronomic advances have resulted in improvement over several orders of magnitude in the nutritional value per acre of modern maize compared to that of teosinte. Some of the most rapid increases have occurred in the past 40 years, both from advances in agronomic practices and, importantly, from the application of modern genetics.

The optimization of bioenergy crops as feedstocks for transportation fuels is in its infancy, but already genomic information and resources are being developed that will be essential for accelerating their domestication. Many of the traits targeted for optimization in potential cellulosic energy crops are those that would improve growth on poor agricultural lands, to minimize competition with food crops over land use.

 

Populus trichocarpa (poplar), the first tree and potential bioenergy crop to have its genome sequenced, illustrates some of the issues and potential of applying genomics to the challenge of optimizing energy crops. The traits for which the genetic underpinnings will be sought in the genomes of bioenergy-relevant plants, such as poplar, include those affecting growth rates, response to competition for light, branching habit, stem thickness and cell wall chemistry. Significant effort will go into maximizing biomass yield per unit land area, because this more than any other factor will minimize the impact on overall land use. One can imagine trees optimized to have short stature to increase light access and enable dense growth, large stem diameter, and reduced branch count to maximize energy density for transport and processing. Trees have evolved with highly rigid and stable cell walls due to heavy selective pressure for long life and an

upright habit. Plants domesticated for energy production, with a crop cycle time of only a few years, would have less need for a rigid cell wall than wild plants with lifetimes of a hundred years or more. Alterations in the ratios and structures of the various macromolecules forming the cell wall are a major target in energy crop domestication to facilitate post-harvest deconstruction at the cost of a less rigid plant.

 

Already, by comparing several of the presently available plant genomes coupled with largescale plant gene function and expression_(that is, furfural and 5-hydroxymethylfurfural) that decrease the overall yield of the fermentation process.

The human need for efficient breakdown of lignocellulosic biomass for biofuel production is relatively new; however, a variety of organisms have evolved to take advantage of this nutrient source, including the free-living organisms and symbiotic animal–microbe consortia invariably present in biomass-rich environments. Increasing our knowledge of the biochemical machinery used by these organisms for the breakdown of biomass offers new avenues for the development of biologically based processes that could potentially accomplish biomass conversion at an industrial scale. Just as plant genomes are providing us with new approaches for accelerating feedstock domestication, genomics of biomass-degrading microbes and microbial consortia offer a new means to enzyme discovery.

Microbial strategies for degrading lignocellulose are diverse, yet our current understanding of the enzymes involved in these processes is limited to a handful of model organisms such as the fungus Trichoderma reesei and the bacterium Clostridium thermocellum. Recent genome sequencing of these two biomass degraders, as well as a host of other cellulolytic species, has expanded our repertoire of known or potential cellulolytic enzymes. The knowledge of biomass degradation pathways is soon to be increased even more by a number of large-scale genomic surveys under way of isolated organisms and microbial communities known to degrade biomass.

Considering the current dependence on acid and heat pre-treatment in the deconstruction of lignocellulose, it is clear that enzymes that are stable and active at low pH values and at high temperatures are of particular value. Thus, enzymes derived from thermophilic and acidophilic organisms known to degrade lignocellulose, such as Caldicellulosiruptor saccharolyticus and Acidothermus cellulolyticus, hold significant promise for industrial processes21. However, the
number of sequenced thermoacidophilic organisms is low, partly because of the significant obstacles in cultivating these organisms in the laboratory.

Many novel enzymes and enzyme systems that have evolved to make use of cellulosic biomass are present in difficult-to-culture microbes22. This is particularly the case for the communities of
microbes inhabiting the guts of lignocellulose-consuming insects and ruminants. Recently, in response to the challenge of studying difficult-to-culture organisms, metagenomic approaches have been developed to access the information encoded in their genomes. This typically involves the high-throughput sequencing of DNA extracted directly from the mixtures of organisms present
in an environment.

 

The metagenomic analysis of a wood-feeding higher termite has recently revealed hundreds of genes encoding carbohydrate-active enzymes,many of which were subsequently shown to be biochemically active. From the perspective of an industrial application, the termite hindgut is too complex a model to mimic because it comprises hundreds of microbial species and lignocellulose-degrading enzymes. However, termites tend to feed on complex diets of several plant species, which translate to more complex hindgut communities and enzyme inventories. Thus, examination of this single community reveals enzymes capable of hydrolysing a broad assortment of chemical bonds. The saccharification of a single uniform cellulosic feedstock,
particularly one engineered to have a simplified cell wall, will presumably require fewer enzymes and less downstream optimization of gene regulation and enzyme activity in a production host.

The availability of a wide range of naturally occurring lignocellulose-degrading enzymes increases the chances of successful enzyme optimization for industrial processes. Optimization of the saccharification process is crucial because the cost of cellulases remains a key barrier to economical production of biofuels3. A more diverse set of candidate enzymes—identified through a combination of conventional cultured microbial genome studies coupled with environmental prospecting methods such as metagenomics—improves the likelihood of obtaining enzymes with activities and stability suited to a variety of industrial processes.
Biofuel production
Although the final steps of cellulosic ethanol production will require much of the same infrastructure developed for the production of sugar- and starch-based ethanol, changes will need to be made to
exploit the diversity of sugars generated from the breakdown of biomass. Whereas the conversion of starch-based feedstock results primarily in hexoses, the products from degradation of lignocellulosic biomass, composed in part of hemicellulose, include large amounts of the pentose sugars D-xylose and L-arabinose. In contrast to the hexose sugars, the pentose sugars cannot be fermented by wild-type Saccharomyces cerevisiae24. Another factor limiting ethanol yield is
the toxicity of ethanol to the fermenting host. Most fermenting organisms such as S. cerevisiae cannot tolerate ethanol concentrations exceeding 25% (v/v)25, resulting in a product that must then be concentrated through distillation. Distillation represents an expensive and energy-intensive step in ethanol production.

 

These limitations in ethanol production have generated significant interest in developing new organisms able to exploit fully the breakdown products of lignocellulosic biomass as well as tolerate the products of fermentation26. Pichia stipitis represents one yeast species of relevance to biofuel research based on its natural ability to ferment the pentose sugar xylose. Its recently sequenced genome revealed insights into the metabolic pathways responsible for this process27,
and investigators are already working on the optimization of this pathway in P. stipitis as well as on the construction of systems for the heterologous expression_(1) high solubility of ethanol in water, which necessitates an energy-intensive distillation step; (2) diminished energy per gallon content of ethanol compared to petroleum; (3) difficulties in the
distribution of ethanol via today’s pipeline infrastructure owing to its hygroscopic nature; and (4) incompatibilities with many current vehicles at higher blending volumes. Thus, work is under way to
enable the synthesis of next-generation biofuel products, such as higher-chain alcohols, alkanes, and other molecules with structures and activities closer to that of petroleum and diesel. Butanol, for example, can be used in conventional engines either in pure form or mixed with petroleum, can be transported by existing pipelines, and has a higher energy content than petroleum31. A number of organisms, such as Clostridium acetobutylicum, produce butanol  from sugars via the ABE (acetone, butanol, ethanol) fermentation and can be manipulated to produce mostly butanol31. E. coli has recently been engineered to produce isobutanol and other alcohols via a non-fermentative pathway that may be more readily adapted to large-scale production, using heterologous expression_(Table 1), information from which will be called on to increase oil production and facilitate plant growth in a broader range of habitats. Oil-producing algae are also under active study as an alternative source for biodiesel production. One of the major advantages of algae is their growth in liquid, negating the issue
of potential competition between food and energy crops for land use. Chlamydomonas reinhardtii is one of a series of algal species whose genomes are becoming available for analysis34. Considering the magnitude of the transportation fuel problem, biodiesel is likely to contribute at least in part to the solution, and genomic research on oil producers will aid in making biodiesel production cheaper and more efficient.

Placing a man on the Moon and sequencing the human genome represented large-scale technologically challenging projects. In both these instances, substantial research and development efforts resulted in technological advances unforeseen at the projects’ initiation. The
strategies that were pioneered in sequencing and interpreting the human genome for the improvement of human health are now poised to be an important contributing technology in the challenge to develop environmentally and socially acceptable alternatives to fossil fuels.