Plant genetic engineering for biofuel production towards affordable cellulosic ethanol

Plant genetic engineering for biofuel production towards affordable cellulosic ethanol

(Parte 1 de 5)

Finite petroleum reserves and the increasing demands for energy in industrial countries have created international unease. For example, the dependence of the United States on foreign petroleum both undermines its economic strength and threatens its national security1. As highly populated countries such as China and India become more industrialized, they too might face similar problems. It is also clear that no country in the world is untouched by the negative environmental effects of petroleum extraction, refining, transportation and use. For these reasons, governments around the world are increasingly turning their attention to biofuels as an alternative source of energy.

The biofuel that is expected to be most widely used around the globe is ethanol, which can be produced from abundant supplies of biomass from all land plants and plant-derived materials, including animal manure, starch, sugar and oil crops that are already used for food and energy. In addition, ethanol has a low toxicity, is readily biodegradable and its use produces fewer air-borne pollutants than petroleum fuel. The growth of feedstock crops for bioethanol production also reduces greenhouse gas levels, mainly because of the use of atmospheric carbon dioxide in photosynthesis. Although the conversion of biomass to ethanol and the burning of ethanol produce emissions, the net effect can be a large reduction in greenhouse gas emissions compared with petroleum fuel, meaning that the use of bioethanol does not contribute to an increase in net atmospheric carbon dioxide2.

Starch- and sugar-derived ethanol already make a relatively small but significant contribution to global energy supplies. In particular, Brazil produces relatively cheap ethanol from the fermentation of sugarcane sugar to supply one quarter of its ground transportation fuel. In addition, the United States produces ethanol from corn grain. However, even if all the corn grain produced in the United States were converted into ethanol, this could only supply about 15% of that country’s transportation fuels. Meeting US fuel requirements using starch would mean that corn grain production must be increased or corn grain be diverted from other uses. For example, 50.8% of total US corn grain production is currently used for livestock feed3, and the conversion of corn grain into ethanol has already increased the prices of meat and dairy products.

The future production and use of ethanol that is obtained from cellulosic matter, supplemented by grain ethanol, has been predicted to decrease the need for petroleum fuel1. The main advantages of using cellulosic matter over starch and sugar for ethanol include the abundant supply of cellulosic biomass as compared with the limited supplies of grain and sugar. In addition, starch and sugar that are used for the production of ethanol compete with food supplies. Therefore, it is advantageous to use non-food crops and the waste from food crops for bioethanol production. Furthermore, the use of cellulosic biomass allows bioethanol production in countries with climates that are unsuitable for crops such as sugarcane or corn. For example, the use of rice

Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824, USA. e‑mail: doi:10.1038/nrg2336

Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol

Mariam B. Sticklen

Abstract | Biofuels provide a potential route to avoiding the global political instability and environmental issues that arise from reliance on petroleum. Currently, most biofuel is in the form of ethanol generated from starch or sugar, but this can meet only a limited fraction of global fuel requirements. Conversion of cellulosic biomass, which is both abundant and renewable, is a promising alternative. However, the cellulases and pretreatment processes involved are very expensive. Genetically engineering plants to produce cellulases and hemicellulases, and to reduce the need for pretreatment processes through lignin modification, are promising paths to solving this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass.

nATUre revIewS | genetics volUMe 9 | jUne 2008 | 433 focuS on global challEngES

© 2008 Nature Publishing Group

Middle lamella b c

Primary wall

Plasma membrane


Cellulose microfibril

Hemicellulose Soluble protein

Middle lamella Primary wall

Celluose Hemicellulose


Nature Reviews | Genetics



Secondary wall (S3)

Secondary wall (S2)

Secondary wall (S1)

Primary wall

Middle lamella

Plasma membrane Secondary wall straw for the production of ethanol is an attractive goal given that it comprises 50% of the word’s agronomic biomass.

Serious efforts to produce cellulosic ethanol on an industrial scale are already underway. notably, in 2006, US president George w. Bush announced the goal of reducing 30% of foreign oil requirements by 2030 by using crop biomass for biofuel production. As a result, the Department of energy announced the funding of three major biofuel centres and the establishment of six cellulosic ethanol refineries, which, when fully operational, are expected to produce more than 130 million gallons of cellulosic ethanol per year3,4, other than the Canadian Iorgen plant, no commercial cellulosic ethanol plant is yet in operation or under construction. However, research in this area is underway and funding is becoming available around the world for this purpose, from both governmental and commercial sources. For example, British Petroleum have donated half a billion dollars to US institutions to develop new sources of energy — primarily biofuel crops.

Presently, several problems face the potential commercial production of cellulosic ethanol. First, the high costs of production of cellulases in microbial bioreactors. Second, and most important, are the costs of pretreating lignocellulosic matter to break it down into intermediates and remove the lignin to allow the access of cellulases to biomass cellulose. These two costs together make the price of cellulosic ethanol about two to three-fold higher than the price of corn grain ethanol. Plant genetic engineering technology offers great potential to reduce the costs of producing cellulosic ethanol. First, all necessary cell-wall-degrading enzymes such as cellulases and hemicellulases could be produced within the crop biomass so there would be no need, or only minimal need, for producing these enzymes in bioreactors. Second, plant genetic engineering technology could be used to modify lignin amount and/or configuration in order to reduce the needs for expensive pretreatment processes. Finally, future research on the upregulation of cellulose and hemicellulose biosynthesis pathway enzymes for increased polysaccharides will also have the potential to increase cellulosic biofuel production.

In this review, I first provide an overview of the process of cellulosic ethanol production, including a brief description of the nature of the plant cell wall as a source of biomass, and the enzymes that are used in the cellulosic conversion process. I then focus on the potential for plant genetic engineering to overcome the challenges described above.

The basics of cellulosic ethanol production Feedstock crops and lignocellulosic biomass. The factors that affect the suitability of potential new feedstock crops around the globe for bioethanol production are complex, and relate to country- and region-specific agricultural practices, market forces, and political as well as biological issues. These factors include land availability, locally accepted cropping systems, and types and forms of transportation fuel. In addition, the current status of a particular species in terms of its development as a crop (for example, the development of breeding strategies) is another important issue; in terms of biology, the feedstock crops that have so far been recommended for conversion to cellulosic ethanol have a high amount of cellulosic biomass. These include corn, rice, sugarcane, fast-growing perennial grasses such as switchgrass and giant miscanthus, and woody crops such as fast-growing poplar and shrub willow5,6. Depending on where they are planted, the ideal characteristics of non-food cellulosic crops are: use of the C4 photosynthetic pathway; long canopy duration; perennial growth; rapid growth in spring (to out-compete weeds); high water-usage efficiency; and possibly partitioning of nutrients to subterranean storage organs in the autumn.

The source of lignocellulosic biomass is the plant cell wall (FIG. 1), which has important roles in determining the structural integrity of the plant, and in defence against pathogens and insects7. The structure, configuration and composition of cell walls vary depending on plant taxa, tissue, age and cell type, and also within each cell wall layer8,9. The basic structure of the primary cell wall is a scaffold of cellulose with crosslinking glycans, and there are two types of primary cell wall, which are classified according to the type of crosslinks. Type I walls are present in dicotyledonous plants and consist of equal

Figure 1 | Plant plasma membrane and cell-wall structure. a | Cell wall containing cellulose microfibrils, hemicellulose, pectin, lignin and soluble proteins. b | Cellulose synthase enzymes are in the form of rosette complexes, which float in the plasma membrane. c | Lignification occurs in the S1, S2 and S3 layers of the cell wall.

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© 2008 Nature Publishing Group amounts of glucan and xyloglucan embedded in a matrix of pectin. Type I walls are found in cereals and other grasses, have glucuronoarabinoxylans as their crosslinking glucans, and lack pectin and structural proteins7. Polysaccharides, such as cellulose, hemicellulose and pectin (BOX 1), are abundant in plant primary cell walls and can be hydrolysed to provide fermentable sugars for bioethanol production. They contain various combinations of constituent sugars, all of which are initially produced from glucose7.

The plant secondary cell wall contains cellulose, hemicellulose and lignin (BOX 1). The cellulose microfibrils of the secondary cell wall are embedded in lignin, and in this context function like steel rods embedded in concrete, but with less rigidity. In tree trunks, there are three layers of secondary cell wall, which are called the S1, S2 and S3 lamellae, resulting from different arrangements of cellulose microfibrils. The S1 layer is the outermost layer, is produced first and contains helices of microfibrils. The S2 and S3 are the inner layers and have no helices.

The cellulosic ethanol production process. To produce cellulosic ethanol, lignocellulosic biomass is harvested from the feedstock crop, compacted (fresh or dry) and transported to a cellulosic ethanol refinery where it is stored, ready for conversion. The biomass is then pretreated with extreme heat and/or chemically in order to break it down into intermediates and remove the lignin; this is followed by detoxification, neutralization and separation into its liquid and solid components. The latter are then hydrolysed using enzymes that are produced in microbial bioreactors from bacteria or fungi. Finally, sugars are separated and fermented to produce ethanol (FIG. 2).

Cell-wall-deconstructing enzymes. Cell-wall polysaccharides can be converted into fermentable sugars through enzymatic hydrolysis using enzymes such as cellulases and hemicellulases (BOX 2). lignin is the main barrier to such conversion as it prevents cell-wall hydrolysis enzymes from accessing polysaccharides10,1. Therefore, heat and/or chemical pretreatment processes are being developed and used to break down cell walls into intermediates and remove lignin to allow the exposure of cellulose to cellulases. These enzymes are produced naturally by a range of microbial species. As biofuels research increases in the twenty-first century, an increasing number of bacteria and fungi will be studied for their ability to convert cell-wall polysaccharides into fermentable sugars for biofuels. Many cell-wall-deconstructing enzymes have been isolated and characterized, and more are under investigation, particularly with the hope of finding more enzymes that can resist higher conversion temperatures and a range of pHs during pretreatment — presently the two most important limiting factors in the production of cellulosic ethanol. At present, commercial cellulases are produced as a combination of microbial enzymes. A future goal is the commercial production and use of hemicellulases to increase the output of five- and six-carbon fermentable sugars. Certain commercial hemicellulases are available, but are not suitable for biofuel production.

Genetic manipulation of feedstock crops Genetic engineering of most food crop species is well established, using either Agrobacterium tumefaciens or gene-gun-mediated gene transfer. Among biomass feedstock crops, rice, maize, sorghum, poplar and switchgrass12 are efficiently transformable at commercially acceptable levels. In terms of other relevant species, Agrobacterium has been known to genetically transform dicotyledonous crops (including fast-growing woody plants such as willow and poplar), which are natural hosts for Agrobacterium. Although Agrobacterium does not infect monocotyledonous plants such as cereals and perennial grasses in nature, certain strains have been shown to transform rice, corn, wheat, barley, sorghum and switchgrass. whether Agrobacterium or gene-gun transformation is used, the main challenge is genotype-nonspecific genetic transformation of these crops: among many species and cultivars, generally only one or two are ideal

Box 1 | Key components of the plant cell wall cellulose Plants produce about 180 billion tons of cellulose per year globally, making this polysaccharide the largest organic carbon reservoir on earth. Cellulose makes up 15–30% of the dry mass of primary and up to 40% of secondary cell walls, where it is found in the form of 30 nm diameter microfibrils. Each microfibril is an unbranched polymer with about 15,0 anhydrous glucose molecules that are organized in β‑1,4 linkages (that is, each unit is attached to another glucose molecule at 180° orientation). The microfibrils are lined up parallel to each other and consist of crystalline regions, within which cellulose molecules are tightly packed. Cellulose also has amorphous or soluble regions, in which the molecules are less compact, but these regions are staggered, making the overall cellulose structure strong. So far, cellulose is the only polysaccharide that has been used for commercial cellulosic ethanol production, probably because it is the only one for which there are commercially available deconstructing enzyme mixtures.

Hemicellulose Cellulose microfibrils are coated with other polysaccharides such as hemicellulose or xyloglucans. All dicotyledonous cell walls and about half of monocotyledonous ones consist mainly of xyloglucans. However, in the commelinoid monocotyledons, such as cereals and other grasses, cell walls mostly consist of glucuronoarabinoxylans. Depending on the plant species, 20– 40% of the plant cell‑wall polysaccharides are hemicellulose. Like cellulose, hemicellulose could be converted into fermentable sugars by enzymatic hydrolysis for the production of cellulosic ethanol.

(Parte 1 de 5)