Environmental Engineering Reference
In-Depth Information
4
In Planta
Production of Cell
Wall Degrading Enzymes
Karen A. McDonald
University of California, Davis
contents
4.1 Introduction ............................................................................................................................ 55
4.2 Plant Biotechnology Approaches and Considerations ............................................................ 59
4.2.1 Stable Nuclear Transformation ................................................................................... 59
4.2.2 Chloroplast Transformation ........................................................................................ 60
4.2.3 Transient Viral Expression ......................................................................................... 60
4.2.4 Transient Agrobacterium-Mediated Expression ......................................................... 60
4.2.5 Plant Promoters ........................................................................................................... 62
4.2.6 Subcellular Targeting/Localization ............................................................................ 63
4.2.7 Optimization of Gene Constructs ............................................................................... 63
4.3 Plant-Made Cell-Wall Degrading Enzymes ........................................................................... 63
4.4 Conclusions ............................................................................................................................. 70
References ........................................................................................................................................ 71
4.1 IntroductIon
As a result of requirements of the Energy Independence and Security Act of 2007, by the year 2022,
36 billion gal of biofuels will need to be produced to meet liquid transportation fuel demand, with at
least 21 billion gal of “advanced biofuels,” defined as renewable fuels derived from non-cornstarch
sources achieving greater than 50% reduction in greenhouse gas (GHG) emission. In meeting this
challenge, cellulosic biofuels will likely be a major contributor because of the resource potential of
cellulosic feedstocks, which are estimated to be over 1 billion dry tons per year in the United States,
sufficient to produce enough biofuels to replace 30% of current demand for transportation fuels
(Perlack et al. 2005). The process of breaking down a complex polysaccharide carbohydrate (such
as starch, cellulose, or hemicellulose) into monosaccharide components that can be fermented into
biofuels is called saccharification. Saccharification of corn starch, alpha-linked glucose polymers,
is relatively easy compared with breaking down the beta-linked glucose polymers that make up the
structurally aligned and hydrogen-bonded cellulose polymers in cellulose microfibrils. In addition,
the biological decomposition of cellulosic biomass presents a formidable challenge because of the
recalcitrance of cellulose microfibrils embedded within the complex, heterogeneous structure of the
plant cell wall composed of cellulose, hemicellulose, and lignan (Figure 4.1). This necessitates not
only costly, energy-intensive, and environmentally detrimental biomass pretreatment steps (usually
involving high temperatures, acids, and/or enzymes) to increase the accessibility and effectiveness
of cellulase enzymes, but also high cellulase enzyme loadings (ratio of enzyme mass to biomass),
currently approximately 100 times the loadings used for corn starch saccharification.
For the enzymatic conversion routes, enzyme preparations (referred to as cellulases), composed of
mixtures of endoglucanses, exoglucanases, hemicellulases, and/or beta-glucosidases, are produced
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