Environmental Engineering Reference
In-Depth Information
hydrolysis, independent of pretreatment and/or addition of exogenous enzymes, expression
in
planta
may produce some benefits, including
• A reduction in process costs and/or exogenous enzyme requirements because of reduction
in the amount of exogenous enzyme needed to achieve the same glucan conversion,
• An increase in biomass digestability,
• Reduced severity requirements for pretreatment, and/or
• Improved yield of the desired biofuel.
This chapter provides an overview of plant biotechnology approaches and considerations for
in
planta
production of cell-wall-degrading enzymes for biofuel applications and provides an updated
review of recent work on the production of plant-made cellulase and hemicellulase enzymes
produced using stable nuclear transformation, chloroplast transformation, and transient expres-
sion. Sainz (2009) and Taylor et al. (2008) also provide recent reviews of plant-expressed glycosyl
hydrolase enzymes in stably transformed plants. Expression of enzymes in other photosynthetic
systems such as moss, algae, cyanobacteria photobioreactors, and plant cell suspension cultures is
also possible but is not included here because these systems would likely not be as scalable or eco-
nomically feasible for biofuel applications.
4.2 Plant BIotechnoloGy aPProaches and consIderatIons
Heterologous enzymes can be expressed in plants from transgenes that have been stably inserted
into the plant nuclear genome or the chloroplast genome, or from expression vectors that are intro-
duced transiently using recombinant plant viruses or recombinant agrobacterial vectors. These
alternative approaches and the implications for plant-produced enzymes for biofuels applications
are outlined below.
4.2.1 S
taBlE
n
uclEar
t
ranSformation
Foreign DNA can be stably inserted in the plant nuclear genome of a plant cell using various meth-
ods such as
Agrobacterium tumefaciens
-mediated transformation, microprojectile bombardment,
electroporation, free DNA uptake under certain conditions, and other forced penetration methods.
However, the transgene is generally inserted at a random position in the plant nuclear genome result-
ing in “position effects” as well as the potential for multiple inserts. Both of these effects can influ-
ence the expression level observed for the heterologous protein, requiring extensive screening to
identify the most productive transgenic line. The efficiency of transformation and the ability to regen-
erate whole transgenic plants depends strongly on the plant species and choice of explant, the trans-
formation method and conditions, selection media/conditions, and regeneration methods. The main
advantages of using stably transformed plants for production of cell-wall degrading and/or modify-
ing enzymes is that once a transgenic line is established, the foreign gene is passed to subsequent
generations allowing for relatively inexpensive, easily scalable, long-term production of the enzymes
within field-grown plants. The main disadvantages are the long time frames (typically 6 months
to several years) required to establish the high expressing transgenic line, the potential negative
impact of the transgenes and/or transgenic crop in the environment, concerns about the transfer of
transgenes from genetically modified (GM) to non-GM relatives through cross-fertilization, and/or
the possibility of introduction of the GM material into the food/feed supply chain. Wolt (2009) pro-
vides a recent review on environmental risk assessment for transgenic biofuels crops. For transgenic
plants expressing cell-wall-degrading or cell-wall-modifying enzymes, it is particularly important to
ensure that these enzymes are not active on their host cell walls to avoid detrimental effects on plant
growth and viability. Previous work on heterologous expression of cell-wall-deconstructing enzymes
in plants has focused on production in stably transformed plants in which the enzyme is produced
