Environmental Engineering Reference
In-Depth Information
To increase productivity of heterologous proteins, plant viral amplicon expression systems have
been developed (see Gleba et al. 2007, for a recent review). In these systems, replication-competent
plant viral components, typically from tobacco
mosaic virus (TMV) [e.g., the TMV RNA-based
overexpression (TRBO) system (Lindbo 2007)] or cucumber mosaic virus (CMV) [e.g., such as
the CMViva system (Sudarshana et al. 2006)], are inserted in the T-DNA to enable the cellular
machinery to amplify the number of copies of the transgene during replication of the viral RNAs.
Expression levels of recombinant proteins using viral amplicon expression systems often greatly
exceed (~10- to 100-fold) that which is possible using traditional constitutive promoters such as the
cauliflower mosaic virus (CaMV 35S) promoter. Harvested leaves (Plesha et al. 2009), individual
leaves on intact plants (Sudarshana et al. 2006), or even entire plants (Gleba et al. 2005; Marillonnet
et al. 2005) can be infiltrated and infected this way, making the process useful for rapid evaluation
of novel enzymes as well as rapid, scalable production of cellulase and hemicellulase enzymes.
Co-infiltration with
Agrobacterium
containing genes capable of suppressing the plant's innate abil-
ity to recognize and shut down foreign gene expression (i.e., gene-silencing suppressors) has also
proven useful to enhance target protein production in the CMViva and TRBO systems.
In addition to the expression technology (stable nuclear, stable chloroplast, transient viral, or
transient agroinfiltration) method used, there are several factors that also influence the enzyme
productivity, activity, stability, and ease of recovery. These include the type of plant promoter, sub-
cellular targeting/localization, design of the gene construct, post-translational modifications, and
gene silencing.
4.2.5 p
lant
p
romotErS
For many biofuel applications, high-level expression of the heterologous enzymes is generally the
goal, particularly for applications such as production of plant-made additive enzymes. An effi-
cient plant transgene expression system, consisting of a promoter, targeting signal peptide, target
gene that has been optimized for expression in the plant host, and transcription terminator, is
essential for high-level production of the target enzyme(s). The choice of promoter system signifi-
cantly influences the production yield by affecting the transcription rate of the target gene. Plant
promoters can be divided into several categories: constitutive, tissue-specific, developmentally
regulated, and inducible promoters. Constitutive promoters directly drive the expression of the
target gene in all tissues and are largely independent of developmental factors. An example is the
well known CaMV 35S promoter. Constitutive expression of a recombinant protein could result
in an additional metabolic burden during plant cell growth and hence reduce the plant growth
rate. The characteristics of the recombinant protein might also affect the plant cells' physiology
because of intrinsic toxic properties of the product on the host cells or interference with host cell
metabolism.
Inducible promoters are modulated by the presence of specific external factors or compounds
such as light, temperature, wounding, or the concentrations of metal ions, alcohols, steroids, herbi-
cides, etc. Such regulated expression systems are advantageous because they allow the plant growth
and protein production phases to be independently optimized. This approach is particularly attrac-
tive when product synthesis is deleterious to plant growth and/or viability. Furthermore, because
expression of a foreign gene linked to an inducible promoter can be induced at a specific stage dur-
ing the plant growth, there is less potential for post-transcriptional gene silencing (PTGS) found in
gene expression systems that use constitutive promoters in transgenic plants (Vaucheret et al. 2001;
Vaucheret and Fagard 2001). Various inducible promoters have been developed for use in plants (for
example, see Boetti et al. 1999; Zuo and Chua 2000; Huang et al. 2001; Padidam 2003; Tang et al.
2004). For field production of lignocellulolytic enzymes, chemically inducible promoters are likely
to be costly and may have negative environmental impacts depending on the system. However,
chemically inducible promoters may have use in applications in which enzyme expression is initi-
ated after harvest in a production facility.
