Environmental Engineering Reference
In-Depth Information
constitutively but is sequestered in a subcellular compartment away from the cell wall or is not active
under growth conditions (see recent review by Sticklen, 2008). Alternatively, the transgene could be
under the control of a chemically or environmentally inducible promoter.
4.2.2 c
hloroplaSt
t
ranSformation
Chloroplast genetic engineering utilizes site-specific homologous recombination to insert the trans-
gene into the chloroplast genome. Because there are approximately 100 chloroplasts per cell and each
chloroplast contains approximately 100 identical copies of the chloroplast genome, this approach
has the potential to generate stable transformants that have tens of thousands of copies of the trans-
gene. High copy number, combined with the absence of epigenetic effects such as gene silencing,
have led to very high expression levels (>46% TSP) of heterologous proteins using this approach
although most of the studies have been done in tobacco. Because the chloroplast is maternally inher-
ited, there is reduced potential for gene flow and biosafety is enhanced. Other advantages of this
approach, particularly for large-scale production of cell-wall-modifying/degrading enzymes, is the
ease of transgene stacking to produce multiple enzymes in the same plant and the sequestration of the
enzyme products in the chloroplast where they cannot harm the cell wall during plant growth. One
disadvantage of this approach is that the chloroplast is only capable of bacterial-like processing, so it
is not possible to produce glycosylated enzymes. There is evidence, at least in some cases, that glyco-
sylation of fungal glycosyl hydrolase enzymes is important for stability and activity of the enzymes
(Jeoh et al. 2008). In addition, the high expression levels that have been achieved in transplastomic
plants have often resulted in slow growth and/or other detrimental/lethal effects on the plants.
4.2.3 t
ranSiEnt
v
iral
E
xprESSion
Recombinant plant viruses have been recognized as a very useful means for high level, rapid expres-
sion of foreign proteins in plants (Scholthof et al. 1996). In this approach, recombinant plant virions
(or alternatively infectious RNA) containing the transgene (usually as an insert or as a replacement
of a nonessential viral gene) of interest are used to infect a compatible plant host, typically through
a mechanical process such as rubbing or spraying the virion solution, which also contains an abra-
sive agent such as carborundum or diatomaceous earth, onto plant leaves. The virus infects the
host, and during replication it generates large amounts of viral nucleic acids including the transgene
insert, which may spread cell to cell and systemically throughout the plant within a few days, and
expresses the foreign protein during replication. The major advantages of this approach are that it
is fast (recombinant protein is produced in a matter of a week), very efficient (high transgene copy
number and the ability to spread systemically allow many cells in the plant to act as hosts, not just
those initially infected), and does not require the deployment of transgenic crops in the field (non-
transgenic plants are used as the hosts). It is also a preferred method for production of proteins, such
as cellulases or cell-wall-modifying proteins, that could be detrimental to plant health. However, for
most viruses there is a limitation to the gene insert size of approximately 1 kb. The stability of the
transgene during serial propagation and systemic spread can be an issue, plant gene silencing mech-
anisms can limit recombinant protein production, and biosafety aspects need to be assessed for each
system and production method. Production of multiple enzymes within the same plant host may
require the use of “noncompeting” recombinant viral vectors as described by Giritch et al. (2006).
4.2.4 t
ranSiEnt
A
grobActerium
-m
EdiatEd
E
xprESSion
Another transient plant-based heterologous expression process known as “agroinfiltration” allows
for the rapid, high-level expression of multiple recombinant proteins in harvested plant tissues
as well as tissues of intact plants (Sudarshana et al. 2006; Plesha et al. 2007, 2009). This pro-
cess utilizes
Agrobacterium-
mediated transformation, a highly efficient process for introducing