Plant biotechnology
Molecular farming and metabolic engineering promise a new
generation of high-tech crops
Editorial overview
Pal Maliga and Ian Graham
Current Opinion in Plant Biology 2004, 7:149–151
1369-5266/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2004.01.016
Pal Maliga
Waksman Institute, Rutgers University, 190
Frelinghuysen Road, Piscataway, New Jersey
08854-8020, USA
e-mail: maliga@waksman.rutgers.edu
Pal Maliga’s research group has
developed the technology of plastid
transformation in higher plants using
tobacco as the model system. They
currently use plastid transformation to
characterize the plastid’s transcription
machinery, to understand the rules of
mRNA translation and RNA editing,
and to explore biotechnological
applications.
Ian Graham
CNAP, Department of Biology (Area 7),
University of York, PO Box 373,
York YO10 5YW, UK
e-mail: iag1@york.ac.uk
Ian Graham works on the metabolic
regulation of primary carbon
metabolism. He is particularly
interested in the mechanisms
regulating storage reserve
accumulation and breakdown in
Arabidopsis seed. His group uses
forward and reverse genetic
approaches to identify gene functions
that are potentially useful in the
production of high yields of novel
products in crop plants.
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The first generation of transgenic crops was based on the early tools of plant
genetic engineering. These crops were (and still are) necessarily relatively
unsophisticated and carry only a few simple transgenic traits, such as
herbicide resistance or an insecticidal protein. GM crops have been rapidly
embraced by farmers because of the benefits from lower inputs and
increased productivity and they became cash machines for biotech compa-
nies who had the vision to invest in the new technologies, but the opponents
of GM have been working hard to make the uninformed public suspicious of
transgenic plants. Public opinion on this issue will ultimately be based on
individual consumers doing their own risk–benefit analysis. Unless the
consumer can see and appreciate direct benefits of GM crops then any
perceived risk, no matter how small or misplaced, will outweigh the benefits.
The new areas of molecular farming and metabolic engineering promise
more readily identifiable benefits for consumers in the form of inexpensive
safer medication, more wholesome food and environmentally sustainable
industrial feedstocks. We have commissioned reviews that highlight the
recent successes and the challenges of these emerging areas of plant
biotechnology.
The driving force behind molecular farming is production at costs that are
much lower than those of traditional agriculture. In their review on plant-
based production of biopharmaceuticals, Fischer and colleagues (pp. 152–
158) give an overview of the emerging plant production platforms, including
leafy crops, cereal grain, fruits, vegetables and cultured cells, and their
advantages and disadvantages. The review addresses the various approaches
employed to improve protein yields and problems related to downstream
processing. In addition to references, the review contains links to websites
for an easy follow-up. This review on production systems and related
reviews on target proteins [1,2] communicate the opportunities and excite-
ment in this rapidly developing area.
One of the new production platforms, as reviewed by Franklin and Mayfield
(pp. 159–165), is Chlamydomonas reinhardtii, a unicellular alga. C. reinhardtii is
the only plant in which transformation has been accomplished in all three
DNA-containing genetic compartments (i.e. nucleus, plastids and mitochon-
dria). While study of the photosynthetic machinery in Chlamydomonas
flourishes on the basis of genetic and biochemical approaches [3], biotech-
nological applications in Chlamydomonas have been curiously absent. We
now learn that the problems of expressing recombinant proteins from
nuclear genes relate to the need for codon optimization and the need to
overcome transgene silencing. Codon optimization is also necessary for
the expression of recombinant proteins in Chlamydomonas chloroplasts.
Current Opinion in Plant Biology 2004, 7:149–151
150 Plant biotechnology
This contrasts with the higher-plant chloroplast system,
in which cDNAs from diverse organisms could be
expressed without codon optimization [4]. Now that
the bottlenecks have been identified, and supported
by a genomics effort, the Chlamydomonas system is likely
to become the system of choice for at least some
applications.
Decker and Reski (pp. 166–170) review the expression
of recombinant proteins in the closed moss Physcomitrella
patens bioreactor. Unique features of the moss system are
the opportunities for targeted gene deletion and the
purification of secreted proteins from the culture med-
ium. Targeted gene deletion has been employed to knock
out nuclear genes for glycosylation, the first step towards
the long-term goal of re-engineering the protein modifi-
cation machinery in plants for ‘humanization’ of plant-
produced pharmaceuticals, setting a new standard for all
plant expression systems.
Most therapeutic proteins require some form of posttrans-
lational modification for bioactivity. Plants are preferred
over prokaryotic hosts, such as E. coli, for the expression of
recombinant proteins because most co- and posttransla-
tional protein maturation events, including signal-peptide
cleavage, protein folding, disulfide-bond formation and
glycosylation, are similar in plants and mammals. Gomord
and Faye (pp. 171–181) discuss the similarities in and
differences between posttranslational protein modifica-
tion in plant and mammalian hosts that are relevant to the
posttranslational modification of therapeutic proteins in
plants. They outline new approaches for engineering
posttranslational modification to obtain humanized plant-
made pharmaceuticals in plant hosts.
Plant viral vectors are the system of choice when the
recombinant protein is needed within the shortest pos-
sible period of time. Gleba, Marillonnet and Klimyuk
(pp. 182–188) review recent advances in the use of plant
viral vectors for the expression of recombinant proteins.
They describe vector systems for small-scale ‘desktop’
protein production as well as for production on an indus-
trial scale. The trend is to incorporate some of the viral
functions into the plant nuclear genome to improve
protein expression. The new virus vectors are safer, as
they are able to function only in a genetically modified
host and do not yield mature viral particles that are
capable of secondary infection. Gleba et al. discuss
RNA-mediated gene silencing and the interference of
viral vectors with the host’s biosynthetic processes as
factors that affect protein yield.
The benefits of lower production costs in plants are
obvious and, in some cases, plants may be the only
practical way to produce a certain medication, but nobody
wants these benefits at the price of finding bioactive
molecules in their food [5]. Thus, the search for new
Current Opinion in Plant Biology 2004, 7:149–151
contained production systems and the tightening up of
transgene containment in existing systems are ongoing.
Mascia and Flavell (pp. 189–195) give an industry per-
spective of safe and acceptable strategies for producing
foreign molecules in plants. The most effective of these
should be genetic containment, in which the seed of the
production plant does not germinate unless treated with a
chemical or, better yet, does not contain a viable embryo.
Reading this sentence may bring back memories of the
discussions about the conflict of interest between the
subsistence farmers who want to replant the seed that
they save and biotech companies who want to protect
their hybrid seed with ‘terminator technologies’. How-
ever, ensuring the safety of the food supply through the
genetic containment of plants that produce foreign mole-
cules and providing seed for subsistence farmers should
be compatible goals.
Shifting attention from recombinant proteins to meta-
bolic engineering introduces a new set of challenges.
Foremost among these is a need for better understanding
of basic metabolic processes. Our current understanding
of plant metabolism is primarily based on a concept of
fairly independent linear biochemical pathways that are
controlled by one or a few regulatory enzymes. In reality,
plant metabolism is a complex web of interacting path-
ways and processes, which equip the plant with a sig-
nificant degree of plasticity that is essential for survival
in an ever-changing environment. What controls flux
through a given pathway is not always obvious. There-
fore, even though a metabolic-engineering approach to
increase the amount of a specific intermediate or product
of a known biochemical pathway often appears relatively
straightforward at first sight, the result can be quite
unpredictable. Trethewey (pp. 196–201) highlights the
‘trial and error’ nature of many metabolic-engineering
programmes. He argues for the greater adoption of a
systems-biology approach, involving extensive meta-
bolic profiling, rather than limiting analysis to metabo-
lites that are associated with a particular pathway.
Trethewey emphasises the impracticality, owing to the
diversity of chemical species, of developing profiling
technologies that measure all of the metabolites in a
sample and calls for the further development of targeted
technologies for better coverage. Such technologies will
provide valuable information for both the identification
of target genes and evaluating the consequences of
manipulating such target genes on the metabolic web.
Such information will almost certainly be required for the
regulatory approval of new crops and products that result
from metabolic engineering.
Rather than attempting to manipulate flux through a
biochemical pathway by altering levels of individual
enzymes, a more effective approach may be to target
regulatory proteins, such as transcription factors, that
control the transcription of the pathway genes. Such an
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approach is based on the assumption that a common
mechanism is responsible for the coordinate regulation
of genes from a given pathway. There is much to be
learned: of the approximately 1700 Arabidopsis transcrip-
tion factors only about 7% have been genetically char-
acterized [6]. Broun (pp. 202–209) reviews our current
knowledge of transcription factors that regulate metabolic
pathways and considers their utility as tools for metabolic
engineering. A recent notable success of this approach has
been the use of two regulatory proteins from maize to
engineer elevated levels of health-beneficial flavonols in
tomato fruit. Global transcript profiling shows that the
overexpression of pathway regulators often also affects
genes that are unrelated to the target pathway including,
for example, transporters that are possibly associated with
metabolite partitioning and storage. This could lead to
the identification of new targets for engineering and also
serves to remind us of the potential pleiotropic effects
that can complicate the use of such an approach.
Although engineering one or a few steps in a biochemical
pathway does not always achieve the desired outcome,
successes have been achieved by this approach. Among
these, the recent progress on the genetic modification of
crops to produce novel starches with improved function-
ality is one of the most notable. Jobling (pp. 210–218)
describes the recent advances in our understanding of
starch biosynthesis that have led to the rational design of
approaches to modify the composition and structure of
starch granules. These approaches have led to the devel-
opment of potato starch that has elevated freeze-thaw
stability, thus avoiding the need for chemical crosslinking
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Editorial overview Maliga and Graham 151
and stabilisation with associated benefits to consumers
and the environment; of high-amylose potato starch that
does not swell when heated; and of modified amylopectin
starch that gelatinises at a very low temperature. Under-
standing the complex process of starch biosynthesis,
including how the individual enzymes interact (possibly
as complexes), will lead to opportunities for additional
rational design.
The limited selection of topics covered in this plant
biotechnology section gives only a glimpse of the new
generation of high-tech crops that are in the production
pipeline and the science that underpins them. Many of
these crops will benefit consumers directly, being the
source of medicine or improved food, giving high-tech
GM crops the positive image that they deserve.
References
1. Ma JKC, Drake PMW, Christou P: The production of
recombinant pharmaceutical proteins in plants. Nat Rev Genet
2003, 4:794-805.
2. Carter JE, Langridge WH: Plant-based vaccines for protection
against infectious and autoimmune diseases. Crit Rev Plant Sci
2002, 21:93-103.
3. Rochaix JD: Chlamydomonas, the model system for studying
the assembly and dynamics of photosynthetic complexes.
FEBS Lett 2002, 529:34-38.
4. Maliga P: Plastid transformation in higher plants. Annu Rev
Plant Biol 2004, 55:289-313.
5. Ellstrand NC: Going to ‘‘great lengths’’ to prevent the escape of
genes that produce speciality chemicals. Plant Physiol 2003,
132:1770-1774.
6. Riechmann JL, Ratcliffe OJ: A genomic perspective on plant
transcription factors. Curr Opin Plant Biol 2000, 3:423-434.
Current Opinion in Plant Biology 2004, 7:149–151