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

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 companies 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 plantbased 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 excitement 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 mitochondria). While study of the photosynthetic machinery in Chlamydomonas flourishes on the basis of genetic and biochemical approaches [3], biotechnological 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

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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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 medium. 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 modification machinery in plants for ‘humanization’ of plantproduced pharmaceuticals, setting a new standard for all plant expression systems. Most therapeutic proteins require some form of posttranslational modification for bioactivity. Plants are preferred over prokaryotic hosts, such as E. coli, for the expression of recombinant proteins because most co- and posttranslational 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 modification 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 plantmade pharmaceuticals in plant hosts. Plant viral vectors are the system of choice when the recombinant protein is needed within the shortest possible 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 industrial 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 perspective 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’. However, ensuring the safety of the food supply through the genetic containment of plants that produce foreign molecules and providing seed for subsistence farmers should be compatible goals. Shifting attention from recombinant proteins to metabolic 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 pathways and processes, which equip the plant with a significant degree of plasticity that is essential for survival in an ever-changing environment. What controls flux through a given pathway is not always obvious. Therefore, 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 metabolic profiling, rather than limiting analysis to metabolites 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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Editorial overview Maliga and Graham 151

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 transcription factors only about 7% have been genetically characterized [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 functionality 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 development of potato starch that has elevated freeze-thaw stability, thus avoiding the need for chemical crosslinking

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. Understanding 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. Carter JE, Langridge WH: Plant-based vaccines for protection against infectious and autoimmune diseases. Crit Rev Plant Sci 2002, 21:93-103. Rochaix JD: Chlamydomonas, the model system for studying the assembly and dynamics of photosynthetic complexes. FEBS Lett 2002, 529:34-38. Maliga P: Plastid transformation in higher plants. Annu Rev Plant Biol 2004, 55:289-313. Ellstrand NC: Going to ‘‘great lengths’’ to prevent the escape of genes that produce speciality chemicals. Plant Physiol 2003, 132:1770-1774. Riechmann JL, Ratcliffe OJ: A genomic perspective on plant transcription factors. Curr Opin Plant Biol 2000, 3:423-434.

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Current Opinion in Plant Biology 2004, 7:149–151

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