Molecular Biotechnology: Principles and Applications - Glick, B. R., and Pasternak, J. J. 2002

Molecular Biotechnology of Microbiological Systems
Plant Genetic Engineering: Applications
Plants as Bioreactors

Plants produce vast amounts of biomass and are straightforward to cultivate, making it a logical step to develop Transgenic Plants capable of synthesizing commercially valuable Proteins and chemicals. Unlike recombinant Bacteria, which require large bioreactors, highly skilled personnel, and expensive equipment, growing crops demands minimal financial investment and unskilled labor. The primary challenge in using plants as bioreactors lies in extracting the introduced Gene product from the bulk plant tissue and comparing the production costs of the target protein between transgenic plants and microorganisms. Experimental systems have already been established to produce Monoclonal Antibodies, functional antibody fragments, and poly-ß-hydroxybutyrate—a polymer utilized in the manufacturing of biodegradable Materials.

Antibodies

The production of Introduction/42.html">Antibodies and Their fragments using transgenic plants offers several advantages over synthesis in recombinant microbial Cells. Plant transformation is stable, with foreign DNA integrating almost irreversibly into the plant genome, whereas most microorganisms are transformed using Plasmids that can be lost during prolonged or large-scale Fermentation. Furthermore, the Processing and folding of foreign proteins in plants closely mirror those in animal cells, whereas bacteria struggle with the processing, folding, and post-translational modification of eukaryotic proteins. Additionally, large-scale plant cultivation is cost-effective and unconstrained by the limitations of fermentation processes. Finally, conditions can be engineered to target the synthesis of foreign proteins in seeds, where their structural integrity is preserved for extended periods.

Polymers

Large-scale bacterial synthesis of poly-ß-hydroxybutyrate, a polymer used to produce biodegradable plastics, is quite costly. Consequently, investigating whether this polymer could be produced via transgenic plants was of considerable interest. In bacteria such as Alcaligenes eutrophus, Poly-β-hydroxybutyrate is synthesized from acetyl-CoA in a three-stage pathway catalyzed by three Enzymes (see Fig. 12.22), the genes for which form a single Operon. Because plants cannot process polycistronic operon transcripts, each gene was cloned individually and integrated into the chloroplast DNA of Arabidopsis thaliana. METABOLISM/14.html">Chloroplasts were selected because prior experiments demonstrated that cytoplasmic synthesis yielded low quantities of the polymer and often resulted in stunted plant growth. Moreover, chloroplasts are capable of accumulating another biopolymer, starch.

DNA fragments encoding the chloroplast transit peptide of the pea ribulose-1,5-bisphosphate carboxylase small subunit were fused to each of the three poly-β-hydroxybutyrate genes, and each gene was placed under the transcriptional control of the cauliflower mosaic virus 35S promoter. These genes were introduced into A. thaliana plants using Ti plasmid-based binary vectors. Two transgenic lines, each carrying a different foreign gene, were crossed to generate plants containing two foreign genes integrated into their chloroplast DNA. This dual-gene transgenic line was subsequently crossed with a plant carrying the third foreign gene, and progeny containing all three bacterial poly-β-hydroxybutyrate genes were selected. In mature leaves of certain transgenic plants expressing all three bacterial genes, over 1 mg of poly-β-hydroxybutyrate per gram of fresh leaf tissue was synthesized. This achievement marks a crucial first step toward developing crops capable of producing high yields of poly-β-hydroxybutyrate.

Foreign Proteins Accumulating in Seeds

Oleosins, or oil body proteins, are found in the seeds of various plant species. Highly hydrophobic in nature, they stabilize oil bodies as discrete structures, while their N- and C-terminal domains are more hydrophilic than the internal region and extend into the aqueous Cytoplasm. Genetic Engineering offers a strategy to construct recombinant fusion proteins comprising an oleosin and a Water-soluble protein (Fig. 18.19). These recombinant proteins accumulate within oil bodies, facilitating relatively straightforward purification. Because the water-soluble protein is exposed to the aqueous environment, it can be cleaved off if necessary. This approach significantly reduces the cost of purifying proteins synthesized in plant systems.

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Fig. 18.19. A recombinant protein composed of an oleosin and a water-soluble protein. The oleosin exhibits a high affinity for the plant seed oil body, while its N- and C-termini, along with the secondary protein, are hydrophilic and exposed to the aqueous environment.