Plant-based material, protein and biodegradable plastic

Fibrous proteins from spiders, proteins with synthetic multiple repeats and mammalian structural proteins such as collagen have been produced in transgenic plants. Recent advances in the production of biodegradable plastic in plants also show the potential of molecular farming for research into and production of materials. Selection of a growing variety of such products, optimization of expression, and the development of effective purification strategies will further promote this growing field of biotechnology. Introduction Plant materials such as cellulose, starch or even latex from rubber trees have traditionally been used for several purposes by mankind. In the past century, the chemical industry opened the way to modify plant-based materials, and products such as cellulose acetate or cellulose nitrate were invented. The development of genetic engineering technology and efficient transformation methods for several crops has opened the way for the genetic modification of traditional plant products; for example, the development of modified starch. Furthermore, interesting proteins originating from animals and novel polymers can now be synthesized in transgenic plants New protein-based materials from plants Naturally occurring proteins often exhibit interesting properties as fibers or adhesives. For example, larvae from insects produce fibrous glues in the form of sericin (produced by Bombyx mori for example Silk proteins from spiders and insects and their production in transgenic plants During evolution, spiders have become highly diverse in production and use of silks (for review see We believe that transgenic plants are powerful protein factories that can overcome the problems mentioned above. Therefore, we constructed stable transgenic tobacco and potato lines to express various synthetic spider-silk genes ranging from 420 to 3600 basepairs in size. The genes were assembled so as to achieve very high homology to the native MaSpI gene from Nephila clavipes (more than 90%). Accumulation of up to 2% spider-silk protein of total soluble protein (TSP) in the endoplasmic reticulum (ER) of tobacco leaves was achieved, and this production was relatively independent of the size of the spider-silk protein. The recombinant spidroins exhibited extreme heat stability. This property, together with resistance against acidification and fractionated ammonium sulfate precipitation, was used to purify the plantproduced spidroins by a simple and efficient procedure A protocol similar to that described by Scheller et al. A new purification strategy has been developed using the unique properties of ELP fusions. First, the spider silk proteins from plants were enriched by boiling. We used heat treatment at 95 8C for 60 min and clearance by centrifugation of tobacco leaf extracts to enrich the spider-silk-ELP fusion protein in the supernatant. For the selective precipitation of the SO1-100XELP, NaCl was added to a final concentration of 2 M and the temperature was raised to 60 8C. Under these conditions, the recombinant spider-silk-elastin fusion proteins aggregated and could be precipitated by centrifugation. Cellular proteins remained in the supernatant. The precipitated recombinant proteins were resolved at a lower temperature and without salt to a final concentration of 1 mg/ml of nearly homogenous product. Dialysis against water and drying led to the formation of silk-proteins in the form of storable membranes. Extraction of 1 kg tobacco leaf resulted in 80 mg pure recombinant spider-silk-elastin protein. Transgenic plants for spider silk and spider-silkelastin fusion proteins were phenotypically indistinguishable from wildtype plants. In addition, the expression of recombinant proteins in second generation plants was comparable to that in first-generation plants. In summary, expression and purification systems for plant-based silk proteins have been developed. Expression in storage organs such as tubers and seeds has been generally shown. The selection of suitable spidroin and fibroin sequences for defined applications and commercialization is still at an early stage. To date, there are no reports of the successful spinning of plant-derived silk proteins. The first example of possible medical use has, however, been reported Production of mammalian-derived structural proteins in plants Elastin is a strong elastic fiber that is present in ligaments and in arterial walls. Synthetic proteins that are constructed from multiple repeats also show elastic properties. Such polymers have also been shown to prevent postsurgical adhesions and scars in rats Collagens form a family of extra-cellular matrix proteins that are detectable in all connective tissues of mammals. They represent 30% of total body weight of proteins. Collagen molecules consist of three polypeptides called a chains, which assemble to form triple helical domains. The collagen a chains contain the repeating triple sequence Gly-X-Y, where X and Y are frequently proline and hydroxyproline residues, respectively. This sequence is necessary for the correct formation of the triple helix Biodegradable plastics and PHB from transgenic plants Until now, the commercial production of PHBs has been restricted by high production costs, making bacterial fermentation of PHB at least five times more expensive than chemical synthesis of polyethylene. Plants might be a suitable alternative for the low-cost production of PHBs. It was calculated that polymer concentrations in plants will need to reach at least 15% of dry weight for economically useful production. The general feasibility of the production of PHB in plants was shown in 1992 in pilot experiments in which PHB synthesis was directed into the cytoplasm of Arabidopsis thaliana. The accumulation of 0.1% of leaf dry weight as PHB was low in comparison to PHB accumulation in bacteria. Nevertheless, the plant-produced PHB formed granules whose size and appearance were comparable to those of the granules formed by bacteria. Unfortunately, these Arabidopsis plants displayed a stunted-growth phenotype, leading to the assumption that the redirection of cytoplasmic acetyl-CoA to PHB production results in insufficient production of isoprenoids and flavoids, which are needed for normal growth Major improvements in PHB accumulation were achieved by targeting the bacterial PHB-biosynthesis pathway to other compartments of plant cells in transgenic plants. In 1994, Nawrath et al. [58] produced constitutive expression of the enzymes of the PHB biosynthetic pathway in chloroplasts of A. thaliana by nuclear transformation and fusion of transit peptides to the three PHB enzymes. They reported PHB accumulation of up to 14% of leaf dry weight. In contrast to the cytoplasm, plastids are compartments in which there is a high flux of acetyl-CoA, which is mainly utilized as precursor for the fatty-acid biosynthesis of membrane lipids. Plants in which the bacterial PHBbiosynthesis pathway was targeted to plastids showed nearly wildtype growth and fertility but, in high PHB producers, chlorosis of the leaves was observed. One crucial issue that was not addressed by Nawrath et al. [58] is whether stable, high-level polymer production in plants could be maintained over many generations. Later, transgenic A. thaliana plants were directly screened for high accumulation of PHB in the plastids of leaves using gas chromatography and mass spectrometry (GC-MS). Accumulation of up to 40% of leaf dry weight as PHB Expression systems for the production of biopolymers in plants: ER Leaves Current Opinion in Plant Biology • Ubiquitous expression and expression of transgenic proteins in storage organs (e.g. tubers, seeds) • Accumulation in 'optimal' compartments (e.g. ER, chloroplast) • Production of plastic-like biomaterials in chloroplasts and peroxisomes The syntheses of PHB in transgenic cotton and flax were examples of introducing a new polymer into existing fibers There is an obvious need to further improve transgenic PHB production by producing higher accumulation without side-effects such as chlorosis, male sterility and growth retardation, but genetic engineering has already yielded the first examples of the production of PHAcopolymers in transgenic plants (SLC-and MCL-PHA). PHB is a highly crystalline polymer whose usefulness is restricted by its physical properties, being relatively stiff and brittle. SLC-and MCL-PHA are less stiff and brittle, even having rubber-like properties, and therefore exhibit a greater potential for commercial applications. In 1999, Slater and colleagues IlvA from E. coli encodes the protein threonine deaminase and converts threonine to 2-ketobutyrate. The endogenous pyruvate dehydrogenase complex catalyzes the synthesis of propionyl-CoA from 2-ketobutyrate. Acetyl-CoA and propionyl-CoA are then further processed and copolymerized into PHBV In contrast to peroxisomal fatty acids in mammals, those in plants can be completely oxidized by the acyl-CoA oxidase (SC-ACOX) to acetyl-CoA (a process known as b-oxidation