Protein Based Biomaterials

Protein-based biomaterials have a wide area of application, ranging from tissue engineering, drug carriers, coatings and glues to elastomers and fibres, dispersants, thickeners and additives to hydrogels. Two important target proteins are described here in detail: fibrous proteins (e.g. spider silk) and non-ribosomally produced poly-amino acids like cyanophycin. Fibrous Proteins

Fibrous proteins contain short blocks of repeated amino acids and can be regarded as elaborate block co-polymers with unique strength-to-weight, adhesive or elastic properties (Hüang et al. 2007; Sanford and Kumar 2005; Scheibel 2005). Well known fibrous proteins are elastin, resilin, collagen, keratin, mussel adhesive proteins and wheat glütenin (Kiick 2007). A hüge combinatorial range is available by combining repeated sequences of the various natural fibrous proteins, or even using synthetic gene sequences or changing the linker elements between the repeated sequences (Holland et al. 2007; Nagapudi et al. 2005).

Natural silk fibres are mainly produced by a variety of silkworms (Altman et al. 2003; Shao and Vollrath 2002) and spiders (Perez-Rigueiro et al. 2003; Rising et al. 2005; Vollrath 2000). The development of novel silk-based fibres has mainly focused on the silks produced by the golden orb-weaving spider Nephila clavipes, which synthesizes different kinds of silks for several purposes, e.g. weaving cocoons, as a dragline or constructing a web (Hinman et al. 2000; Vollrath and Knight 2001). The main focus lays on the silks of the dragline, the main structural web silk and the spider's lifeline, because of their high tensile strength. This is comparable to that of the synthetic superfibre Kevlar, but it additionally shows high elasticity (Tirrell 1996), useful for industrial and medical purposes. Dragline spider silk consists of repeated sequence blocks of various types (Huang et al. 2007). The GGX motif probably forms a p10 helix, while the GPGXX motif is thought to form a p-turn spiral. Effective high-level and stable expression of silk proteins in fast-growing micro-organisms such as yeast and bacteria leads to difficulties, like the formation of inclusion bodies or distinct codon usage. In addition, bacterial production is genetically unstable due to recombination, resulting from the highly repetitive genes encoding the repetitively composed spider silk proteins. Nevertheless synthetic spider silk genes have been successfully expressed in transgenic tobacco, potato and Arabidopsis thaliana plants (Barr et al. 2004; Scheller et al. 2001) under control of the cauliflower mosaic virus (CaMV) promoter and targeted to the endoplasmatic reticulum. Transgenic plants were cultivated in greenhouses and in field trails ( Menassa et al. 2004; Scheller and Conrad 2005). In tobacco and potato leaves up to 2% of total soluble protein (TSP) was observed (Scheller et al. 2001). The expression only in leaf apoplasts of A. thaliana led to spider silk production of 8.5% TSP, whereas targeting to seed endoplasmic reticulum yielded 18% TSP (Yang et al. 2005). The expression levels in plants are close to the level of 10% and 30% TSP reported in Escherichia coli and Pichia pastoris. The combination of spider silk protein and elastin polymer leads to a new biomaterial, which is used for industrial and medical purposes. For that, the expression of synthetic collagen, made from repeats of a motif found in elastin, and also of a chimeric protein composed of silk and elastin domains has been expressed in tobacco or potato (for a review, see van Beilen and Poirier 2008). The extraction of the proteins from 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein (Scheller et al. 2004).

The enhancement of fibrous protein synthesis in plant requires several approaches, for example optimization of the amino acid and tRNA pools for those amino acids, which form the main part of the protein, like glycine and alanine in spider silk. Further possibilities are dislocation to compartments and tissues that are optimal for protein synthesis and storage and also the co-expression of several fibrous proteins as found in natural silk (for a review, see van Beilen and Poirier 2008). In addition to the optimized production of fibrous proteins, the resulted fibre should have characteristics similar to the silk proteins from spider. The properties of silk fibres depend on correct assembly of the different types of proteins by spinning. Recombinant spider silk, obtained from mammalian cells, shows similar toughness to dragline silk, but with a lower tenacity (Lazaris et al. 2002). Non-Ribosomally Produced Poly-Amino Acids

Polymers produced in transgenic plants include polyaminoacids such as poly-g-glutamate, poly-a-aspartate, and poly-e-lysine, which have a wide range of applications, e.g. as dispersants, thickeners or additives to hydrogels (Chang and Swift 1999; Lossl et al. 2003; Oppermann-Sanio et al. 1999; Oppermann-Sanio and Steinbuchel 2002).

Polyaspartate is a soluble, non-toxic and biodegradable polycarboxylate (Tabata et al. 2000) that could replace the non-biodegradable polyacrylates in many industrial, agricultural and medical applications (Joentgen et al. 2001; Oppermann-Sanio and Steinbüchel 2002; Schwamborn 1998; Zotz et al. 2001). Because no polyaspartate-producing organism has been identified up to now, the polymer is chemically synthesized (Schwamborn 1996). However, it can also be obtained from cyanophycin (multi-L-arginyl-poly-L-aspartic acid). Cyanophycin is a cyanobac-terial reserve polymer composed of a poly-a-aspartic acid backbone with arginine residues linked via their a-amino group to the ß-carboxyl group of each aspartate residue (Simon 1976, 1987; Simon and Weathers 1976). Mild hydrolysis of cyano-phycin (Joentgen et al. 2001) results in homo- and copolymers of polyaspartate and L-arginine. The basic amino acid L-arginine has been suggested to be a regulator of some immunological and physiological processes, e.g. being an immune system stimulator (Cen et al. 1999; de Jonge et al. 2002; Li et al. 2007; Nieves and Langkamp-Henken 2002; Popovic et al. 2007; Taheri et al. 2001; Tapiero et al. 2002; Yeramian et al. 2006), agrowth inductor (Lenis et al. 1999; Roth et al. 1995; Wu et al. 2007) or a tumour cell growth inhibitor (Amber et al. 1988; Caso et al. 2004; Flynn et al. 2002). Alternatively, aspartate and arginine from cyanophycin could serve as a starting point for the synthesis of a range of chemicals (Fig. 13.2). Arginine can be converted to 1,4-butanediamine, which can be used for the synthesis of nylon-4,6. Aspartate is converted in several chemicals like 2-amino-1,4-butanediol, 3-aminotetrahydrofuran (analogues of high-volume chemicals used in the polymer industry), fumaric acid (used for polyester resins) and acrylamide (used as a thickener, in manufacturing dyes or in papermaking). Cyanophycin is synthesized via non-ribosomal polypeptide synthesis in many Cyanobacteria (Simon 1987) and some other non-photosynthetic bacteria (Krehenbrink et al. 2002; Ziegler et al. 2002). For cyanophycin synthesis, only one enzyme, the cyanophycin synthetase encoded by cphA, is necessary to catalyse the ATP-depen-dent elongation of a cyanophycin primer by the consecutive addition of L-aspartic acid and L-arginine (Ziegler et al. 2002). In cyanobacteria, the polymer is variable

Fig. 13.2 Potential products derived from the polymer cyanophycin

in length (25-125 kDa), water-insoluble and stored in membrane-less granules (Allen 1984; Simon 1987). As a first step to establish a system for mass production of the polymer in plants, the cyanophycin synthetase gene from Thermosynecho-coccus elongatus BP-1 (cphATe) was incorporated into tobacco and potato plants, with mRNA expression under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (Neumann et al. 2005). The maximum amount of cyanophycin in the cytosol of tobacco leaves was 1.14% of dry weight. However, the cyanophycin-producing plants exhibited phenotypical changes like thickened cell walls, variegated leaves and slow growth. The same was true for the transgenic potato plants containing maximal amounts of 0.24% of dry weight. The clone producing the most cyanophycin did not develop eyes and could not be propagated further. Moreover, in tubers, the presence of cyanophycin could only be demonstrated by electron microscopy. A much higher capacity to produce cyanophycin was achieved by targeting the cyanophycin synthetase to plastids of Nicotiana tabacum (Huhns et al. 2008). Yields of up to 6.8% dry weight in leaves were obtained, without significant disturbance of plant growth and development. However, the line producing the most cyanophycin produced fewer seeds.

When the cphA expression is restricted to tubers, the plant fitness and cyano-phycin production in potato is further enhanced up to 7.5% dry weight, with minimal effects on growth and morphology of the plants.

The improvement of the cyanophycin accumulation in plants may require optimization of the gene sequence, adapted to the target plant and also the optimization of the pathways involved in supplying arginine and aspartic acid.

13.3 Conclusion

The production of biopolymers has proven to be feasible and might contribute to a sustainable agriculture. Nevertheless, application still lies in the far future. Before transgenic plants can be cultivated and used, they must undergo an authorization procedure based on a safety assessment to guarantee their safe usage. Currently, the approval of transgenic plants is an extremely cost and time-demanding process, specific for one event (one plant line derived from a single transformant with one insertion locus of the transgene). These efforts will only be undertaken if the expected gain can exceed the costs. The gain depends on: (i) the potential market of the biopolymer, (ii) the pureness and concentration of the biopolymer in the plants, (iii) the potential reduction of the primary value of the cultivar (e.g. reduction of biomass, starch content or processing quality) and (iv) the isolation costs. Except for the amylopectin potato Amflora, further investigations have to be done for all of the polymers described to optimize these parameters as far as possible, either by modification of the production compartment in the plant, selection of the production cultivar, support of plant health and biomass production, or by optimization of cultivation, harvest, storage and isolation strategies. Further, risk assessment strategies have to be optimized in order to reduce the cost and time for approval without any reduction in safety. This might be done by the development of new and specific techniques for analysis as well as by the acceptance of transgene-and cultivar-specific data to reduce effort for single events. In addition, analysis always has to be hypothesis-driven and the selection of topics addressed has to be restricted to risks specific for the event in question.

Due to the different legal frameworks in most parts of the world, it might be expected that biopolymer-producing transgenic plants will be on the market first in the United States and Canada since their regulations come under existing laws covering seed and pesticide approval as well as food and feed control and the basis for safety assessment of a new transgenic plant is by comparison to known and established plants and products. Most importantly, the final decision for approval is carried out by scientists. Nevertheless, no polymer-producing plants are close to the United States market. In contrast, in Europe, the assessment focuses on the process (i.e. genetic engineering) and the precautionary approach; and the final decision is made by the European Commission in consultation with the Member States. Therefore no transgenic polymer-producing plant has yet been authorized in the European Union, but the potato event Amflora is close to approval. Nevertheless, although the safety assessment for cultivation and for food and feed use by the European Food Safety Authority (EFSA) was completed years ago, this event is still waiting for authorization.

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