Production of renewable polymers from crop plants

Plants produce a range of biopolymers for purposes such as maintenance of structural integrity, carbon storage, and defense against pathogens and desiccation. Several of these natural polymers are used by humans as food and materials, and increasingly as an energy carrier. In this review, we focus on plant biopolymers that are used as materials in bulk applications, such as plastics and elastomers, in the context of depleting resources and climate change, and consider technical and scientific bottlenecks in the production of novel or improved materials in transgenic or alternative crop plants. The biopolymers discussed are natural rubber and several polymers that are not naturally produced in plants, such as polyhydroxyalkanoates, fibrous proteins and poly-amino acids. In addition, monomers or precursors for the chemical synthesis of biopolymers, such as 4-hydroxybenzoate, itaconic acid, fructose and sorbitol, are discussed briefly.     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Cyanophycin production from feather hydrolysate using biotechnological methods

Abstract

Cyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93?h with degradation rate of 96.5%. A concentration of 60?g/L of FH, generated by keratinolytic activity (8?×?10³?U?g^?1L^?1d^?1) within 24?h, was used as the main carbon/peptone source to produce cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1?±?0.4 and 5.3?±?0.3?g/L of cell mass were obtained after 72?h from FH and CA, respectively. Cyanophycin production yielded 1.4?±?0.1g/L for FH with average molecular mass of 28.8 and 1.4?±?0.2 for CA with average molecular mass of 35.3, after 60?h. For the first time, FH generated by biotechnological methods from environmentally problematic, abundant and renewable feather bioresource was successfully used for cyanophycin biopolymer production.     

Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene

The synthesis of cyanophycin, a biodegradable polymer, is directed by cyanophycin synthetase. Polymerase chain reaction (PCR) cloned the gene cphA coding for cyanophycin synthetase from Synechocystis sp. PCC 6803 into pET-21b followed by transformation into two Escherichia coli hosts. The culture conditions for cyanophycin production were investigated, and the molecular weight and compositions of purified cyanophycin were analyzed. The results showed that E. coli BL21-CodonPlus(DE3)-RIL could produce 120 mg cyanophycin per gram dry cell weight in terrific medium. The purified cyanophycin consisted of insoluble and soluble forms at pH 7. The insoluble form had a higher molecular weight (20-32 kDa) than the soluble form (14-25 kDa). Both forms are composed of three major amino acids, aspartic acid, arginine, and lysine, and the insoluble form showed a higher arginine/lysine molar ratio (4.61 ± 0.31) than the soluble form (0.89 ± 0.05). In addition, the nitrogen sources could affect the yields of insoluble and soluble forms of cyanophycin. The medium containing additional lysine could enhance the proportion of the soluble form, but had little effect on the lysine and arginine percentages of both soluble and insoluble forms. The medium containing additional arginine slightly decreased the proportion of soluble form and altered its amino acid composition, with a minimal effect on the lysine and arginine percentages in the insoluble form.     

Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.     

Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design

Elementary mode analysis was applied to simulate conditions for cyanophycin (CGP) biosynthesis and to optimize its production in bacteria. The conclusions from these simulations were confirmed by experiments with recombinant strains of the wild types and polyhydroxyalkanoate (PHA)-negative mutants of Ralstonia eutropha and Pseudomonas putida expressing CGP synthetase genes (cphA) of Synechocystis sp. strain PCC6308 or Anabaena sp. strain PCC7120. In particular, the effects of suitable precursor substrates and of oxygen supply as well as of the capability to accumulate PHA in addition to CGP biosynthesis were investigated. Since CGP consists of the amino acids aspartate and arginine, the tricarboxylic acid cycle (TCC), which provides intermediates for biosynthesis of these amino acids, seems to be important. Excretion of intermediates of the TCC upon cultivation at restricted oxygen supply and conversion of fumarate mainly to malate and to only little succinate in the absence of oxygen indicated that TCC intermediates for arginine and aspartate biosynthesis were provided by the oxidative or reductive parts of the TCC, respectively. The following important conclusions were made from the experiments and the simulations: (i) external arginine additionally supplied to the medium, (ii) oxygen limitation, and (iii) absence of PHA accumulation exerted positive effects on CGP accumulation. These conclusions were utilized to obtain CGP contents in the cells of as high as 17.9% (w x w(-1)) during cultivation of the investigated bacteria at the 30-L scale using mineral salts medium. Such high CGP contents were previously not obtained with these bacteria at a 30-L scale, even if complex media were used.     

Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene ( cph A_Te) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske- cph A_Te, pCP24- cph A_Te, pFNR- cph A_Te and pPsbY- cph A_Te. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY- cph A_Te led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24- cph A_Te and pFNR- cph A_Te constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY- cph A_Te produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T₀) transformants. Specific T₂ plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.     

THE NORMAL AND INDUCED OCCURRENCE OF CYANOPHYCIN INCLUSION BODIES IN SEVERAL BLUE-GREEN ALGAE1

Multi-L-arginyl-poly(L-aspartic acid) [arg-poly(asp)], the polypeptide component of the cyanophycin inclusion body, is found in cells of many blue-green algae. Formation of this material can be induced by a variety of treatments including the addition of excess nitrogen-containing compounds, the addition of specific inhibitors of macromolecular synthesis, and the exclusion of sulfur or phosphorus. Knowledge of the conditions that induce the synthesis of this polypeptide has made possible an ultrastructural survey to determine the presence and cellular location of cyanophycin bodies in a variety of cyanobacteria. Data presented show that certain strains of the unicellular genus Synechococcus Nägeli do not contain arg-poly(asp) under environmental conditions that markedly increase the level of such material in other cyanobacteria. In addition many strains show spatial localization of the cyanophycin bodies under normal growth conditions, and moreover the normal pattern is retained even when massive synthesis of arg-poly(asp) is induced. Finally there is no evidence that these inclusion bodies occur in certain beggiatoan gliding bacteria.     

Cyanophycin-degrading bacteria in digestive tracts of mammals, birds and fish and consequences for possible applications of cyanophycin and its dipeptides in nutrition and therapy

Aims:  To determine the susceptibility of cyanophycin granule polypeptide (CGP) to degradation by several mammalian, avian and fish gut flora.
Methods and Results:  Samples of gut flora were investigated for the occurrence of bacteria capable of CGP degradation. With all samples, a complete anaerobic degradation of CGP was achieved over incubation periods of only 12–48 h at 37°C. CGP-degrading bacteria were detected in all samples, and they occurred in particular high titres in caecum flora from rabbit and sheep and in the digestive tract of carp fish. A total of 62 axenic cultures were isolated. All degraded CGP aerobically, 46 of them degraded CGP also anaerobically over incubation periods ranging from 24 h to 7 days. HPLC analysis revealed that all isolates degraded CGP to its constituting dipeptides. Eight strains were identified by 16S rRNA gene sequencing and were affiliated to the genera Bacillus , Brevibacillus , Pseudomonas , Streptomyces and Micromonospora .
Conclusions:  These data demonstrate for the first time the occurrence of a natural niche for CGP in the digestive tracts of animals.
Significance and Impact of the Study:  The biodegradability of CGP by gut flora provides a first confirmation for the potential applications of CGP and its dipeptides in nutrition and therapy as highly bio-available sources for arginine, lysine, aspartate and possibly also other amino acids.