Engineering of PHB synthesis causes improved elastic properties of flax fibers

Flax stem is a source of fiber used by the textile industry. Flax fibers are separated from other parts of stems in the process called retting and are probably the first plant fibers used by man for textile purposes (1). Nowadays flax cultivation is often limited because of its lower elastic property compared to cotton fibers. Thus the goal of this study was to increase the flax fiber quality using a transgenic approach. Expression of three bacterial genes coding for beta-ketothiolase (phb A), acetoacetyl-CoA reductase (phb B), and PHB synthase (phb C) resulted in poly-beta-hydroxybutyrate (PHB) accumulation in the plant stem. PHB is known as a biodegradable thermoplastic displaying chemical and physical properties similar to those of conventional plastics (i.e., polypropylene). The fibers isolated from transgenic flax plants cultivated in the field and synthesizing PHB were then studied for biomechanical properties. All measured parameters, strength, Young's modulus, and energy for failure of flax fibers, were significantly increased. Thus the substantial improvement in elastic properties of fibers from the transgenic line has been achieved. Since the acetyl CoA, substrate for PHB synthesis, is involved not only for energy production but also for synthesis of many cellular constituents, the goal of this study was also the analysis of those metabolites, which interfere with plant physiology and thus fiber quality. The analyzed plants showed that reduction in lignin, pectin, and hemicellulose levels resulted in increased retting efficiency. A significant increase in phenolic acids was also detected, and this was the reason for improved plant resistance to pathogen infection. However, a slight decrease in crop production was detected.     

Biochemical,mechanical, and spectroscopic analyses of genetically engineered flax fibers producing bioplastic (poly‐β‐hydroxybutyrate)

The interest in biofibers has grown in recent years due to their expanding range of applications in fields as diverse as biomedical science and the automotive industry. Their low production costs, biodegradability, physical properties, and perceived eco‐friendliness allow for their extensive use as composite components, a role in which they could replace petroleum‐based synthetic polymers. We performed biochemical, mechanical, and structural analyses of flax stems and fibers derived from field‐grown transgenic flax enriched with PHB (poly‐β‐hydroxybutyrate). The analyses of the plant stems revealed an increase in the cellulose content and a decrease in the lignin and pectin contents relative to the control plants. However, the contents of the fibers' major components (cellulose, lignin, pectin) remain unchanged. An FT‐IR study confirmed the results of the biochemical analyses of the flax fibers. However, the arrangement of the cellulose polymer in the transgenic fibers differed from that in the control, and a significant increase in the number of hydrogen bonds was detected. The mechanical properties of the transgenic flax stems were significantly improved, reflecting the cellulose content increase. However, the mechanical properties of the fibers did not change in comparison with the control, with the exception of the fibers from transgenic line M13. The generated transgenic flax plants, which produce both components of the flax/PHB composites (i.e., fibers and thermoplastic matrix in the same plant organ) are a source of an attractive and ecologically safe material for industry and medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.     

Lignin deficiency in transgenic flax resulted in plants with improved mechanical properties

Flax (Linum usitatissimum L.) is a very important source of natural fibres used by the textile industry. Flax fibres are called lignocellulosic, because they contain mainly cellulose (about 70%), with hemicellulose, pectin and lignin. Lignin is a three-dimensional polymer with a high molecular weight, and it gives rigidity and mechanical resistance to the fibre and plant. Its presence means the fibres have worse elastic properties than non-lignocellulosic fibres, e.g. cotton fibres, which contain no lignin. The main aim of this study was to produce low-lignin flax plants with fibres with modified elastic properties. An improvement in the mechanical properties was expected. The used strategy for CAD down-regulation was based on gene silencing RNAi technology. Manipulation of the CAD gene caused changes in enzyme activity, lignin content and in the composition of the cell wall in the transgenic plants. The detected reduction in the lignin level in the CAD-deficient plants resulted in improved mechanical properties. Young's modulus was up to 75% higher in the generated transgenic plants (CAD33) relative to the control plants. A significant increase in the lignin precursor contents and a reduction in the pectin and hemicellulose constituents was also detected. A decrease in pectin and hemicellulose, as well as a lower lignin content, might lead to improved extractability of the fibres. However, the resistance of the transgenic lines to Fusarium oxysporum was over two-fold lower than for the non-transformed plants. Since Fusarium species are used as retting organisms and had been isolated from retted flax, the increased sensitivity of the CAD-deficient plant to F. oxysporum infection might lead to improved flax retting.     

Synthesis of poly[(R)-3-hydroxybutyric acid) in the cytoplasm of Pichia pastoris under oxygen limitation

We have constructed a tandem gene expression cassette containing three Ralstonia eutropha poly[(R)-3-hydroxybutyrate] (PHB) synthesis genes under the control of the Pichia pastoris glyceraldehyde-3-phosphate promoter and the green fluorescent protein (Gfp) under the control of the P. pastoris alcohol oxidase promoter. The inducible Gfp reporter protein has been used to rapidly isolate transformed strains with two copies of the entire expression cassette. The isolated strain exhibits Gfp induction kinetics that is twice as fast as that of the strains isolated without cell sorting. In addition, the sorted strains exhibited higher PHB contents in preliminary screening experiments. PHB synthesis was characterized in more detail in the sorted strain and was found to be dependent on culture conditions. It was observed that the specific PHB synthesis rate was dependent on the carbon source utilized and that the conditions of oxygen stress lead to increased fractional PHB content. When this strain is cultivated on glucose under oxygen-limited conditions, the cultures accumulated ethanol during the initial growth phase and then consumed the ethanol for the accumulation of PHB and biomass. While PHB was not synthesized during initial growth on glucose, significant levels of PHB were synthesized when ethanol was subsequently consumed. PHB was also synthesized under aerobic conditions when ethanol was the only carbon source. During growth on ethanol, the specific growth rate of the culture was reduced under oxygen-limited conditions but the specific PHB synthesis rate was relatively unaffected. Thus, the high accumulation of PHB which exceeded 30% of the cell dry weight appears to be the consequence of the decreased biomass growth rate under severe oxygen limitation.     

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.