Journal: New biotechnology
Despite the fact that a thorough, lengthy and costly evaluation of genetically engineered (GE) crop plants (including compositional analysis and toxicological tests) is imposed before marketing some European citizens remain sceptical of the safety of GE food and feed. In this context, are additional tests necessary? If so, what can we learn from them? To address these questions, we examined data from 60 recent high-throughput “-omics” comparisons between GE and non-GE crop lines and 17 recent long term animal feeding studies (longer than the classical 90-day subchronic toxicological tests), as well as 16 multigenerational studies on animals. The “-omics” comparisons revealed that the genetic modification has less impact on plant gene expression and composition than that of conventional plant breeding. Moreover, environmental factors (such as field location, sampling time, or agricultural practices) have a greater impact than transgenesis. None of these “-omics” profiling studies has raised new safety concerns about GE varieties; neither did the long-term and multigenerational studies on animals. Therefore, there is no need to perform such long term studies in a case-by-case approach, unless reasonable doubt still exists after conducting a 90-day feeding test. In addition, plant compositional analysis and “-omics” profiling do not indicate that toxicological tests should be mandatory. We discuss what complementary fundamental studies should be performed and how to choose the most efficient experimental design in order to assess risks associated with new GE traits. The possible need to update the current regulatory framework is discussed.
In the global context of increased concerns for our environment, the use of bioplastics as a replacement for existing petroleum-based polymers is an important challenge. Indeed, bioplastics hardly meet economical and technical constraints. One, of the most promising among currently studied bioplastics, is the polyhydroxyalkanoate (PHA). To circumvent the economical issue for this particular biopolymer one solution can be the enhancement of the overall productivity by the improvement of the nutritional medium of the microorganism producing the biopolymer. Thus, several nutrition media, supplemented or not with sodium glutamate, were tested for the growth and the PHA production by Cupriavidus necator DSM 545 strain. The most efficient for the biomass and the PHA production improvement were found to be the Luria broth (LB) and the Bonnarme’s media, both supplemented with 10g/L sodium glutamate. Hence the overall productivity was 33 times enhanced comparing to traditional cultivation methods. These results open a new route for the PHA production by C. necator which appears to be more suitable on a rich, or enriched, medium with no limiting factors.
Society is fundamentally ambivalent to the use of plastics. On the one hand, plastics are uniquely flexible materials that have seen them occupy a huge range of functions, from simple packing materials to complex engineering components. On the other, their durability has raised concerns about their end-of-life disposal. When that disposal route is landfill, their invulnerability to microbial decomposition, combined with relatively low density and high bulk, means that plastics will occupy increasing amounts of landfill space in a world where available suitable landfill sites is shrinking. The search for biodegradable plastics and their introduction to the marketplace would appear to be a suitable amelioration strategy for such a problem. And yet the uptake of biodegradable plastics has been slow. The term biodegradable itself has entered public controversy, with accidental and intended misuse of the term; the intended misuse has led to accusations and instances of “greenwashing”. For this and other reasons standards for biodegradability and compostability testing of plastics have been sought. An environmental dilemma with more far-reaching implications is climate change. The need for rapid and deep greenhouse gas (GHG) emissions cuts is one of the drivers for the resurgence of industrial biotechnology generally, and the search for bio-based plastics more specifically. Bio-based has come to mean plastics based on renewable resources, but this need not necessarily imply biodegradability. If the primary purpose is GHG emissions savings, then once again plastics durability can be a virtue, if the end-of-life solution can be energy recovery during incineration or recycling. The pattern of production is shifting from the true biodegradable plastics to the bio-based plastics, and that trend is likely to persist into the future. This paper looks at aspects of the science of biodegradable and bio-based plastics from the perspective of policy advisers and makers. It is often said that the bioplastics suffer from a lack of a favourable policy regime when compared to the wide-ranging set of policy instruments that are available on both the supply and demand side of biofuels production. Some possible policy measures are discussed.
Nucleic Acid sequencing is the mainstay of biological research. There are several generations of DNA sequencing technologies that can be well characterized through their nature and the kind of output they provide. Dideoxy terminator sequencing developed by Sanger dominated for 30 years and was the workhorse used for the Human Genome Project. In 2005 the first 2(nd) generation sequencer was presented with an output order of magnitude higher than Sanger sequencing and dramatically decreased cost. We are now at the dawn of 3(rd) generation with nanopore systems that are being developed for DNA sequencing. Meanwhile the field is also broadening into applications that complement 1(st), 2(nd) and 3(rd) generation sequencing systems to get high resolution genetic information. The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) consortium funded by the European Commission under FP7 has made great contributions to the development of new nucleic acid analysis methodology.
The yeast P. pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, i.e. glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05h(-1) and 0.16h(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources have a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges between 50 -70%. At high growth rate the methanol is completely dissimilated to CO2 by the direct pathway, in the two conditions of highest methanol content.
Soil microbial community composition and activity could be affected by suitable manipulation of the environment they live in. If correctly applied such an approach could become a very effective way to remediate excess of chemicals. The concentration of nitrogen, especially nitrate deriving from agricultural managements, is generally found to increase in water flow. Therefore, by forcing the water flow through a buffer strip specifically designed and possibly afforested with suitable plant species, may result effective in reducing high nitrogen contents. The management of a riparian buffer may definitely affect the soil microbial activities, including denitrification, as well as the composition of the community. The present study reports on the changes occurred in terms of denitrifying microbial community composition, as compared to that of a neighbouring agricultural area, as a consequence of hydraulic management coupled to the suspension of farming practices and to the development of the woody and herbaceous vegetation. With this aim, denitrification was repeatedly measured and the data obtained were related to those deriving from a specific analysis of bacterial groups involved in denitrification. nirK, encoding for nitrite reductase, an enzyme essential for the conversion of nitrite to nitric oxide and considered the key step in the denitrification process, was chosen as the target gene. The main results obtained indicated that denitrification activity changes in riparian buffer as compared to agricultural soil and it is strongly influenced by carbon availability and soil depth. Although no significant differences on the community composition between superficial (0-15cm) and medium (40-55cm) layers were observed, the nirK-type denitrifier community was shown to significantly differ between riparian and agricultural soils in both surface and medium layers.
Biodegradable polymers are currently viable alternatives to traditional synthetic polymers. For instance, polyhydroxybutyrate (PHB) is intracellularly produced and accumulated by Bacillus species, among others. This study reports several wild-type Bacillus strains with the ability to accumulate PHB using raw glycerol from biodiesel production as the sole carbon source. Out of 15 strains from different sources, B. megaterium B2 was selected as the most promising strain for further statistical optimization of the medium composition. Plackett-Burman and central composite designs were used to establish key variables and optimal culture conditions for PHB production using both 250-mL shake flasks and a 7.5-L bioreactor. Temperature and concentrations of glycerol and Na2HPO4 are the experimental variables with the most significant influence on PHB production by B2. After 14h of fermentation in shake flasks with optimized medium, B2 produced 0.43g/L of PHB with a 34% accumulation in the cells. In contrast, under the same conditions, a maximum PHB concentration of 1.20g/L in the bioreactor was reached at 11 h. These values correspond to a 48% and 314% increase in PHB production compared to the initial culture conditions. These results suggest the potential of B2 as a PHB producer using raw glycerol, which is an inexpensive, abundant and readily available carbon source.
Autotransporters represent one of the most popular anchoring motifs used to display peptides, proteins or enzymes on the cell surface of a Gram-negative bacterium. Applications range from vaccine delivery to library screenings to biocatalysis and bioremediation. Although the underlying secretion mechanism is supposed to be available in most, if not all, Gram-negative bacteria, autotransporters have to date almost exclusively been used for surface display on Escherichia coli. However, for their utilisation beyond a laboratory scale, in particular for biocatalysis, host bacteria with specific features and industrial applicability are required. A few groups have addressed this issue and demonstrated that bacteria other than E. coli can also be used for autotransporter based surface display. We summarise these studies and discuss opportunities and challenges that arise from surface display of recombinant proteins using the autotransporter pathway in alternative hosts.
Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of E. coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.
Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.