The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Improved production of echinenone and canthaxanthin in transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme.

Echinenone and canthaxanthin are important carotenoid pigments with food and industrial applications. Biosynthesis of echinenone and/or canthaxanthin is catalyzed by ß-carotene ketolase (CrtO), with ß-carotene as the substrate. In this study, we generated transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme and evaluated the productivity of both pigments. Normal (BG11 medium, 30 °C) and osmotic stress (BG11 medium supplemented with 0.4 M mannitol, 30 °C) conditions were used for cultivation. As compared to control strain, production of echinenone and canthaxanthin in transgenic strain were respectively increased by more than 16 % and 80 %, under either normal or osmotic stress conditions. Especially upon the stress condition, higher proportion of echinenone and canthaxanthin in total pigments was achieved, which should be beneficial for downstream separation and purification. In addition, transgenic strain showed drought tolerance and could revive from desiccation treatment after rewetting. Thus, this study provided technical clues for production of both pigments in engineered cyanobacteria as well as for cyanobacterial anhydrobiotic engineering.

Rapid and Effective Electroporation Protocol for Nannochloropsis oceanica.

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 µF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.

The EU regulatory framework on genetically modified organisms (GMOs).

The European Union (EU) legislation on genetically modified organisms (GMOs) aims to ensure a high level of protection for human, animal and environmental health and a well-functioning EU internal market. The framework regulates the release of GMOs into the environment and their use as, or in, food and feed. It has three main pillars: pre-market authorisation based on a prior risk assessment, traceability and labelling. Within this legal framework, the EU has authorised the placing on the market of 118 GMOs so far. These have been obtained through long-standing techniques of genetic modification, namely transgenesis. Following the adoption of the GMO legislation, new techniques of genetic modification, including new mutagenesis techniques, have been developed, which have raised questions regarding the applicability of the GMO legislation and attracted a lot of attention from stakeholders and the general public. This article provides an overview of EU GMO legislation and implementation of the EU Court of Justice ruling on organisms obtained by mutagenesis techniques, issued in July 2018. It also updates on the recent initiatives by the European Commission and EU Member States on new developments in biotechnology. The manuscript is based on the author's contribution at the OECD Conference on Genome Editing, Applications in Agriculture, Implications for Health, Environment and Regulation held in Paris on 28-29 June 2018. It is complemented with updated information.

Agriculture R&D Implications of the CJEU's Gene-Specific Mutagenesis Ruling.

On 25 July 2018, the Court of Justice of the European Union (CJEU) ruled that gene-specific mutagenesis must be regulated as genetically modified organism (GMO) technologies. However, the costs to agricultural research and development (R&D) innovation will be staggering, not to mention the brain drain to other countries. As a result, Europe can now be known as the deathplace of agricultural breeding innovations.

A Welcome Proposal to Amend the GMO Legislation of the EU.

Is the European Union (EU) regulatory framework for genetically modified organisms (GMOs) adequate for emerging techniques, such as genome editing? This has been discussed extensively for more than 10 years. A recent proposal from The Netherlands offers a way to break the deadlock. Here, we discuss how the proposal would affect examples from public plant research.

In-vivo monitoring of infectious diseases in living animals using bioluminescence imaging.

Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However, the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative. Dedicated bioluminescence imaging (BLI) cameras can record the light emitted from living animals in real time allowing non-invasive, longitudinal monitoring of the anatomical location and growth of infectious microorganisms as measured by strength of the BLI signal. BLI technology has been used to follow bacterial infections in traumatic skin wounds and burns, osteomyelitis, infections in intestines, Mycobacterial infections, otitis media, lung infections, biofilm and endodontic infections and meningitis. Fungi that have been engineered to be bioluminescent have been used to study infections caused by yeasts (Candida) and by filamentous fungi. Parasitic infections caused by malaria, Leishmania, trypanosomes and toxoplasma have all been monitored by BLI. Viruses such as vaccinia, herpes simplex, hepatitis B and C and influenza, have been studied using BLI. This rapidly growing technology is expected to continue to provide much useful information, while drastically reducing the numbers of animals needed in experimental studies.

Frontline Science: Leishmania mexicana amastigotes can replicate within neutrophils.

Cutaneous leishmaniasis is a neglected tropical disease, causing a spectrum of clinical manifestations varying from self-healing to unhealing lesions that may be very difficult to treat. Emerging evidence points to a detrimental role for neutrophils during the first hours following infection with many distinct Leishmania species (spp.) at a time when the parasite is in its nonreplicative promastigote form. Neutrophils have also been detected at later stages of infection in unhealing chronic cutaneous lesions. However, the interactions between these cells and the replicative intracellular amastigote form of the parasite have been poorly studied. Here, we show that Leishmaniamexicana amastigotes are efficiently internalized by neutrophils and that this process has only a low impact on neutrophil activation and apoptosis. In neutrophils, the amastigotes were found in acidified vesicles. Furthermore, within cutaneous unhealing lesions, heavily infected neutrophils were found with up to 6 parasites per cell. To investigate if the amastigotes could replicate within neutrophils, we generated photoconvertible fluorescent parasites. With the use of flow cytometry imaging and time-lapse microscopy, we could demonstrate that a subset of parasites replicated within neutrophils. Overall, our data reveal a novel role for neutrophils that can act as a niche for parasite replication during the chronic phase of infection, thereby contributing to disease pathology.

Microbial and genetically engineered oils as replacements for fish oil in aquaculture feeds.

As the global population grows more of our fish and seafood are being farmed. Fish are the main dietary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, but these cannot be produced in sufficient quantities as are now required for human health. Farmed fish have traditionally been fed a diet consisting of fishmeal and fish oil, rich in n-3 LC-PUFA. However, the increase in global aquaculture production has resulted in these finite and limited marine ingredients being replaced with sustainable alternatives of terrestrial origin that are devoid of n-3 LC-PUFA. Consequently, the nutritional value of the final product has been partially compromised with EPA and DHA levels both falling. Recent calls from the salmon industry for new sources of n-3 LC-PUFA have received significant commercial interest. Thus, this review explores the technologies being applied to produce de novo n-3 LC-PUFA sources, namely microalgae and genetically engineered oilseed crops, and how they may be used in aquafeeds to ensure that farmed fish remain a healthy component of the human diet.

Farming and public goods production in Caenorhabditis elegans populations.

The ecological and evolutionary dynamics of populations are shaped by the strategies they use to produce and use resources. However, our understanding of the interplay between the genetic, behavioral, and environmental factors driving these strategies is limited. Here, we report on a Caenorhabditis elegans-Escherichia coli (worm-bacteria) experimental system in which the worm-foraging behavior leads to a redistribution of the bacterial food source, resulting in a growth advantage for both organisms, similar to that achieved via farming. We show experimentally and theoretically that the increased resource growth represents a public good that can benefit all other consumers, regardless of whether or not they are producers. Mutant worms that cannot farm bacteria benefit from farming by other worms in direct proportion to the fraction of farmers in the worm population. The farming behavior can therefore be exploited if it is associated with either energetic or survival costs. However, when the individuals compete for resources with their own type, these costs can result in an increased population density. Altogether, our findings reveal a previously unrecognized mechanism of public good production resulting from the foraging behavior of C. elegans, which has important population-level consequences. This powerful system may provide broad insight into exploration-exploitation tradeoffs, the resultant ecoevolutionary dynamics, and the underlying genetic and neurobehavioral driving forces of multispecies interactions.

Population modification of Anopheline species to control malaria transmission.

Vector control strategies based on population modification of Anopheline mosquitoes may have a significant role in the malaria eradication agenda. They could consolidate elimination gains by providing barriers to the reintroduction of parasites and competent vectors, and allow resources to be allocated to new control sites while maintaining treated areas free of malaria. Synthetic biological approaches are being used to generate transgenic mosquitoes for population modification. Proofs-of-principle exist for mosquito transgenesis, the construction of anti-parasite effector genes and gene-drive systems for rapidly introgressing beneficial genes into wild populations. Key challenges now are to develop field-ready strains of mosquitoes that incorporate features that maximize safety and efficacy, and specify pathways from discovery to development. We propose three pathways and a framework for target product profiles that maximize safety and efficacy while meeting the demands of the complexity of malaria transmission, and the regulatory and social diversity of potential end-users and stakeholders.

Vibrio natriegens as a fast-growing host for molecular biology.

A rapidly growing bacterial host would be desirable for a range of routine applications in molecular biology and biotechnology. The bacterium Vibrio natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min. We report the development of genetic tools and methods to engineer V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes.

Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, ß-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

Principles for designing synthetic microbial communities.

Advances in synthetic biology to build microbes with defined and controllable properties are enabling new approaches to design and program multispecies communities. This emerging field of synthetic ecology will be important for many areas of biotechnology, bioenergy and bioremediation. This endeavor draws upon knowledge from synthetic biology, systems biology, microbial ecology and evolution. Fully realizing the potential of this discipline requires the development of new strategies to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities. Here, we review recent experimental, analytical and computational advances to study and build multi-species microbial communities with defined functions and behavior for various applications. We also highlight outstanding challenges and future directions to advance this field.

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

Cloning of new toxic genes from Bacillus thuringiensis (Bt) and construction of Bt engineered strains are two key strategies for bio-control of coleopteran pests in agriculture and forestry. In this study, we cloned a new cry3Aa-type gene, cry3Aa8, from wild Bt strain YC-03 against coleopteran, and constructed a Bt engineered strain, ACE-38, containing insecticidal protein-encoding gene cry3Aa8. The engineered strain, with almost four times of Cry3Aa yield compared with strain YC-03, was an antibiotic marker-free strain. Though no selective pressure was presented in the medium, cry3Aa8 in the engineered strain ACE-38 remained stable. The yield of Cry3Aa by strain ACE-38 reached 2.09 mg/ml in the optimized fermentation medium. The activity of strain ACE-38 against Plagiodera versicolora was tested, and the LC50 of ACE-38 cultures in the optimized fermentation medium was 1.13 µl/ml. Strain ACE-38 is a non-antibiotic Bt engineered strain with high Chrysomelidae toxicity and exhibits good fermentation property. The modified indigenous site-specific recombination system constructed in this study might be useful for the construction of Bt engineered strains containing genes that cannot be expressed in the indigenous site-specific recombination system using plasmid pBMB1205R.

Multilayered genetic safeguards limit growth of microorganisms to defined environments.

Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping 'safeguards'-engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome-to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10(-12). The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species.