Autoluminescent Plants

Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression

Engineering the fatty acid synthesis pathway in Synechococcus elongatus PCC 7942 improves omega-3 fatty acid production

Background: The microbial production of fatty acids has received great attention in the last few years as feedstock for the production of renewable energy. The main advantage of using cyanobacteria over other organisms is their ability to capture energy from sunlight and to transform CO2 into products of interest by photosynthesis, such as fatty acids. Fatty acid synthesis is a ubiquitous and well-characterized pathway in most bacteria. However, the activity of the enzymes involved in this pathway in cyanobacteria remains poorly explored. Results: To characterize the function of some enzymes involved in the saturated fatty acid synthesis in cyanobacteria, we genetically engineered Synechococcus elongatus PCC 7942 by overexpressing or deleting genes encoding enzymes of the fatty acid synthase system and tested the lipid profile of the mutants. These modifications were in turn used to improve alpha-linolenic acid production in this cyanobacterium. The mutant resulting from fabF overexpression and fadD deletion, combined with the overexpression of desA and desB desaturase genes from Synechococcus sp. PCC 7002, produced the highest levels of this omega-3 fatty acid. Conclusions: The fatty acid composition of S. elongatus PCC 7942 can be significantly modified by genetically engineering the expression of genes coding for the enzymes involved in the first reactions of fatty acid synthesis pathway. Variations in fatty acid composition of S. elongatus PCC 7942 mutants did not follow the pattern observed in Escherichia coli derivatives. Some of these modifications can be used to improve omega-3 fatty acid production. This work provides new insights into the saturated fatty acid synthesis pathway and new strategies that might be used to manipulate the fatty acid content of cyanobacteria.Work in the FDLC laboratory was financed by the Spanish Ministry of Economy and Competitivity (MINECO) Grant BFU2014-55534-C2-1-P. MSM. was recipientof a Ph.D. fellowship (BES-2012-057387) from MINECO

Why are chlorinated pollutants so difficult to degrade aerobically? Redox stress limits 1,3-dichloroprop-1-ene metabolism by Pseudomonas pavonaceae

Chlorinated pollutants are hardly biodegradable under oxic conditions, but they can often be metabolized by anaerobic bacteria through organohalide respiration reactions. In an attempt to identify bottlenecks limiting aerobic catabolism of 1,3-dichloroprop-1-ene (1,3-DCP; a widely used organohalide) in Pseudomonas pavonaceae, the possible physiological restrictions for this process were surveyed. Flow cytometry and a bioluminescence reporter of metabolic state revealed that cells treated with 1,3-DCP experienced an intense stress that could be traced to the endogenous production of reactive oxygen species (ROS) during the metabolism of the compound. Cells exposed to 1,3-DCP also manifested increased levels of D-glucose-6-P 1-dehydrogenase activity (G6PDH, an enzyme key to the synthesis of reduced NADPH), observed under both glycolytic and gluconeogenic growth regimes. The increase in G6PDH activity, as well as cellular hydroperoxide levels, correlated with the generation of ROS. Additionally, the high G6PDH activity was paralleled by the accumulation of D-glucose-6-P, suggesting a metabolic flux shift that favours the production of NADPH. Thus, G6PDH and its cognate substrate seem to play an important role in P. pavonaceae under redox stress caused by 1,3-DCP, probably by increasing the rate of NADPH turnover. The data suggest that oxidative stress associated with the biodegradation of 1,3-DCP reflects a significant barrier for the evolution of aerobic pathways for chlorinated compounds, thereby allowing for the emergence of anaerobic counterparts.Fil: Nikel, Pablo Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Pérez Pantoja, Danilo. Consejo Superior de Investigaciones Científicas. Centro Nacional de Biotecnología; EspañaFil: de Lorenzo, Víctor. Consejo Superior de Investigaciones Científicas. Centro Nacional de Biotecnología; Españ