Bioengineered Bugs

The stability and superior metal bioremediation ability of genetically engineered Deinococcus radiodurans cells, expressing a non-specific acid phosphatase, PhoN in high radiation environment has already been established. The lyophilized recombinant DrPhoN cells retained PhoN activity and uranium precipitation ability. Such cells also displayed an extended shelf life of 6 months during storage at room temperature and showed surface associated precipitation of uranium as well as other metals like cadmium. Lyophilized cells, immobilized in polyacrylamide gels could be used for uranium bioprecipitation in a flow through system resulting in 70% removal from 1mM input uranium solution and a loading of 1 g uranium/g dry weight cells...

Diagnostic molecular biology is arguably the fastest growing area in current laboratory-based medicine. Growth of the so called 'omics' technologies has, over the last decade, led to a gradual migration away from the 'one test, one pathogen' paradigm, toward multiplex approaches to infectious disease diagnosis, which have led to significant improvements in clinical diagnostics and ultimately improved patient care.

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem...

Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements...

The methylotropic yeast Pichia pastoris has been used for more than two decades to successfully produce a large number of recombinant proteins. Currently, a wide variety of auxotrophic and drug based selection markers are employed to screen for clones expressing the protein of interest. For most proteins an increased copy number of the integrated plasmid results in higher levels of expression, but these multi-copy integrants can be unstable due to the propensity of P. pastoris for homologous recombination. Here we describe a multi-copy selection system based on ade1 and ade2 auxotrophic parent strains and the respective attenuated markers with truncated promoter regions...

Numerous mobile genetic elements (MGE) are associated with the human gut microbiota and collectively referred to as the gut mobile metagenome. The role of this flexible gene pool in development and functioning of the gut microbial community remains largely unexplored, yet recent evidence suggests that at least some MGE comprising this fraction of the gut microbiome reflect the co-evolution of host and microbe in the gastro-intestinal tract. In conjunction, the high level of novel gene content typical of MGE coupled with their predicted high diversity, suggests that the mobile metagenome constitutes an immense and largely unexplored gene-space likely to encode many novel activities with potential biotechnological or pharmaceutical value, as well as being important to the development and functioning of the gut microbiota...

Human trisomy 21 is the most frequent live-born human aneuploidy and causes a constellation of disease phenotypes classified as Down syndrome, which include heart defects, myeloproliferative disorder, cognitive disabilities and Alzheimer-type neurodegeneration. Because these phenotypes are associated with an extra copy of a human chromosome, the genetic analysis of Down syndrome has been a major challenge. To complement human genetic approaches, mouse models have been generated and analyzed based on evolutionary conservation between the human and mouse genomes...

Plants express genes that encode enzymes that catalyse reactions to form plant secondary metabolites in specific cell types. However, the mechanisms of how plants decide their cellular metabolic fate and how cells diversify and specialise their specific secondary metabolites remains largely unknown. Additionally, whether and how an established metabolic program impacts genome-wide reprogramming of plant gene expression is unclear. We recently isolated PAP1-programmed anthocyanin-producing (red) and -free (white) cells from Arabidopsis thaliana; our previous studies have indicated that the PAP1 expression level is similar between these two different cell types...

Bifidobacteria and lactobacilli are widely exploited as health-promoting bacteria in many functional foods. However, the molecular mechanisms as to how these bacteria positively impact on host health are far from completely understood. For this reason these microorganisms represent a growing area of interest with respect to their genomics, molecular biology and genetics. Recent genome sequencing of a large number of strains of bifidobacteria and lactobacilli has allowed access to the complete genetic makeup of representative members of these bacteria...

Whereas the great majority of the current vaccines are delivered through the parenteral route, mucosal administration has been increasingly considered for controlling infection and preventing disease. Mucosal vaccination can trigger both humoral and cell-mediated protection, not only at the targeted mucosal surface, but also systemically. In this regard, nasal vaccination has shown great potential. The live attenuated strain of Bordetella pertussis, BPZE1, is particularly attractive and promising as a nasal vaccine delivery vector of heterologous antigen vaccine candidates...

Leishmania parasites cause a variety of devastating diseases in tropical areas around the world. Due to the lack of vaccines and limited availability of drugs, new therapeutic targets are urgently needed. A variety of genetic tools have been developed to investigate the complex biology of this parasite and its interactions with the host. One of the main techniques is the generation of knock-out parasites via targeted gene replacement, a process that takes advantage of the parasites ability to undergo homologous recombination...

For even the best-studied species, there is a large gap in their representation in the protein databank (PDB) compared to within sequence databases. Typically, less than 2% of sequences are represented in the PDB. This is partly due to the considerable experimental challenge and manual inputs required to solve three dimensional structures by methods such as X-ray diffraction and multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in comparison to high-throughput sequencing. This gap is made even wider by the high level of redundancy within the PDB and under-representation of some protein categories such as membrane-associated proteins which comprise approximately 25% of proteins encoded in genomes...

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect...

In addition to our previous study on the effect of fnr gene knockout on the metabolism in Escherichia coli under aerobic conditions (Kumar and Shimizu, Microb Cell Fact 2011), here we further investigated the effect of fnr gene knockout on the metabolism under micro-aerobic condition based on gene expressions, enzyme activities and intracellular metabolic fluxes. The objective of the present research is to clarify the metabolic regulation mechanism on how the culture environment, such as oxygen level, affects the cell metabolism in relation to gene expressions, enzyme activities and fluxes via global regulators such as Fnr and ArcA/B systems...

Organisms rarely live as isolated species and usually show symbiosis in nature. As natural selection is not simple in symbiosis, the establishment and development of symbiosis is still unclear. Insight can be gained by not only retracing the history of well-developed natural symbiotic relationships, but also by observing the development of nascent symbiosis. By using synthetic symbiosis composed of two previously noninteracting populations, we can observe the establishment and its development. We have recently simulated the establishment of nascent symbiosis using two genetically engineered auxotrophic strains of Escherichia coli...

No abstract text is available yet for this article.

A key challenge in the area of determining how the microbiome communicates with the host's karyome is deciding which microbial functions should be studied. Ideally we would wish to look at functions which are not only important to the microbial host, but which also play roles in host physiology. Selecting the key microbial functions is essential to developing robust strategies to either promote or demote them, with the aim to enhancing host health. This commentary argues that the bottom-up approach is not providing the necessary gene-set from which we can start to develop a robust core microbiome and in fact we should adopt a top-down strategy in order to indentify the functions that are important and need further study...

5-Aminolevulinic acid (ALA) has recently attracted significant attentions due to its potential applications in many diverse fields. The majority of engineered ALA producers are based on the whole cell catalysis, supplemented with succinate and glycine as precursors. Recently, we succeeded in producing ALA directly from inexpensive glucose, through re-constructing the native C5 pathway of ALA synthesis in Escherichia coli. Herein, we further discuss ALA production by manipulating the C5 and C4 pathways in Escherichia coli through the strategy of metabolic engineering...

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols...

Genome-scale metabolic network reconstruction can be used for simulating cellular behaviors by simultaneously monitoring thousands of biochemical reactions, and is therefore important for systems biology studies in microbes. However, the labor-intensive and time-consuming reconstruction process has hindered the progress of this important field. Here we present a web server, MrBac (Metabolic network Reconstructions for Bacteria), to streamline the network reconstruction process for draft genome-scale metabolic networks and to provide annotation information from multiple databases for further curation of the draft reconstructions...