A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

<p>Abstract</p> <p>Background</p> <p>FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.</p> <p>Results</p> <p>We have developed a synthetic metabolic pathway in <it>E. coli </it>that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O₂levels eliminate growth. This pathway forms the basis for a genetic selection for O₂tolerance. Genetically selected hydrogenases did not show improved stability in O₂and in many cases had lost H₂production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.</p> <p>Conclusions</p> <p>Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient <it>in vivo </it>assays to aid in the engineering of synthetic H₂metabolism. Our results also indicate a H₂-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H₂-activating catalysts.</p

Insulation of a synthetic hydrogen metabolism circuit in bacteria

<p>Abstract</p> <p>Background</p> <p>The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits <it>in vivo </it>has not been pursued.</p> <p>Results</p> <p>Here we show that a synthetic hydrogen-producing electron transfer circuit in <it>Escherichia coli </it>can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.</p> <p>Conclusions</p> <p>Through the construction and characterization of a synthetic metabolic circuit <it>in vivo</it>, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.</p

Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low

A BioBrick compatible strategy for genetic modification of plants

Background: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. Results: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. Conclusions: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.Molecular and Cellular Biolog

Towards a Synthetic Chloroplast

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices

A survey of the microbial community in the rhizosphere of two dominant shrubs of the Negev Desert highlands, Zygophyllum dumosum (Zygophyllaceae) and Atriplex halimus (Amaranthaceae), using cultivation-dependent and cultivation-independent methods.

UnlabelledPremise of the studyPlant roots comprise more than 50% of the plant's biomass. Part of that biomass includes the root microbiome, the assemblage of bacteria and fungi living in the 1-3 mm region adjacent to the external surface of the root, the rhizosphere. We hypothesized that the microorganisms living in the rhizosphere and in bulk soils of the harsh environment of the Negev Desert of Israel had potential for use as plant-growth-promoting bacteria (PGPB) to improve plant productivity in nutrient-poor, arid soils that are likely to become more common as the climate changes. •MethodsWe used cultivation-dependent methods including trap experiments with legumes to find nitrogen-fixing rhizobia, specialized culture media to determine iron chelation via siderophores and phosphate-solubilizing and cellulase activities; cultivation-independent methods, namely 16S rDNA cloning and sequencing; and also community-level physiological profiling to discover soil microbes associated with the Negev desert perennials Zygophyllum dumosum and Atriplex halimus during the years 2009-2010. •Key resultsWe identified a number of PGPB, both epiphytes and endophytes, which fix nitrogen, chelate iron, solubilize phosphate, and secrete cellulase, as well as many other bacteria and some fungi, thereby providing a profile of the microbiomes that support the growth of two desert perennials. •ConclusionWe generated a snapshot of the microbial communities in the Negev Desert, giving us an insight in its natural state. This desert, like many arid environments, is vulnerable to exploitation for other purposes, including solar energy production and dry land farming

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia