Cyanobacterial Alkanes Modulate Photosynthetic Cyclic Electron Flow to Assist Growth under Cold Stress.

All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons at concentrations similar to chlorophyll. Recently, two universal but mutually exclusive hydrocarbon production pathways in cyanobacteria were discovered. We engineered a mutant of Synechocystis sp. PCC 6803 that produces no alkanes, which grew poorly at low temperatures. We analyzed this defect by assessing the redox kinetics of PSI. The mutant exhibited enhanced cyclic electron flow (CEF), especially at low temperature. CEF raises the ATP:NADPH ratio from photosynthesis and balances reductant requirements of biosynthesis with maintaining the redox poise of the electron transport chain. We conducted in silico flux balance analysis and showed that growth rate reaches a distinct maximum for an intermediate value of CEF equivalent to recycling 1 electron in 4 from PSI to the plastoquinone pool. Based on this analysis, we conclude that the lack of membrane alkanes causes higher CEF, perhaps for maintenance of redox poise. In turn, increased CEF reduces growth by forcing the cell to use less energy-efficient pathways, lowering the quantum efficiency of photosynthesis. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacterial thylakoid membranes: they regulate redox balance and reductant partitioning in these oxygenic photosynthetic cells under stress.

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the Pcpc560 or the Ptrc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.

Rapid construction of metabolic models for a family of Cyanobacteria using a multiple source annotation workflow.

BACKGROUND: Cyanobacteria are photoautotrophic prokaryotes that exhibit robust growth under diverse environmental conditions with minimal nutritional requirements. They can use solar energy to convert CO2 and other reduced carbon sources into biofuels and chemical products. The genus Cyanothece includes unicellular nitrogen-fixing cyanobacteria that have been shown to offer high levels of hydrogen production and nitrogen fixation. The reconstruction of quality genome-scale metabolic models for organisms with limited annotation resources remains a challenging task. RESULTS: Here we reconstruct and subsequently analyze and compare the metabolism of five Cyanothece strains, namely Cyanothece sp. PCC 7424, 7425, 7822, 8801 and 8802, as the genome-scale metabolic reconstructions iCyc792, iCyn731, iCyj826, iCyp752, and iCyh755 respectively. We compare these phylogenetically related Cyanothece strains to assess their bio-production potential. A systematic workflow is introduced for integrating and prioritizing annotation information from the Universal Protein Resource (Uniprot), NCBI Protein Clusters, and the Rapid Annotations using Subsystems Technology (RAST) method. The genome-scale metabolic models include fully traced photosynthesis reactions and respiratory chains, as well as balanced reactions and GPR associations. Metabolic differences between the organisms are highlighted such as the non-fermentative pathway for alcohol production found in only Cyanothece 7424, 8801, and 8802. CONCLUSIONS: Our development workflow provides a path for constructing models using information from curated models of related organisms and reviewed gene annotations. This effort lays the foundation for the expedient construction of curated metabolic models for organisms that, while not being the target of comprehensive research, have a sequenced genome and are related to an organism with a curated metabolic model. Organism-specific models, such as the five presented in this paper, can be used to identify optimal genetic manipulations for targeted metabolite overproduction as well as to investigate the biology of diverse organisms.

Synthetic biology of cyanobacteria: unique challenges and opportunities.

Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria's potential for these functions, but unfortunately tool development for these organisms has lagged behind that for S. cerevisiae and E. coli. While these organisms may in many cases be more difficult to work with as "chassis" strains for synthetic biology than certain heterotrophs, the unique advantages of autotrophs in biotechnology applications as well as the scientific importance of improved understanding of photosynthesis warrant the development of these systems into something akin to a "green E. coli." In this review, we highlight unique challenges and opportunities for development of synthetic biology approaches in cyanobacteria. We review classical and recently developed methods for constructing targeted mutants in various cyanobacterial strains, and offer perspective on what genetic tools might most greatly expand the ability to engineer new functions in such strains. Similarly, we review what genetic parts are most needed for the development of cyanobacterial synthetic biology. Finally, we highlight recent methods to construct genome-scale models of cyanobacterial metabolism and to use those models to measure properties of autotrophic metabolism. Throughout this paper, we discuss some of the unique challenges of a diurnal, autotrophic lifestyle along with how the development of synthetic biology and biotechnology in cyanobacteria must fit within those constraints.

Reconstruction and comparison of the metabolic potential of cyanobacteria Cyanothece sp. ATCC 51142 and Synechocystis sp. PCC 6803.

Cyanobacteria are an important group of photoautotrophic organisms that can synthesize valuable bio-products by harnessing solar energy. They are endowed with high photosynthetic efficiencies and diverse metabolic capabilities that confer the ability to convert solar energy into a variety of biofuels and their precursors. However, less well studied are the similarities and differences in metabolism of different species of cyanobacteria as they pertain to their suitability as microbial production chassis. Here we assemble, update and compare genome-scale models (iCyt773 and iSyn731) for two phylogenetically related cyanobacterial species, namely Cyanothece sp. ATCC 51142 and Synechocystis sp. PCC 6803. All reactions are elementally and charge balanced and localized into four different intracellular compartments (i.e., periplasm, cytosol, carboxysome and thylakoid lumen) and biomass descriptions are derived based on experimental measurements. Newly added reactions absent in earlier models (266 and 322, respectively) span most metabolic pathways with an emphasis on lipid biosynthesis. All thermodynamically infeasible loops are identified and eliminated from both models. Comparisons of model predictions against gene essentiality data reveal a specificity of 0.94 (94/100) and a sensitivity of 1 (19/19) for the Synechocystis iSyn731 model. The diurnal rhythm of Cyanothece 51142 metabolism is modeled by constructing separate (light/dark) biomass equations and introducing regulatory restrictions over light and dark phases. Specific metabolic pathway differences between the two cyanobacteria alluding to different bio-production potentials are reflected in both models.

Upregulation of plasmid genes during stationary phase in Synechocystis sp. strain PCC 6803, a cyanobacterium.

We analyzed DNA microarrays to identify highly expressed genes during stationary-phase growth of Synechocystis sp. PCC 6803. Many identified genes are on endogenous plasmids, with copy numbers between 0.4 and 7 per chromosome. The promoters of such genes will be useful for synthetic biology applications with this phototrophic host.

Comprehensive analysis of the Brassica juncea root proteome in response to cadmium exposure by complementary proteomic approaches.

Indian mustard (Brassica juncea L.) is known to both accumulate and tolerate high levels of heavy metals from polluted soils. To gain a comprehensive understanding of the effect of cadmium (Cd) treatment on B. juncea roots, two quantitative proteomics approaches--fluorescence two-dimensional difference gel electrophoresis (2-D DIGE) and multiplexed isobaric tagging technology (iTRAQ)--were implemented. Several proteins involved in sulfur assimilation, redox homeostasis, and xenobiotic detoxification were found to be up-regulated. Multiple proteins involved in protein synthesis and processing were down-regulated. While the two proteomics approaches identified different sets of proteins, the proteins identified in both datasets are involved in similar biological processes. We show that 2-D DIGE and iTRAQ results are complementary, that the data obtained independently using the two techniques validate one another, and that the quality of iTRAQ results depends on both the number of biological replicates and the number of sample injections. This study determined the involvement of enzymes such as peptide methionine sulfoxide reductase and 2-nitropropane dioxygenase in alternatives redox-regulation mechanisms, as well as O-acetylserine sulfhydrylase, glutathione-S-transferase and glutathione-conjugate membrane transporter, as essential players in the Cd hyperaccumation and tolerance of B. juncea.