Within the wide biodiversity that is found in the bacterial world, Cyanobacteria represents a unique phylogenetic group that is responsible for a key metabolic process in the biosphere - oxygenic photosynthesis - and that includes representatives exhibiting complex morphologies. Many cyanobacteria are multicellular, growing as filaments of cells in which some cells can differentiate to carry out specialized functions. These differentiated cells include resistance and dispersal forms as well as a metabolically specialized form that is devoted to N(2) fixation, known as the heterocyst. In this Review we address cyanobacterial intercellular communication, the supracellular structure of the cyanobacterial filament and the basic principles that govern the process of heterocyst differentiation.

Compartmentalized function through cell differentiation in filamentous cyanobacteria

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

Synthetic biology in cyanobacteria engineering and analyzing novel functions.

Cyanobacteria and algae are becoming increasingly attractive cell factories for producing renewable biofuels and chemicals due to their ability to capture solar energy and CO2 and their relatively simple genetic background for genetic manipulation. Increasing research efforts from the synthetic biology approach have been made in recent years to modify cyanobacteria and algae for various biotechnological applications. In the article, we critically review recent progresses in developing genetic tools for characterizing or manipulating cyanobacteria and algae, the applications of genetically modified strains for synthesizing renewable products such as biofuels and chemicals. In addition, the emergent challenges in the development and application of synthetic biology for cyanobacteria and algae are also discussed.

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Application of synthetic biology in cyanobacteria and algae.

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

/pdf/broad-host-range-vector-system-for-synthetic-biology-and-3i8h001its.pdf

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

Freshwater cyanobacteria produce several bioactive secondary metabolites with diverse chemical structure, which may achieve high concentrations in the aquatic medium when cyanobacterial blooms occur. Some of the compounds released by cyanobacteria have allelopathic properties, influencing the biological processes of other phytoplankton or aquatic plants. These kinds of interactions are more easily detectable under laboratory studies; however their ecological relevance is often debated. Recent research has discovered new allelopathic properties in some cyanobacteria species, new allelochemicals and elucidated some of the allelopathic mechanisms. Ecosystem-level approaches have shed some light on the factors that influence allelopathic interactions, as well as how cyanobacteria may be able to modulate their surrounding environment by means of allelochemical release. Nevertheless, the role of allelopathy in cyanobacteria ecology is still not well understood, and its clarification should benefit from carefully designed field studies, chemical characterization of allelochemicals and new methodological approaches at the "omics" level.

Allelopathy in freshwater cyanobacteria

http://www.csun.edu/~mls42367/Summers_papers/%20%20Argueta%20et%20al.pdf

Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme.

Akinetes are spore-like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild-type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild-type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real-time quantitative PCR and subsequent testing for akinete-specific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete-expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP-binding cassette (ABC)-type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete-expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.

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Kamile Yuksek

Papers

Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme.

Identification of Nostoc punctiforme akinete-expressed genes using differential display.