Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology.

Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P(rnpB) and variants of P(rbcL), and a version of the promoter P(trc) with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

Design, engineering, and construction of photosynthetic microbial cell factories for renewable solar fuel production.

There is an urgent need to develop sustainable solutions to convert solar energy into energy carriers used in the society. In addition to solar cells generating electricity, there are several options to generate solar fuels. This paper outlines and discusses the design and engineering of photosynthetic microbial systems for the generation of renewable solar fuels, with a focus on cyanobacteria. Cyanobacteria are prokaryotic microorganisms with the same type of photosynthesis as higher plants. Native and engineered cyanobacteria have been used by us and others as model systems to examine, demonstrate, and develop photobiological H(2) production. More recently, the production of carbon-containing solar fuels like ethanol, butanol, and isoprene have been demonstrated. We are using a synthetic biology approach to develop efficient photosynthetic microbial cell factories for direct generation of biofuels from solar energy. Present progress and advances in the design, engineering, and construction of such cyanobacterial cells for the generation of a portfolio of solar fuels, e.g., hydrogen, alcohols, and isoprene, are presented and discussed. Possibilities and challenges when introducing and using synthetic biology are highlighted.

Cyanobacterial hydrogenases: diversity, regulation and applications.

Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an uptake enzyme found in N(2)-fixing strains, and a bidirectional one present in both non-N(2)-fixing and N(2)-fixing strains. The uptake hydrogenase (encoded by hupSL) catalyzes the consumption of the H(2) produced during N(2) fixation, while the bidirectional enzyme (hoxEFUYH) probably plays a role in fermentation and/or acts as an electron valve during photosynthesis. hupSL constitute a transcriptional unit, and are essentially transcribed under N(2)-fixing conditions. The bidirectional hydrogenase consists of a hydrogenase and a diaphorase part, and the corresponding five hox genes are not always clustered or cotranscribed. The biosynthesis/maturation of NiFe-hydrogenases is highly complex, requiring several core proteins. In cyanobacteria, the genes that are thought to affect hydrogenases pleiotropically (hyp), as well as the genes presumably encoding the hydrogenase-specific endopeptidases (hupW and hoxW) have been identified and characterized. Furthermore, NtcA and LexA have been implicated in the transcriptional regulation of the uptake and the bidirectional enzyme respectively. Recently, the phylogenetic origin of cyanobacterial and algal hydrogenases was analyzed, and it was proposed that the current distribution in cyanobacteria reflects a differential loss of genes according to their ecological needs or constraints. In addition, the possibilities and challenges of cyanobacterial-based H(2) production are addressed.

Design and construction of a photobioreactor for hydrogen production, including status in the field.

Several species of microalgae and phototrophic bacteria are able to produce hydrogen under certain conditions. A range of different photobioreactor systems have been used by different research groups for lab-scale hydrogen production experiments, and some few attempts have been made to upscale the hydrogen production process. Even though a photobioreactor system for hydrogen production does require special construction properties (e.g., hydrogen tight, mixing by other means than bubbling with air), only very few attempts have been made to design photobioreactors specifically for the purpose of hydrogen production. We have constructed a flat panel photobioreactor system that can be used in two modes: either for the cultivation of phototrophic microorganisms (upright and bubbling) or for the production of hydrogen or other anaerobic products (mixing by "rocking motion"). Special emphasis has been taken to avoid any hydrogen leakages, both by means of constructional and material choices. The flat plate photobioreactor system is controlled by a custom-built control system that can log and control temperature, pH, and optical density and additionally log the amount of produced gas and dissolved oxygen concentration. This paper summarizes the status in the field of photobioreactors for hydrogen production and describes in detail the design and construction of a purpose-built flat panel photobioreactor system, optimized for hydrogen production in terms of structural functionality, durability, performance, and selection of materials. The motivations for the choices made during the design process and advantages/disadvantages of previous designs are discussed.

Hydrogen production by the engineered cyanobacterial strain Nostoc PCC 7120 ΔhupW examined in a flat panel photobioreactor system.

Nitrogenase based hydrogen production was examined in a ΔhupW strain of the filamentous heterocystous cyanobacterium Nostoc PCC 7120, i.e., cells lacking the last step in the maturation system of the large subunit of the uptake hydrogenase and as a consequence with a non-functional uptake hydrogenase. The cells were grown in a developed flat panel photobioreactor system with 3.0L culture volume either aerobically (air) or anaerobically (Ar or 80% N2/20% Ar) and illuminated with a mixture of red and white LED. Aerobic growth of the ΔhupW strain of Nostoc PCC 7120 at 44µmolar photons m(-2)s(-1) PAR gave the highest hydrogen production of 0.7mL H2 L(-1)h(-1), 0.53mmol H2 mg chlorophyll a(-1)h(-1), and a light energy conversion efficiency of 1.2%. Anaerobic growth using 100% argon showed a maximal hydrogen production of 1.7mLL(-1)h(-1), 0.85mmol per mg chlorophyll a(-1) h(-1), and a light energy conversion efficiency of 2.7%. Altering between argon/N2 (20/80) and 100% argon phases resulted in a maximal hydrogen production at hour 128 (100% argon phase) with 6.2mL H2L(-1)h(-1), 0.71mL H2 mg chlorophyll a(-1)h(-1), and a light energy efficiency conversion of 4.0%. The highest buildup of hydrogen gas observed was 6.89% H2 (100% argon phase) of the total photobioreactor system with a maximal production of 4.85mL H2 L(-1)h(-1). The present study clearly demonstrates the potential to use purpose design cyanobacteria in developed flat panel photobioreactor systems for the direct production of the solar fuel hydrogen. Further improvements in the strain used, environmental conditions employed, and growth, production and collection systems used, are needed before a sustainable and economical cyanobacterial based hydrogen production can be realized.

Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium.

BACKGROUND: Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library). RESULTS: We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis. CONCLUSIONS: The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.

Synthetic biology in cyanobacteria engineering and analyzing novel functions.

Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.

Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera Roseobacter, Ruegeria and Leisingera.

A heterotrophic, antibiotic-producing bacterium, strain T5(T), was isolated from the German Wadden Sea, located in the southern region of the North Sea. Sequence analysis of the 16S rRNA gene of this strain demonstrated close affiliation with Roseobacter gallaeciensis BS107(T) (99 % similarity), but the results of genotypic (DNA-DNA hybridization and DNA G + C content) and phenotypic characterization revealed that strain T5(T) represents a novel species. The novel organism is strictly aerobic, Gram-negative, rod-shaped, motile and forms brown-pigmented colonies. Strain T5(T) produces the antibiotic tropodithietic acid throughout the exponential phase which inhibits the growth of bacteria from different taxa, as well as marine algae. Strain T5(T) requires sodium ions and utilizes a wide range of substrates, including oligosaccharides, sugar alcohols, organic acids and amino acids. The DNA G + C content is 55.7 mol%. Comparative 16S rRNA gene sequence analysis revealed that strains T5(T) and Roseobacter gallaeciensis BS107(T) group with Leisingera methylohalidivorans as their closest described relative within the Roseobacter clade (97.9 and 97.6 % sequence similarity, respectively) and with Ruegeria algicola (96.6 and 96.5 % similarity, respectively) of the Alphaproteobacteria. Comparison of strains T5(T) and Roseobacter gallaeciensis BS107(T) with Roseobacter denitrificans and Roseobacter litoralis showed striking differences in 16S rRNA gene sequence similarities, chemical composition, pigmentation, presence of bacteriochlorophyll a and antibiotic production. On the basis of these results, it is proposed that Roseobacter gallaeciensis is reclassified as the type species of a new genus, Phaeobacter, as Phaeobacter gallaeciensis comb. nov. (type strain BS107(T) = CIP 105210(T) = ATCC 700781(T) = NBRC 16654(T) = DSM 17395(T)). Strain T5(T) (=LMG 22475(T) = DSM 16374(T)) is proposed as the type strain of a novel species of this genus, Phaeobacter inhibens sp. nov. At the same time, emended descriptions are provided of the genera Roseobacter, Ruegeria and Leisingera, as well as reclassifying Ruegeria algicola as the type species of a new genus, Marinovum, with the name Marinovum algicola comb. nov.

Antibiotic production by a Roseobacter clade-affiliated species from the German Wadden Sea and its antagonistic effects on indigenous isolates.

A strain affiliated with the Roseobacter clade and producing a new antibiotic named tropodithietic acid (L. Liang, Ph.D. thesis, University of Göttingen, Göttingen, Germany, 2003) was isolated from the German Wadden Sea. The compound showed strong inhibiting properties with respect to marine bacteria of various taxa and marine algae. Antibiotic production was found to occur during the complete growth phase. Strain mutants without antagonistic properties appeared several times spontaneously.