Environmental modulation of exopolysaccharide production in the cyanobacterium Synechocystis 6803.

Microorganisms produce extracellular polymeric substances (EPS, also known as exopolysaccharides) of diverse composition and structure. The biochemical and biophysical properties of these biopolymers enable a wide range of industrial applications. EPS from cyanobacteria are particularly versatile as they incorporate a larger number and variety of building blocks and adopt more complex structures than EPS from other organisms. However, the genetic makeup and regulation of EPS biosynthetic pathways in cyanobacteria are poorly understood. Here, we measured the effect of changing culture media on titre and composition of EPS released by Synechocystis sp. PCC 6803, and we integrated this information with transcriptomic data. Across all conditions, daily EPS productivity of individual cells was highest in the early growth phase, but the total amount of EPS obtained from the cultures was highest in the later growth phases due to accumulation. Lowering the magnesium concentration in the media enhanced per-cell productivity but the produced EPS had a lower total sugar content. Levels of individual monosaccharides correlated with specific culture media components, e.g. xylose with sulfur, glucose and N-acetyl-galactosamine with NaCl. Comparison with RNA sequencing data suggests a Wzy-dependent biosynthetic pathway and a protective role for xylose-rich EPS. This multi-level analysis offers a handle to link individual genes to the dynamic modulation of a complex biopolymer. KEY POINTS: • Synechocystis exopolysaccharide amount and composition depends on culture condition • Production rate and sugar content can be modulated by Mg and S respectively • Wzy-dependent biosynthetic pathway and protective role proposed for xylose-rich EPS.

Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.

Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis.

The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexes mediate cotranslational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of unchaperoned protein in the aqueous phase. Here, we used giant unilamellar vesicles encapsulating minimal translation components to systematically interrogate the requirements for insertion of the model protein proteorhodopsin (PR) - a structurally ubiquitous membrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for cotranslational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a "pretranslocon" era, and they offer new opportunities for generating artificial cells.

Environmental Regulation of PndbA600, an Auto-Inducible Promoter for Two-Stage Industrial Biotechnology in Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase (ndbA) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.

Engineering Mannitol Biosynthesis in Escherichia coli and Synechococcus sp. PCC 7002 Using a Green Algal Fusion Protein.

The genetic engineering of microbial cell factories is a sustainable alternative to the chemical synthesis of organic compounds. Successful metabolic engineering often depends on manipulating several enzymes, requiring multiple transformation steps and selection markers, as well as protein assembly and efficient substrate channeling. Naturally occurring fusion genes encoding two or more enzymatic functions may offer an opportunity to simplify the engineering process and to generate ready-made protein modules, but their functionality in heterologous systems remains to be tested. Here we show that heterologous expression of a fusion enzyme from the marine alga Micromonas pusilla, comprising a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase, leads to synthesis of mannitol by Escherichia coli and by the cyanobacterium Synechococcus sp. PCC 7002. Neither of the heterologous systems naturally produce this sugar alcohol, which is widely used in food, pharmaceutical, medical, and chemical industries. While the mannitol production rates obtained by single-gene manipulation were lower than those previously achieved after pathway optimization with multiple genes, our findings show that naturally occurring fusion proteins can offer simple building blocks for the assembly and optimization of recombinant metabolic pathways.