A synthetic biology approach to engineering living photovoltaics

The ability to electronically interface living cells with electron accepting scaffolds is crucial for the development of next-generation biophotovoltaic technologies. Although recent studies have focused on engineering synthetic interfaces that can maximize electronic communication between the cell and scaffold, the efficiency of such devices is limited by the low conductivity of the cell membrane. This review provides a materials science perspective on applying a complementary, synthetic biology approach to engineering membrane–electrode interfaces. It focuses on the technical challenges behind the introduction of foreign extracellular electron transfer pathways in bacterial host cells and past and future efforts to engineer photosynthetic organisms with artificial electron-export capabilities for biophotovoltaic applications. The article highlights advances in engineering protein-based, electron-exporting conduits in a model host organism, E. coli, before reviewing state-of-the-art biophotovoltaic technologies that use both unmodified and bioengineered photosynthetic bacteria with improved electron transport. A thermodynamic analysis is used to propose an energetically feasible pathway for extracellular electron transport in engineered cyanobacteria and identify metabolic bottlenecks amenable to protein engineering techniques. Based on this analysis, an engineered photosynthetic organism expressing a foreign, protein-based electron conduit yields a maximum theoretical solar conversion efficiency of 6–10% without accounting for additional bioengineering optimizations for light-harvesting

Binding of the RNA chaperone Hfq to the type IV pilus base is crucial for its function in Synechocystis sp PCC 6803

This work was supported by the DFG priority program SPP1258 Sensory and Regulatory RNAs in Prokaryotes (Wi-2014/3-1, 3-2) to A.W. D.J.N. was supported by a Queen Mary college studentship

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane

Defining motility in the Staphylococci

The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions

A Synthetic Biology Approach to Engineering Living Photovoltaics.

The ability to electronically interface living cells with electron accepting scaffolds is crucial for the development of next-generation biophotovoltaic technologies. Although recent studies have focused on engineering synthetic interfaces that can maximize electronic communication between the cell and scaffold, the efficiency of such devices is limited by the low conductivity of the cell membrane. This review provides a materials science perspective on applying a complementary, synthetic biology approach to engineering membrane-electrode interfaces. It focuses on the technical challenges behind the introduction of foreign extracellular electron transfer pathways in bacterial host cells and the past and future efforts to engineer photosynthetic organisms with artificial electron-export capabilities for biophotovoltaic applications. The article highlights advances in engineering protein-based, electron-exporting conduits in a model host organism, E. coli, before reviewing state-of-the-art biophotovoltaic technologies that use both unmodified and bioengineered photosynthetic bacteria with improved electron transport capabilities. A thermodynamic analysis is used to propose an energetically feasible pathway for extracellular electron transport in engineered cyanobacteria and identify metabolic bottlenecks amenable to protein engineering techniques. Based on this analysis, an engineered photosynthetic organism expressing a foreign, protein-based electron conduit yields a maximum theoretical solar conversion efficiency of 6-10% without accounting for additional bioengineering optimizations for light-harvesting

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria