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The last few years have witnessed an exponential increase in the number of bacteria that can be used as microbial cell platforms in practical applications. The microorganisms which are the easiest to manipulate genetically (i.e., the so-called "model" bacteria, such as Escherichia coli or Bacillus subtilis) are often not adequate to perform given biotechnological applications (e.g., harsh oxidations or dehalogenation reactions). Contemporary Synthetic Biology endeavors rely on the adoption of specific bacterial chasses for plugging-in and -out genetic circuits and engineer new-to-Nature functionalities. Against this background, environmental bacteria, such as Pseudomonas strains, constitute ideal starting points to design flawless microbial cell platforms, since these microorganisms are pre-endowed with a number of metabolic and stress-endurance traits that are optimal for biotechnological needs. Recent developments on the taming of P. putida for biotechnological applications will be discussed in the context of Synthetic Biology strategies for [i] re-designing the metabolic architecture of central carbon catabolism and [ii] manipulating catalytic biofilms through Synthetic Morphology approaches. [mehr]

Cells usestructure to catalyze and facilitate the chemical reactions of metabolism. Thisprinciple is exemplified by the process of carbon dioxide assimilation inphotosynthetic cyanobacteria, which coordinate myriad biochemical components inspace and time, in order to achieve a single physiological goal – convert solarpower into fixed chemical energy. An essential player in this process is thecarboxysome, a protein-based organelle composed of an icosahedral proteinshell, which encapsulates the enzymes RuBisCO and carbon anhydrase within a~100 nm structure. Despite knowledge of the overall structure of thecarboxysome, much less is known about the molecular interactions driving itsself-assembly and how this process is capable of occurring in the complex invivo environment. Here, I describe our biochemical efforts to elucidate amechanistic picture of how the carboxysome assembles and functions in the cell.At the same time, our lab is also interested in developing a holistic pictureof how a coordinated physiology emerges from the many different proteinactivities, including the carboxysome and numerous transporters, found incyanobacteria. To this end, I present our recent efforts at reconciling theseactivities using a mathematical reaction-diffusion model of carbon dioxideassimilation. Finally, a general challenge to studying physiology is the lowthroughput of assays for quantifying metabolism. I therefore conclude with ourefforts at using protein engineering for constructing fluorescent metabolitebiosensors and enabling high-throughput studies of metabolism. [mehr]

Bacterial microcompartments are widespread metabolic modules found in at least 23 bacterial phyla. The carboxysome is the best studied bacterial microcompartment and serves to illustrate the basic principles of bacterial micrcompartment structure and function. It consists of a selectively permeable protein shell encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Cyanobacteria depend on the carboxysome to concentrate carbon dioxide near RuBisCO while minimizing the wasteful side reaction with oxygen. The carboxysome functions as an organelle but, in contrast to eukaryotic organelles, is composed entirely of protein; thousands of protein subunits self-assemble to form this ~300 MDa complex. Many bacteria assemble architecturally-related types of organelles for diverse catabolic functions. Our studies suggest that the principles of carboxysome structure, function and assembly likely extend to other bacterial microcompartments and provide the foundation for design and construction of synthetic nanoreactors based on bacterial microcompartment architecture. [mehr]

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