Mukhopadhyay:Research

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Host Engineering for solvent tolerant microbes Efflux Pumps provide a direct mechanism to alleviate product toxicity (Image prepared by Everett Kaplan (JBEI)) Many petrochemical compounds that are candidates for microbial production are also solvent-like in nature. Examples of such compounds are fuels and other bulk chemicals such as precursors for polymers and plastics. For these compounds, two aspects impede the efficiency of microbial production. One is their inherent solvent like nature that results in toxicity towards the microbe. Second is product inhibition due to intracellular accumulation. We have used both systems and synthetic biology to successfully investigate the role of cellular transporters and other tolerance genes, towards improving biofuel tolerance and production in Escherichia coli. Using simple but effective competition based strategies we identified heterologous pumps that bestowed tolerance against representative biogasoline, biodiesel and biojetfuel candidates. We have also used functional genomics data to identify tolerance bestowing genes and used them successfully to increase both tolerance and production levels. Transporters specifically stand out as an ideal tolerance mechanism, as they also serve to export a final product. Building upon the discovery of various transport systems, we are now exploring several avenues to optimize the use of transporters in microbial host engineering. Strategies that improve the efficiency of a transporter (e.g. via directed evolution) have allowed us to bypass the necessity to overexpress these high burden systems. Optimization of expression systems has also proven to be important in maximizing the benefits from a specific pump in a fuel production strain. As we study mechanisms of solvent tolerance we also understand more about the molecular basis for toxicity and stress response. This work is done as the Engineering team at the Joint BioEnergy Institute. Signal Transduction and survival in metal reducing bacteria Map of genes regulated by Response Regulators in D. vulgaris (From Rajeev et al 2011) Signaling systems are critical to bacteria in enabling them to continually monitor their environment and respond appropriately to any changes. The numbers and types of signaling systems a microbe possesses is an indication both of the variability of its environment as well as its ability to perceive and fine-tune its response to diverse signals. As part of the ENIGMA Scientific Focus Area, we are studying signaling systems in microbes present in DOE-relevant sites. Desulfovibrio vulgaris is a model sulfate-reducing bacterium with a vast array of uncharacterized signaling and regulatory systems. Our group has developed and optimized an in vitro microarray-based DAP-chip (or seq) method to determine gene targets for bacterial response regulators and used this method to reveal regulatory networks by determining the gene targets for almost all (twenty-four) D. vulgaris two component response regulators that function via transcriptional control. Our study led to the discovery of a complex regulatory network around the central carbon metabolic pathway of lactate uptake and oxidation, which is under the control of lactate-sensing, nitrite-sensing, and phosphate-sensing two-component systems. Currently, we are characterizing cyclic-di-GMP based signaling pathways, the role of which has not been examined in sulfate-reducing bacteria. To this end, we have identified one cyclic-di-GMP-modulating response regulator that impacts biofilm formation, and one that impacts planktonic growth. In collaboration with other ENIGMA researchers, we are also examining sigma54-dependent one-component systems, and unique tungstate-responsive transcription factors. Other ongoing experiments involve using transposon mutant pools to determine genes required for fitness in limiting nutrient conditions as often found in the environment, as well as genes that are required for motility and chemotaxis. The microCLeAN G-agent mineralization project This project focuses on the development of bacterial platforms for the bioremediation of phosphonate ester containing nerve agents. We introduce catabolic enzyme pathways into E. coli to mineralize sarin, a phosphonate eseter, into basic phosphorus and carbon units that can be utilized for the organism’s own growth. The current focus is on engineering esterases for efficient hydrolysis of diverse phosphonate esters. In collaboration with with Dr. Margaret Brown in Prof. Jay Keasling’s lab, we hope to generate a strain that cal fully catabolize Sarin - using it both as its phosphate and carbon source. Signaling and gene regulation in dominant cyanobacteria in Desert soil crusts A gift of Microcoleus culture from the Garcia-Pichel group to start off our project Desert soil crusts are living systems, primarily microbial, that cover large areas of our planet. Complex enough to survive extreme conditions and simple enough to be studied using state of the art technologies, these microbial communities provide invaluable systems to evaluate the impact of climate change on carbon flux. The cyanobacterium that is the dominant organism in these crusts is being sequenced at JGI as is the entire desert soil crusts community. These organisms encode elegant signal transduction and response regulatory systems that are at the core of the ability of these microbes to respond and survive in their ecosystems. The study of these signaling mechanisms and the corresponding response provides the molecular level assessment of important biogeochemical activities that will be utilized for improved climate models.