Microbial host engineering for robust production platforms
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 use both systems and synthetic biology to investigate the role of cellular transporters and other tolerance genes, towards improving biofuel tolerance and production in a range of microbial hosts. We have identified export pumps as an ideal category of proteins that not only bestow tolerance against solvent like compounds but also improve production.
Our projects revolve around the discovery of carbon uptake and tolerance mechanisms, optimizing protein function and expression of these systems as well as integrating multiple phenotypes in the same strains. We also continue to develop tools for microbial strain engineering that are broadly applicable across multiple bacterial and fungal strains.
- Reider Apel A, d’Espaux L, Wehrs M, et al A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae NAR 2016
- Frederix M, et al Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass. Green Chemistry 2016
- Mukhopadhyay A Tolerance engineering in bacteria for the production of advanced biofuels and chemicals Trends in Microbiology 2015
Signal Transduction and response regulation in bacteria
Signaling systems are critical to bacteria in enabling them to continually monitor the environment and respond appropriately to any changes. The numbers and types of signaling systems a microbe possesses is an indication, both the variability of its environment, as well as its ability to perceive and fine-tune its response to diverse signals. We study signaling systems in microbes important for elemental cycling and a variety of environmental impacts, such as nitrate, sulfate and metal homeostasis.
We have examined all two components signaling systems in the model sulfate reducer Desulfovibrio vulgaris Hildenborough. Two component signaling in this organism spans transcriptionally modulated, cyclic-di-GMP modulated as well as chemotaxis modulated via direct protein-protein interactions. To examine transcriptionally acting response regulators in bacteria, we developed the DAP chip method. This enabled the evaluation of all DNA binding RRs in D. vulgaris and revealed unique regulatory clusters and interactions. It also led to the discovery of new motifs and provided the fundamental understanding of all genes that are targeted by two component signaling in this bacterium.
We have now developed a sequencing based DAP-seq to examine signaling in a range of environmentally important organisms. Our recent focus is on denitrifying bacteria such as Pseudomonas stutzeri along with many other isolates from environments such as the Oakridge Field research site.
- Garber ME, Rajeev L, et al. . Multiple signaling systems target a core set of transition metal homeostasis genes using similar binding motifs. Molecular Microbiology 2018
- Rajeev L, et al. Regulation of nitrite stress response in Desulfovibrio vulgaris Hildenborough, a model sulfate-reducing bacterium J Bact. 2015
- Rajeev L, et al. Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium. Genome Biology 2011
Discovery of genes on mobile genetic elements from natural isolates
- Kothari A, Wu Y-W, Charrier M, Rajeev L, Rocha AM, Paradis CJ, Hazen TC, Singer SW, Mukhopadhyay A Plasmid DNA analysis of pristine groundwater microbial communities reveal extensive presence of metal resistance genes BioRxIv 2017 doi: 10.1101/113860
Examination of impact of small molecules on microbial, plant microbial and metazoan systems
Completed Research (since 2007)
The microCLeAN G-agent mineralization project
In this DARPA funded project we developed a bacterial platform for the bioremediation of phosphonate ester containing nerve agents. We introduced catabolic enzyme pathways into E. coli to mineralize sarin, a phosphonate ester, into simple phosphorus and carbon units that can be utilized for the organism’s own growth. The mineralization of the carbonaceous part of Sarin is now published. We hope to publish the mineralization of the phosphonate part of soon.
Discovery of Alkane utilization genes
We examined the alkane utilizing ability of a newly sequenced microbe Acinetobacter venetianus RAG1. The RNAseq evaluation of this organism cultivated under various carbon sources relevealed the key genes involved in Alkane import and metabolism.
Signaling and gene regulation in dominant cyanobacteria in Desert soil crusts
We examined the physiology of Microcoleus vaginatus, the dominant cyanobacerium in desert soil crusts as it emerges from and reenters long term desiccation by simulating rainfall, diurnal cycles and dry-down. Read our paper in the ISME journal.
Functional Genomics in the model sulfate reducing bacterium Desulfovobrio vulgaris Hildenborough
We conducted a large set of studies for the sulfate reduced D. vulgaris to examine its response to salt, pH, oxygen, nitrate, nitrite as well as its adaptation to several of these stresses, its syntrophic interaction with methanogens and its cellular state in a biofilm.