Metabolic Engineering, Genomics and Computational Biology in Clostridia and E. coli for biofuels and biorefining applications
In the prokaryotic projects, the focus is to develop and use genomic and metabolic engineering tools, both experimental and computational, in order to understand the genomic basis of cellular differentiation (sporulation) and physiology of clostridia and generate strains suitable for applications in biorefining. Clostridia are anaerobic, endospore-forming prokaryotes (bacteria) that include strains of importance to human and animal health (the genus includes several important pathogens like Clostridium botulinum, C. tetani, C. perfrigens, and C. difficile) and physiology, cellulose degradation, solvent (butanol, acetone and ethanol) production and bioremediation. In addition to transcriptional analysis by DNA microarrays, and using Clostridium acetobutylicum as a model organism, we develop new experimental (e.g., ChIP-on-chip, that is, chromatin immunoprecipitation on a "chip" (DNA microarray)) and computational tools that will allow to develop a detailed understanding of the genetic networks (including the determination of the large regulons of important transcription factors) and signal-transduction that underlies the complex phenotypes of sporulation, stress responses, motility, chemotaxis, and germination. The laboratory is working to develop efficient chromosomal integration tools in clostridia, necessary for both functional genomic studies as well as strain development. Important applied goals include the understanding of the cellular and molecular basis of solvent and carboxylic-acid toxicity, and the generation of tolerant strains for bioprocessing (to produce biofuels) and bioremediation applications. Another goal is the generation of stable, asporogenous solvent overproducing strains for economical solvent production from renewable resources.
Our laboratory is also interested in the principles and tools necessary to develop complex microbial phenotypes. Such phenotypes include tolerant phenotypes. The issue of tolerance of cells to a variety of stressful conditions is an important problem both fundamentally and practically. For example, the ability of cells to withstand "stressful" bioprocessing conditions without loss of productivity is a most significant goal. Such conditions include: toxic substrates, accumulation of toxic products & byproducts, high or low pH, or high salt concentrations as encountered in most applications for the production of chemicals and biofuels as well as in bioremediation applications. The difficulty -but also the intellectual and biotechnological challenge - is that the desirable phenotypic trait is determined by several genes or a complex regulatory circuit. Complex phenotypes are also encountered when one desires to develop a de novo capability or pathway in a particular cell type. For example, how do cells put together the regulatory elements of a sequence of genes to make a pathway or program possible? Yes, it is an evolutionary process, but if we are to "imitate" the process, what would we do? What tools could one possibly use and strategies to facilitate the development of complex phenotypes in microbial cells? From omics-based analysis to synthesis, all selection based, or hybrid? Knowledge-based and mechanistic or not? Conceptual strategies, and multi-library-based tools are the focus current efforts in the lab. The development of tolerant phenotypes is pursued in both solventogenic, butyric-acid clostridia (C. acetobutylicum) as well as in the important platform organism E. coli.
An important area of research involving a complex phenotype is to develop by genetic and high-throughput (HT) means clostridia strains capable of effectively and quickly utilize directly the the two important components of cellulosics, semi-cellulose (xylans) and cellulose. The complexity of the phenotype calls for a HT exploration of the possibilities for achieving this important goal, which would significantly simplify bioprocessing into a single step (termed Consolidated Biorpocessing) of cellulosics utilization and fermentation into desirable products such as biofuels (butanol and other 4-C products, ethanol, and 3-C chemicals).
An important activity of the lab involves the development of computational biology and bioinformatic tools for mining our genomic data, for discovering new regulatory patterns and molecules (small RNAs), and for building models that would predict the behavior of key metabolic patterns and simple phenotypes, and guide the experimentation towards achieving complex phenotypes such as those discussed above. The development of genome-scale metabolic and differentiation models is an important goal towards achieving more complex predictive capabilities.
