476130 Harnessing Diverse Microorganisms for Biochemical Production
Biochemical production of compounds offers advantages over conventional synthesis due to the high specificity and diversity of biological catalysts, reactions occurring under labile conditions, and the use of renewable substrates. Metabolic engineering of microbes has primarily focused on modifying the model organisms E. coli and S. cerevisiae due to their fast and robust growth on inexpensive fermentation broths and the wealth of genetic tools available to them. Heterologous pathway addition in these platform hosts has had mixed success, often with natural producers reaching higher titers due to improper recombinant enzyme folding and regulation. Additionally, other traits should be considered before selecting a production host, such as substrate utilization, endogenous pathways, product toxicity, secretion of product or enzymes, growth temperature, susceptibility to contamination, pathogenicity of host, and growth environment. These factors have led to the development of alternative hosts for chemical production, including C. glutamicum (amino acid synthesis), Synechocystis sp. (CO2 autotroph), C. thermocellum (consolidated biomass processing), and B. subtilis (GRAS certified, carotenoids, protein secretion), just to name a few. Further, advances in synthetic biology and –omics techniques have enabled the rapid modification and characterization of microbes. Our laboratory will use these methods and develop additional tools to harness the diversity of microorganisms for biochemical production. Specifically, we aim to 1) engineer organisms that are uniquely tolerant to reaction conditions, 2) use protein engineering and synthetic biology tools to enhance enzyme production and regulation in these hosts, and 3) discover novel pathways and organisms through bioprospecting to expand the range of chemicals to be synthesized and traits to be selected from.
Product toxicity and removal are two long-standing challenges to bioprocess engineering. Co-solvent culturing aims to alleviate both problems by stripping off products as they are generated, sequestering them from the organism and eliminating them from further conversion. We plan to use sustainable solvents and organisms that grow under these unique reaction conditions to produce compounds of interest, such as aldehydes and alkanes, that are highly toxic to cells and have high volatility. In addition to conventional metabolic engineering to increase biochemical production, we will develop new tools to enhance enzyme expression and create gene knockouts in non-model organisms. In particular, we will develop protein-folding reporters to assess and enhance heterologous enzyme stability in non-model hosts. Combinatorial libraries and metabolic flux analysis will be used to balance enzyme expression. CRISPR-based tools will be created for gene editing and chromosomal integration of pathways. Our lab will seek to not only modify organisms that have already been cultured, but to discover new hosts from environmental samples with unique characteristics to enhance biochemical production. Through sequencing and annotation of pools of organisms from specific environments, we hope to find novel enzymes for both production and remediation of compounds of interest. We aim to study the mechanisms that give rise to the unique traits in these organisms to further improve them for specific processes or transfer them to other species of interest. Through this work, we hope to create a set of design rules for utilizing microbes for chemical production as well as put together a pipeline of methods that is needed to systematically discover, characterize and engineer an organism of interest.
I hope to use teaching of courses such as kinetics, separations, control theory and bioprocess engineering as well as outreach with high school students and teachers to share and further develop our study of biochemical engineering of microorganisms. Additionally, I plan to develop new courses focused around hand-on design of biological systems and implement team-based projects in classes I teach.
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