Understanding Gene Regulatory Networks in Hypersaline-adapted Archaea: Toward Synthetic Biology for Industrial Applications

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The project seeks to understand how gene circuits function and evolve in microorganisms living under extreme conditions. This knowledge will be used to enable the construction of synthetic prototype strains that efficiently produce biodegradable plastic from inexpensive feedstock. Synthetic biology has yet to explore the use of archaeal genetic circuitry and metabolic pathways despite their potential value for applications in industrial chemical and biofuel production. Archaea are single-celled microbes that thrive at the limits of life, found in deep-sea hydrothermal vents under high pressure and temperature, saturated salt lakes, and polar icecaps. Survival in these environments requires unique genetic and metabolic strategies, the natural chemical byproducts of which are attractive to industry (biofuels, biodegradable plastics). Strategies that engineer archaeal gene circuits or swap these circuits between archaeal strains to boost chemical production are attractive for biomanufacturing of fuels and chemicals. The NSF funded research will also contribute to education, training, and outreach at the high school and undergraduate levels. Specifically, the PI will collaborate with teachers to continue offering weeklong science immersion courses for high school students from North Carolina School of Science and Math. This public high school in Durham, NC, draws the top students from each congressional district in the state, providing even representation across cultural and socioeconomic backgrounds. The PI's ongoing "Introduction to Systems Biology" will engage biology and engineering undergraduates in collaborative active learning projects to build and analyze gene networks from data generated through the research. Several interested students from the class, together with HBCU summer students recruited through established Duke programs, will engage in research projects in the PI's lab. Through these activities, high school and undergraduate students will contribute directly to generating and analyzing research data. This work will engage students in research earlier in their careers and retain them in STEM fields. These activities are expected to have lasting effects on the recruitment and retention of researchers, especially from underrepresented groups.TECHNICAL DESCRIPTION: Synthetic biology has yet to explore the use of archaeal transcriptional systems and metabolic pathways despite their potential value for applications in industry and biofuel production. The hypersaline-adapted group of archaeal microbes, hereafter referred to as halophiles, hold significant promise because they naturally produce chemicals attractive to industry (isoprenoid lipids for fuels, polyhydroxyalkanoate for biodegradable plastics). Strategies that engineer halophile gene regulatory networks (GRNs) or swap gene circuits between halophile strains are attractive for biofuel or other industrial applications. However, before halophile networks can be customized and controlled, additional basic understanding of GRNs and transcription factor (TF) function is required. This plan has three objectives: (1) Characterize the topology, dynamics, and phenotypic output of small-scale GRN motifs that regulate stress and metabolic responses in halophiles. GRN motifs known to regulate important metabolic pathways and extreme stress resistance across four related halophile species using time course gene expression and TF-DNA binding measurements. (2) Build predictive genome-scale statistical models for each halophile to quantify and compare GRN motif architecture and dynamics. Data generated from objective 1 will be integrated into predictive models at two levels of detail: small-scale dynamical models and genome-scale gene regulatory interaction network models. Models will be compared across organisms. (3) Test model predictions in proof-of-principle synthetic biology experiments. Model tests will include three stages of molecular biology experiments: promoter-reporter fusions to test predictions regarding TF-cis-regulatory sequence interactions, high-resolution time course gene expression experiments, and building prototype synthetic biology circuits for increasing production of polyhydroxyalkanoate in halophiles. The products of this research will include predictive GRN models for four related species and prototype synthetic circuits. These gene circuits will test model predictions and increase the production of biodegradable plastic in halophiles. In the long term, the PI aims to exploit halophile GRNs to extend options for biotechnology and bioenergy.