Host Engineering Group
Microbial life forms are an inexhaustible source of beneficial compounds ranging from pharmaceuticals to fuels. However, in their native state, microbes produce miniscule quantities of these molecules. Metabolic engineering efforts start with the introduction of one or more heterologous pathways in a host microbe for the production of desired compounds such as advanced biofuels. Subsequent optimization of first generation engineered strains requires the application of mathematical and synthetic biology tools, activities, which comprise the core research areas of the Host Engineering Department of JBEI. Overarching goals of host engineering are to: (1) enhance production titers of engineered strains; (2) develop resistant phenotypes towards toxic pretreatment inhibitors; (3) develop systems level understanding of phenotypic improvements to combine multiple traits in a single strain; and, (4) expand the knowledgebase generated from model systems to new microbes.
Local and Global Optimization
Currently, we are examining the following hosts for lignocellulose-based advanced biofuels: Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus acidocaldarius. These microbes share common characteristics of having sequenced and annotated genomes, well-established genetic tools, and decades of research describing their inherent metabolism. Our goal is to exploit these traits to optimize pathways engineered for biofuel production in these organisms. To achieve this, we are employing either flux-based mathematical models or the existing knowledgebase in conjunction with publicly available online tools. For the latter we employ local (pathway specific) modifications or global (genome-specific) changes to realize the common goals of improved production and enhanced resistance to growth inhibitors encountered in the pretreatment of lignocellulose. Beyond improving the aforesaid phenotypes our goal is to gain an understanding of the underlying cellular processes enabling these improvements.
Evolutionary Engineering
Local and global optimization efforts work best for traits such as titer improvement. Other traits such as enhanced resistance to growth inhibitors require simultaneous manipulation of a large number of genes. To accomplish, this we are exploiting the natural process of evolutionary selection using state-of-the-art chemostat bioreactors. As the name suggests chemostats enable maintenance of non-changing conditions in the bioreactor. Therefore, minute incremental additions of growth inhibitors to the growth medium can be used to generate resistant populations in a few growth cycles. To further speed up the evolutionary process, we have engineered strains with ten times the mutation frequency of wild type E. coli. Resistant populations, which are generated, are further characterized using ultra-high throughput sequencing platforms in collaboration with the Joint Genome Institute. Together, these provide powerful tools to generate and study resistant populations for the development of a suitable host chassis.
Engineering An Autotrophic Chassis for Advanced Biofuel Production
The direct sequestration and subsequent conversion of carbon-di-oxide to compounds of commercial interest is the next frontier in microbial metabolic engineering. As mentioned above one of our goals in the Host Engineering Department is to extend knowledge gained from research on E. coli and S. cerevisiae to several other microbes. As part of the 'Electrofuels' program of the Advanced Research Project Agency - Energy (ARPA-E) funded effort, we are engineering the Ralstonia eutropha chassis for the production of advanced biofuels. R. eutropha is an aerobic bacterium that grows with CO2 as the sole carbon source and H2 as the sole electron donor. A complementary goal of the chassis engineering effort is to make the system compatible with novel Mo-polypyridine catalysts that can convert water to hydrogen in neutral aqueous media. These catalysts are being developed in a collaborative effort with UC Berkeley. The resulting engineered chemolithoautotrophic chassis will provide a transformational new source of renewable liquid transportation fuels that extends beyond biomass-derived substrates.
