Engineering Bacterial Microcompartments in Clostridium autoethanogenum to Overcome Bottlenecks in Sustainable Production of Synthetic Rubber
Brett Palmero1*, Rebecca Ostien2, Nolan Kennedy1, Joanna Cogan2, Heidi Schindel2, Carolyn Mills1, Elizabeth Johnson1, Alex Mueller2, Fungmin (Eric) Liew2, Michael Köpke2, and Danielle Tullman-Ercek1
1Northwestern University; and 2LanzaTech
To investigate bacterial microcompartments in Clostridium autoethanogenum and engineer them to compartmentalize synthetic metabolic pathways.
One promising route to sustainable bioproduction of fuels and chemicals is the engineering of organisms such as acetogens to efficiently convert abundant and low-cost gases containing carbon monoxide or carbon dioxide and hydrogen to desirable, value-added products at high efficiency and low cost. This approach not only provides an avenue for repurposing greenhouse gases (GHG), but also minimizes the use of harsh chemicals and hazardous byproducts common in petroleum-based processes. However, many biochemicals are not yet produced biologically due to roadblocks in the cellular biosynthesis process. These roadblocks can include intermediate toxicity, redox imbalances, and loss of product to off-pathway reactions. In nature, these issues are often alleviated using spatial organization strategies, such as sequestration in organelles. In bacteria, such organization often occurs in protein-based organelles known as bacterial microcompartments (MCPs).
The team will investigate the native regulation, assembly, and function of MCPs in the industrially relevant nonmodel host C. autoethanogenum. In the C. autoethanogenum genome, two unique gene clusters have been identified as putative MCP operons. These putative operons contain sequences encoding possible hexamers, trimers, pentamers, and enzyme encapsulation sequences. The team tested potential inducers of these operons and found that some of these small molecules were consumed by C. autoethanogenum. RNAseq data show that these same small molecules transcriptionally activate the MCP operons. MCP formation in these conditions was corroborated by electron microscopy of C. autoethanogenum, which shows distinctive polyhedral shapes within the cells, indicative of MCP formation.
Beyond understanding the native function of these putative MCP operons, the engineering goal is to sequester key biosynthesis enzymes from two distinct metabolic pathways into MCPs to make compounds involved in rubber production. Specifically, researchers aim to showcase the power of enzyme encapsulation in an MCP for reducing toxicity and product losses to side reactions for these pathways. Towards enabling heterologous enzyme encapsulation in these new MCP systems, 16 C. autoethanogenum reporter strains were generated with different putative encapsulation peptides fused to sfGFP. Fluorescence microscopy shows that 11 of these 16 sfGFP-encapsulation peptide fusions exhibit punctate fluorescence upon MCP induction indicating successful encapsulation of the fluorescent reporter within MCPs. These results will pave the way for encapsulating biosynthesis enzymes for rubber production in future years and enable the cost-efficient production of chemicals currently derived from petroleum.
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022180.