Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Engineering Bacterial Microcompartments in Clostridium autoethanogenum to Overcome Bottlenecks in Sustainable Production of Synthetic Rubber

Authors:

Danielle Tullman-Ercek2*, Rebecca Ostien1, Nolan Kennedy2, Brett Palmero2, Joanna Cogan1, Heidi Schindel1, Carolyn Mills2, Elizabeth Johnson2, Alex Mueller1, Fungmin (Eric) Liew1, Michael Köpke1

Institutions:

1LanzaTech; 2Northwestern University

URLs:

Goals

To investigate bacterial microcompartments in Clostridium autoethanogenum and engineer them to compartmentalize synthetic metabolic pathways.

Abstract

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).

We will investigate the native regulation, assembly, and function of MCPs in the industrially relevant non-model host C. autoethanogenum. In the C. autoethanogenum genome, two unique gene clusters have been identified as putative operons encoding sets of proteins required for MCP formation. These putative operons express a variety of possible MCP shell proteins and encapsulation peptides that target enzymes into the MCP. We tested potential inducers of these operons and found that some of these small molecules were consumed by C. autoethanogenum; RNA sequencing data showed 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. We also used cell-free protein synthesis to produce putative MCP shell proteins from C. autoethanogenum and observed self-assembly of large structures, visible under light microscopy.

Beyond understanding the native function of these putative MCP operons, our engineering goal is to sequester key biosynthesis enzymes from two distinct metabolic pathways into MCPs to make compounds involved in rubber production. Specifically, we 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 Superfolder green fluorescent protein (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 demonstrate the potential for encapsulating biosynthesis enzymes and enable the cost-efficient production of chemicals that are currently derived from petroleum.

Funding Information

This material is based upon work supported by the U.S. DOE, Office of Science, BER program, GSP under Award Number DE-SC0022180.