(450b) Enhanced Synthetic Biochemistry Systems Enabled By Improvements in Bacillus subtilis Spore-Display

Currently, there are two production platforms available to metabolic engineers: (1) cellular systems that utilize recombinant production organisms such as Escherichia coli and (2) cell-free systems which utilize isolated enzymatic pathways to produce compounds of interest. Both platforms have their own advantages and limitations. Cellular systems have proven scalability but have limited yield due to competing cellular metabolism. Meanwhile, cell-free systems can achieve near-theoretical yields but their scalability is hindered by a lack of enzyme stability. A synthetic biochemistry system that maintains the high yield and metabolic minimalism of cell-free systems while increasing enzyme stability would be highly advantageous. In this presentation, we present progress towards creating an enhanced synthetic biochemistry platform using Bacillus subtilis spore-display.

B. subtilis spore-display is an increasingly powerful enzyme immobilization strategy. For spore-display, a gene of interest is fused to a spore coat protein and, after sporulation, becomes anchored to the spore surface. Spores are a uniquely robust microbial phenotype and are metabolically inactive, meaning they have little intrinsic catalytic activity. When enzymes are displayed on spores they often show increased stability and can be easily purified by centrifugation. These qualities make spores particularly attractive for use as a self-assembling enzyme immobilization technology as they enable catalyst recycling. While previous studies have demonstrated spore-based cell-free metabolic engineering as a proof-of-concept, the technology is new, not well-developed, and has limited modularity.

There are at least 44 known spore-coat proteins that could potentially act as anchors for spore-display without affecting spore formation. But only 18 of these have previously been tested. To improve the modularity of spore-display for use in synthetic biochemistry systems we set out to identify optimal anchor proteins. In collaboration with the DOE Joint Genome Institute, we designed 88 synthetic DNA constructs that would allow us to test the suitability of each of the 44 spore coat proteins to act as either N or C-terminal fusion partners for the tetrameric enzyme beta-glucuronidase (GusA). From this screen, we have identified additional spore coat proteins that are capable of acting as enzyme fusion anchors and characterized their surface-availability. We are now completing work on increasing enzyme display density and building more complex synthetic biochemistry systems using spore-display.