Team:Santa Clara/OurDefenseSystem
Our Defense System
Of all of the researched mechanisms, the one that stood out as the best candidate was E. coli’s membrane composition change that incorporates cyclopropane fatty acids into the membrane. This mechanism was found to be the single most impactful acid resistant mechanism in this enterobacterium. It involves the increased expression of the cyclopropane fatty acid (CFA) synthase which will catalyze the addition of the methyl group to a monounsaturated fatty acid (UFA), using S-adenosyl methionine (SAM) as a methyl donor. The CFA synthase is a cytosolic protein that reversibly associates with the membrane. These modified phospholipids then incorporate into the membrane and lead to the acid resistant phenotype. The protruding carbon creates a more sterically hindered for weak acids attempting to pass through the membrane.
Schematic of cyclopropane fatty acid defense system.
Chemical reactions involved in CFA formation.
Rationale behind choosing the CFA system
The CFA acid resistance system offers several advantages over the other systems that we identified in nature:
- Requires no major alterations in metabolism
- Does not use precious cellular resources as transporters
- Only requires two cytosolic proteins
- Offers versatility for use in other organisms because the cell membrane is well conserved even across gram positive and negative bacterium
- Found to be the single most effective acid resistance system in E. coli
- Not found in any other organism that has been documented
We sought to create a robust system that could be implemented in all bacterium so that regardless of the chassis chosen for a bioreactor, it would be able to increase its aciduricity. We also wanted to find a way to boost the acid resistance of the organism through the addition of a new system opposed to the upregulation of an endogenous system. By adding a new system, we think it should be possible to greatly increase the acid resistance of an organism. Our plan is to create a BioBrick including both the CFA synthase and SAM synthetase so that during cell line engineering in preparation for use in a bioreactor these genes can be tossed in the mix to increase their acid resistance.