Re-Engineered β-Oxidation Reversal for the Synthesis of ω-Functionalized Products in Escherichia coli

Title:

Re-engineered β-oxidation reversal for the synthesis of Ï?-functionalized products in Escherichia coli

Abstract:

Due to its potential for high product yields, the newly developed β-oxidation reversal pathway is an efficient platform for the production of a wide range of industrially important fuels and chemicals. This pathway has been demonstrated to produce fatty acids and alcohols of varying chain length through the utilization of acetyl-CoA as the primer with termination by thioesterase or acyl-CoA reductase/alcohol dehydrogenase respectively. While these product classes contain many important fuels and chemicals, they are limited to compounds with a non-functionalized omega end as a result of using acetyl-CoA as the primer. Diversification of product classes with varying functionality can be achieved through the use of various functionalized priming molecules. The modular nature of a β-oxidation reversal can be exploited for this purpose through the use of core enzymatic pathway components along with activation and termination enzymes which are shown to function with Ï?-functionalized intermediates of interest.

The work reported here demonstrates the re-engineering of the reverse β-oxidation pathway to enable the utilization of Ï?-functionalized primers and synthesis of Ï?-functionalized products through identifying and utilizing enzymes capable of functioning with required intermediates. This was demonstrated through the production of adipic acid, a six-carbon α, Ï?-diacarboxylic acid with high industrial importance, using Ï?-carboxylated succinyl-CoA as the primer. The re-designed pathway consisting of selected core enzymatic modules for condensation between succinyl-CoA and acetyl-CoA and subsequent dehydrogenation, dehydration, and reduction steps resulted in adipic acid production in vivo when combined with suitable activation enzymes for the supply of succinyl-CoA and thioesterases for termination. Adipic acid titer of up to 0.25 g/L was achieved after further deletions of competing pathways. In addition to adipic acid, we are exploiting this re-engineered β-oxidation reversal for the synthesis of a wide-range Ï?-functionalized products.