(462b) Epathbrick Directed Modular Pathway Engineering for Improved Fatty Acids Production in E. Coli

As an emerging discipline, synthetic biology is becoming increasingly important to design, construct and optimize metabolic pathways for producing biofuels and pharmaceuticals in genetically tractable organisms. To date, a number of synthetic biology approaches have been applied to metabolic pathway optimization including modification of plasmid copy number, promoter strength and gene codon usage.  Nevertheless, most of these approaches are not modular and require time-consuming work tweaking the individual pathway components. From an engineering perspective, we need a more efficient approach that streamlines the process of pathway construction and optimization. Based on central pathway architectures, E.coli fatty acids pathway were decomposed into three modules: (I) the upper module includes the glycolytic pathway encoded by pgk, gapA, aceE, aceF and lpdA; (II) the intermediary module includes the acetyl-CoA carboxylation pathway encoded by accA, accB, accC, accD; (III) the lower module includes the fatty aicds formation pathway encoded by fabA, fabD, fabG, fabI and tesA’ (or other plant fatty-acyl thioesterase). By balancing the expression level of the three module pathway, we have successfully constructed an E. coli recombinant strain exhibiting 25-fold increase in fatty acids production. It was found that fatty acids composition was dramatically changed as a result of expressing different fatty acyl-ACP thioesterase. Expressing the E coli native thioesterase (tesA’) shifted the fatty acids to short-chain C14:0; whereas expressing the plant-derived fatty acyl-ACP thioesterase (BnFatA, CnFatB2, EGOTE) shifted the major fatty acids to palmitic acid compared with the wild type strain.