Campos-Magana, M. A.; Moreno-Paz, S.; Martins dos Santos, V. A. P.; Garcia-Morales, L.; Suarez-Diez, M.

Combinatorial approaches in metabolic engineering enable the optimization of multigene pathways, thereby improving product titers. However, the optimization of complex metabolic pathways is hindered by their multiple interactions. Testing all possible combinations of suitable genetic parts is often prevented by the large number of possible variants. Alternatively, we use statistical design of experiments and linear modeling to collect important information for the optimization without testing every possible combination. The shikimate pathway is an example of a complex metabolic pathway involved in the production of aromatic compounds. In this study, we explore the impact of the modulation of the expression levels of all the genes in the shikimate and para-aminobenzoic acid (pABA) biosynthesis pathways for pABA production in Pseudomonas putida. We used this approach to select 14 representative strains from a total of 512 possible combinations. We obtained a range of product titers from 2 to 186.2 mg/l. This information was used to guide a second round of strain construction to further increase the production to 232.1 mg/l. Using this strategy, we demonstrate that aroB expression, encoding 3-dehydroquinate synthase, is a significant limiting factor in the production of pABA.