Engineering Pseudomonas taiwanensis for efficient chorismate-based production of mono- and dihydroxybenzoates

Aromatics have many important applications in modern society but are traditionally produced in non-sustainable processes from fossil resources. Whole-cell biocatalysis bears great potential to provide a variety of aromatics from renewable carbon sources, thereby offering a more sustainable alternative. In this context, chorismate, the end product of the shikimate pathway, is an important biosynthetic hub compound that serves as precursor of a multitude of industrially relevant aromatics. Here, we screened several pathways for chorismate-derived bioproduction of five different mono- and dihydroxybenzoates in tyrosine-overproducing <i>Pseudomonas taiwanensis</i> GRC3Δ5-TYR1. Subsequently, twelve different modifications targeting the bifunctional chorismate mutase/prephenate dehydratase PheA were screened to reduce flux from chorismate to phenylalanine and tyrosine, thereby further enhancing the production of 2-hydroxy- and 2,3-dihydroxybenzoate without causing an auxotrophy. An auxotrophic Δ<i>pheA</i> strain served as benchmark control. Most promising modifications were subsequently also evaluated for 3-hydroxy-, 4-hydroxy- and 2,5-dihydroxybenzoate production demonstrating increased yields. Replacing the native <i>pheA</i> gene with the unmodified homolog from <i>Escherichia coli</i> was the most beneficial, enabling an increased production of up to 38.2% when combined with <i>attTn7</i>::<i>P</i> _<i>14g</i> <i>-SmCH-IV</i>. With this modification, the highest production was achieved for 4-hydroxybenzoate resulting in titers of 3.59 mM and a yield of 20.9% (Cmol/Cmol) from glucose. However, the impact of the respective <i>pheA</i> modification varies with the applied production module, further emphasizing the strong interplay with the production host's metabolism.