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

Metabolic Engineering Communications, vol. 22, pp. e00273

Abstract

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 Pseudomonas taiwanensis 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 pheA 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 pheA gene with the unmodified homolog from Escherichia coli was the most beneficial, enabling an increased production of up to 38.2% when combined with attTn7 :: P 14g -SmCH-IV . 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 pheA modification varies with the applied production module, further emphasizing the strong interplay with the production host’s metabolism. • Chorismate-derived hydroxybenzoate production in P. taiwanensis CHOR platform • Mutations in pheA improved production without causing auxotrophies • High-yield production of five different hydroxybenzoates from glucose and glycerol

Authors 5

1. Franziska Kofler corresponding

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

2. Tobias Schwanemann Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

   iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

3. Nadine Teófilo da Silva

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

4. Nick J. P. Wierckx corresponding

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

5. Benedikt Wynands

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany