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

TL;DR

Scientists engineered a bacterium to efficiently produce various aromatic compounds from renewable sources, offering a sustainable alternative to traditional fossil-based methods.

Contribution

The study introduces a modified Pseudomonas taiwanensis strain that enhances the production of hydroxybenzoates without causing metabolic deficiencies.

Findings

Modifications to the pheA gene increased the production of 2-hydroxy- and 2,3-dihydroxybenzoate without causing auxotrophy.

Replacing the native pheA gene with the Escherichia coli homolog improved 4-hydroxybenzoate production to 3.59 mM with a 20.9% yield from glucose.

The impact of pheA modifications varied depending on the production module, highlighting the interplay with host metabolism.

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 ΔpheA 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::P14g-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.

Image 1

•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.

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.

Figures