Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies

Abstract

The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

Keywords: Shikimate pathway; aromatics; metabolic engineering; metabolic modelling; strain construction.

Figure 1

Major branch point intermediates and products associated with and derived from the shikimate pathway by means of metabolic engineering.

Figure 2

Reported genetic modifications that significantly improve flux to and through the shikimate pathway in the respective context. Only feedback-resistant enzymes and overexpression targets are included, knockouts have not been respected. Highlighted in blue are yeast (in most cases S. cerevisiae) genes, green E. coli genes, orange C. glutamicum genes, and purple Z. mobilis genes. With the exception of aroL (applied in S. cerevisiae), tyrC (applied in E. coli) and aroG^fbr (applied in C. glutamicum) the respective overexpression targets have not been applied outside their native organism. Major enzymes are indicted to the left of the linear part of the pathway; DAHPS, 3-deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthase; DHQS, 3-dehydroquinate synthase; DHQ, 3-dehydroquinate dehydratase; SDH, shikimate 5-dehydrogenase; SK, shikimate kinase; EPSPS, 5-enolpyruvylshikimate 3-phosphate synthase; CS, chorismate synthase.