Alteration of the Route to Menaquinone towards Isochorismate‐Derived Metabolites

Abstract

Chorismate and isochorismate constitute branch‐point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli . We propose a model for the binding of isochorismate to the active site of MenD ((1 R ,2 S , 5 S ,6 S )‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α‐ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double‐variant Asn117Arg–Leu478Thr preferentially converts (5 S ,6 S )‐5,6‐dihydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐ trans ‐CHD), the hydrolysis product of isochorismate, with a >70‐fold higher ratio than that for the wild type. The single‐variant Arg107Ile uses (5 S ,6 S )‐6‐amino‐5‐hydroxycyclohexa‐1,3‐diene‐1‐carboxylate (2,3‐ trans ‐CHA) as substrate with >6‐fold conversion compared to wild‐type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L −1 ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild‐type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

“…Identification of such as yet enigmatic enzymes might lead to the amino derivatives being recognised as important metabolic branching points contributing to a much larger biosynthetic diversity, as was proposed for the biosynthesis of stravidins (18), echinosporins (39), and SEPHCHC derivatives (36)(37)(38) prepared with variants of MenD. 32,39,140 In addition, the elucidation of the tundrenone (40) biosynthetic pathway suggests an undescribed C-C coupling reaction utilising chorismate as a substrate (Fig. 6).…”

“…5). 31,32,139 Moreover, Fries et al reported the preparative in vivo production of SEPHCHC, (iso)SDHCHC, and (iso)SAHCHC using engineered E. coli strains. Rationally designed MenD variants, based on a docking model with '1,6-dihydroisochorismate' as a surrogate intermediate, increased the yields of fermentations and in vitro reactions significantly (6-and 23-fold).…”

“…The occurrence of some of these rationally designed variants in nature, detected by protein sequence network analysis, suggests that further chorismate-converting enzymes -in particular enzymes acting on the amino derivatives -await discovery. 32 13. Is there a hidden chorismateconverting enzyme potential encoded in natural products?…”

“…In light of this, it is likely that further unprecedented enzyme families will be discovered. 32,46,50 Reconsidering Fig. 1, the products accessible from (iso)chorismate (1 and 2) by enzymatic and non-enzymatic reactions are not fully covered for the corresponding amino derivatives ADIC (3) and ADC (4).…”

“…Many of these have been used as building blocks for the chemoenzymatic synthesis of more complex compounds which are challenging to produce via standard organic synthesis. 10,11,[31][32][33] Examples of such biocatalytic applications are given in Section 12.…”