(S,S)-2,3-Dihydroxy-2,3-dihydrobenzoic Acid: Microbial Access with Engineered Cells of Escherichia coli and Application as Starting Material in Natural-Product Synthesis

• Published in: Chemistry : a European journal 2003-09, Vol.9 (17), p.4188-4196
• Main Authors: Franke, Dirk, Lorbach, Volker, Esser, Simon, Dose, Christian, Sprenger, Georg A., Halfar, Markus, Thömmes, Jörg, Müller, Rolf, Takors, Ralf, Müller, Michael
• Format: Article
• Language: English
• Subjects: arene diols; Benzoates - chemistry; Benzoates - isolation & purification; Benzoates - metabolism; bioorganic chemistry; carbohydrate mimetics; chorismate; Chorismic Acid - analogs & derivatives; Chorismic Acid - metabolism; Cyclohexanecarboxylic Acids - chemistry; Cyclohexanecarboxylic Acids - isolation & purification; Cyclohexanecarboxylic Acids - metabolism; enzyme catalysis; Escherichia coli; Escherichia coli - cytology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Esterification; Fermentation; Hydrolases - genetics; Hydrolases - metabolism; Intramolecular Transferases - genetics; Intramolecular Transferases - metabolism; shikimate; Stereoisomerism
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.200204265

Cyclohexadiene‐trans‐5,6‐diols such as (S,S)‐2,3‐dihydroxy‐2,3‐dihydrobenzoic acid (2,3‐trans‐CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic‐pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over‐expressed in E. coli strains with a deficiency of entA, which encodes 2,3‐dihydroxybenzoate synthase. A 30‐fold increase in the corresponding EntB/EntC enzyme activities affects the accumulation of 2,3‐trans‐CHD in the cultivation medium. Although the strains did not contain deletions in chorismate‐utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by‐products. Fermentation of these strains in a 30 L pH‐controlled stirred tank reactor showed that 2,3‐trans‐CHD could be obtained in concentrations of up to 4.6 g L−1. This demonstrates that post‐chorismate metabolites are accessible on a preparative scale by using techniques of metabolic‐pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion‐exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3‐trans‐CHD as an example, we established short syntheses towards new carbasugar derivatives. Cyclohexadien‐trans‐diole wie beispielsweise (5S,6S)‐Dihydroxycyclohexa‐1,3‐diencarbonsäure (2,3‐trans‐CHD) haben sich als wichtige chirale Ausgangsverbindungen zur Synthese pharmakologisch aktiver Substanzen, insbesondere zur Synthese von Carbazuckern und Aminocarbazuckern erwiesen. Unter Verwendung von Techniken der Stoffflussderegulation wurden die Escherichia coli‐Gene entB und entC, kodierend für Isochorismatase und Isochorismatsynthase, in E. coli‐Stämmen überexpremiert, welche eine Defizienz von entA, kodierend für 2,3‐Dihydroxybenzoatsynthase, hatten. Eine 30‐fache Steigerung der entsprechenden Enzymaktivitäten EntB/EntC bewirkte die Bildung von 2,3‐trans‐CHD im Kultivierungsmedium. Obwohl die Stämme keine Mutationen in den chorismatverwendenden Biosynthesewegen zu den aromatischen Aminosäuren besaßen, wurde weder Chorismat noch ein anderes metabolisches Intermediat als Nebenprodukt gefunden. Fermentationen mit diesen Stämmen in einem pH‐geregelten 30 L Rührkesselreaktor zeigten, dass 2,3‐trans‐CHD in Konzentrationen bis zu 4.6 g L−1 er