title: (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.
abstract: 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.
authors.affiliation: Institut für Biotechnologie 2, Forschungszentrum Jülich GmbH 52425 Jülich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Franke
authors.forename: Volker
authors.initials: V
authors.lastname: Lorbach
authors.forename: Simon
authors.initials: S
authors.lastname: Esser
authors.forename: Christian
authors.initials: C
authors.lastname: Dose
authors.forename: Georg A
authors.initials: GA
authors.lastname: Sprenger
authors.forename: Markus
authors.initials: M
authors.lastname: Halfar
authors.forename: Jörg
authors.initials: J
authors.lastname: Thömmes
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.forename: Ralf
authors.initials: R
authors.lastname: Takors
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
mesh_terms: D001565:Benzoates / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002827:Chorismic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D004951:Esterification; D005285:Fermentation; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D001565:Benzoates; D003509:Cyclohexanecarboxylic Acids; D029968:Escherichia coli Proteins; D006867:Hydrolases; C064012:isochorismatase; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D002827:Chorismic Acid