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Scheme 6.4.2. Chorismate and isochorismate as branch points toward the synthesis of many different primary and secondary metabolites.

is formed from chorismate in one catalytic step, are ideal starting materials for different functionalized aromatic cell products [3]. Thus, biosynthetically both chorismate and isochorismate are characterized by their diversity-oriented bias. Because of their high functionalization these molecules are also very interesting substances chemically and biochemically, not only in terms of possible aromatiza-tion reactions and their products, but also use of the carboxylic and diol functionality and, especially, the cyclohexadiene system as reactive groups for diversity-oriented modifications.

In this contribution we would like to show the production and chemical use of chorismate-derived compounds as an example of the development of microbial production pathways resulting in highly flexible building blocks, which themselves should be applicable in diversity-oriented syntheses. Not only can naturally occurring metabolites be chosen as target molecules, non-natural compounds can also be produced.

514 | 6.4 Microbially Produced Functionalized Cyclohexadiene-trans-diols 6.4.3

Microbial Production of 2,3-trans-CHD

Functionalized cyclohexadiene-trans-diols (trans-CHD; dihydroxydihydrobenzenes) closely related to chorismate or isochorismate have been chosen as primary target molecules for the metabolic pathway engineering approach [4] (Box 24). The corresponding cyclohexadiene-cis-diols (cis-CHD) are known and established as valuable precursors for the production of natural products and bioactive materials. They are readily accessible via whole-cell bioconversion starting from aromatic compounds [5]. Mainly because of the limited availability of trans-CHD [6], their use as chiral synthetic building blocks is rare. All chemical preparations are characterized by tedious multistep syntheses [7]. Attempts by several working groups to prepare trans-CHD from cis-CHD have been characterized by low overall yield [8]. As an alternative, homochiral diols with the trans configuration from bacterial sources have been described as metabolites involved in metabolic pathways derived from the shikimate pathway, therefore, trans-CHD should, in principle, be accessible by metabolic engineering. Using techniques of metabolic pathway engineering in E. coli, J. W. Frost et al. were able to produce shikimate and quinate in concentrations up to 71 g L-1 and 13 g L-1, respectively, using a whole-cell process in a stirred-tank reactor [9].

Pioneering work in the field of bacterial production of trans-CHD has been conducted by Leistner and co-workers, who used strains of Klebsiella pneumoniae as biological hosts [10]. Mutants with expressed plasmid-encoded genes (entC and/or entB) and defects in the postchorismate pathways have been shown to produce the two trans-CHD 1 and 2 in concentrations of up to 200 mg L-1. (S,S)-2,3-Dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD, 2) is the immediate hydrolytic product of isochorismate and occurs as an intermediate in the biosynthesis of the iron chela-tor enterobactin. In two steps chorismate is converted through isochorismate syn-thase (encoded by the gene entC) and isochorismatase (encoded by entB) to give 2,3-trans-CHD 2. This compound is intracellularly transformed in an aromatization step catalyzed by 2,3-dihydroxybenzoate synthase (encoded by entA; Scheme 6.4.3) to give 2,3-dihydroxybenzoate 3.

For our purpose we decided to use Escherichia coli K-12 as a safe and well-characterized host strain for microbial production of trans-CHD. For production of 2,3-trans-CHD 2 the metabolic flux toward catechol 3 has to be barred and the flux toward the desired compound should be increased. E. coli strains with a defective gene entA (entA-) were transformed with plasmids containing entB/entC. This led to production of 2,3-trans-CHD 2 and excretion of the product into the culture medium [11, 12].

In the production of 2, strains with entA--mutation have long-term stability and production rates are high. Typical cultivation of 2,3-trans-CHD 2 is performed at pH 6.8 and 37 °C in a stirred-tank reactor with glucose feeding in a fed-batch mode. Microbial production over a process time of 40 h affords 92 g 2 from 690 g glucose monohydrate in a 20-L cultivation experiment. This corresponds to a molar yield of 17% [13]. Advantageously, 2,3-trans-CHD 2 is the major product with no glucose shikimate pathway

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