Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives.

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1-3g/L in test tube-scale culture.

In vitro production and purification of isochorismate using a two-enzyme cascade.

Combining the isochorismate synthase EntC and the chorismatase FkbO in a sequential enzyme cascade provides a useful system for the biocatalytic production and subsequent purification of isochorismate from an isochorismate/chorismate mixture. FkbO has a strict preference for chorismate - isochorismate is not accepted as a substrate - therefore the enzyme can be used to selectively hydrolyse chorismate, leading to the chiral building block 3,4-dihydroxycyclohexa-1,5-dienecarboxylate. This simplifies the final purification step, as isochorismate is much easier to separate from the chorismate hydrolysis products than from chorismate itself. The presented procedure starts with an optimised method for purifying chorismate from Escherichia coli culture supernatants, which is followed by conversion into isochorismate with the isochorismate synthase EntC, removal of the remaining chorismate by FkbO and a final purification step using an automated flash chromatography system. Isochorismate was isolated in up to 20% yield and >95% purity from chorismate, and has been characterised with respect to its degradation and suitability as a substrate in enzyme assays.

Small scale biosynthesis and purification of gram quantities of chorismic acid.

This paper details improvements in the biosynthetic method of purification of chorismic acid using Klebsiella pneumoniae 62-1. New growth and accumulation conditions yielded 4 L of accumulation medium containing approximately 0.5 g/L of chorismate from 2 L of bacterial growth. Improvements in the handling and extraction procedures produced yields of approximately 2 g of 75 to 90% chorismic acid. A new recrystallization procedure yielded chorismic acid 97% pure by weight (using epsilon 275 = 2630 M-1 cm-1), and 99.8% pure by enzymatic conversion. These results represent a three- to five-fold increase in yield over average published values.

Bulk purification of isochorismic acid by low-pressure octadecyl (C18) reverse-phase liquid chromatography.

Partially purified isochorismate synthase (EC 5.4.99.6) from Klebsiella pneumoniae 62-1 was used to produce bulk quantities (3.4-6.8 mg) of isochorismate from chorismate. A new, preparative, low-pressure liquid chromatographic method for the purification of isochorismate was used; a (1.0 X 13.0 cm) octadecyl (C18) reverse-phase column with a discontinuous, stepped methanol gradient as eluent. The recovery of isochorismate was quantitative and its purity was verified by HPLC using a butyl (C4) reverse-phase column. This chromatographic method is superior to those previously described.

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway.

[5-3H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB3H4 and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem. 15, 428-435). The [5-3H]shikimate has been converted enzymatically to [5-3H]chorismate and [5-3H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatographic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.