Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture

Abstract

Production of toluene cis-glycol (TCG) was investigated using recombinant Escherichia coli strains that express toluene dioxygenase under the tac promoter. E. coli TG2 was selected as the host for the recombinant plasmid, pTAC365, because the TCG yield was 64% higher than with strain JM105 as the host. By using fed-batch culture, TCG production could be improved by 4.4-fold compared with batch cultures, with a toluene vapor feed. A further improvement of 1.2-fold was obtained by using a two-liquid phase culture system, but the improvement was 1.6-fold when the feed rate of toluene vapor was reduced by 75%. When the period of growth in fed-batch culture was extended and the reduced vapor feed was used, the maximum TCG concentration increased to 4 g/l, an overall improvement of 10-fold compared with the batch culture system used initially. However, product consumption was observed during the late stages of fed-batch growth and in stationary phase, resulting in the formation of o-cresol, 3-methylcatechol and benzyl alcohol. We conclude that the key to optimizing TCG production by recombinants is to prolong growth of the cells to ensure sustained growth-linked product formation, and to optimize the supply of the toxic substrate, toluene, but further work is needed to eliminate by-product formation.