Bromination of L-tryptophan in a Fermentative Process With Corynebacterium glutamicum

Abstract

Brominated compounds such as 7-bromo-l-tryptophan (7-Br-Trp) occur in Nature. Many synthetic and natural brominated compounds have applications in the agriculture, food, and pharmaceutical industries, for example, the 20S-proteasome inhibitor TMC-95A that may be derived from 7-Br-Trp. Mild halogenation by cross-linked enzyme aggregates containing FAD-dependent halogenase, NADH-dependent flavin reductase, and alcohol dehydrogenase as well as by fermentation with recombinant <i>Corynebacterium glutamicum</i> expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from <i>Lechevalieria aerocolonigenes</i> have recently been developed as green alternatives to more hazardous chemical routes. In this study, the fermentative production of 7-Br-Trp was established. The fermentative process employs an l-tryptophan producing <i>C. glutamicum</i> strain expressing <i>rebH</i> and <i>rebF</i> from <i>L. aerocolonigenes</i> for halogenation and is based on glucose, ammonium and sodium bromide. <i>C. glutamicum</i> tolerated high sodium bromide concentrations, but its growth rate was reduced to half-maximal at 0.09 g L^-1 7-bromo-l-tryptophan. This may be, at least in part, due to inhibition of anthranilate phosphoribosyltransferase by 7-Br-Trp since anthranilate phosphoribosyltransferase activity in crude extracts was half-maximal at about 0.03 g L^-1 7-Br-Trp. Fermentative production of 7-Br-Trp by recombinant <i>C. glutamicum</i> was scaled up to a working volume of 2 L and operated in batch and fed-batch mode. The titers were increased from batch fermentation in CGXII minimal medium with 0.3 g L^-1 7-Br-Trp to fed-batch fermentation in HSG complex medium, where up to 1.2 g L^-1 7-Br-Trp were obtained. The product isolated from the culture broth was characterized by NMR and LC-MS and shown to be 7-Br-Trp.