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 Corynebacterium glutamicum expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes 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 C. glutamicum strain expressing rebH and rebF from L. aerocolonigenes for halogenation and is based on glucose, ammonium and sodium bromide. C. glutamicum 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 C. glutamicum 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.
Highlights
Brominated tryptophan is typically not found in free form in Nature, but as a biosynthetic precursor in complex structures that for example occur in sponges and lower marine invertebrates (Bittner et al, 2007)
The effect of the substrate NaBr and the product 7-Br-Trp on growth of C. glutamicum was assessed when various concentrations of these compounds were added upon inoculation of C. glutamicum wild type to CGXII minimal medium with 40 g L−1 glucose
Heterologous expression of the genes rebH for FAD-dependent halogenase and rebF for NADH-dependent flavin reductase from the reb cluster of L. aerocolonigenes to enable regioselective chlorination of Trp at the 7 position (Nishizawa et al, 2005) in a Trp overproducing C. glutamicum strain (Purwanto et al, 2018) provided the basis for the development of fermentative processes for chlorination (Veldmann et al, 2019) and bromination of Trp
Summary
Brominated tryptophan is typically not found in free form in Nature, but as a biosynthetic precursor in complex structures that for example occur in sponges and lower marine invertebrates (Bittner et al, 2007). Free unprotected halotryptophans including 7-Br-Trp and 7-chloro-L-tryptophan (7-Cl-Trp) can serve as substrates for Pd-catalyzed crosscoupling reactions (Willemse et al, 2017) for example in the Suzuki-Miyaura cross-coupling in order to attach an aryl, heteroaryl, or alkenyl substituent to the indole ring (Roy et al, 2008). For this reaction, 7-Br-Trp is preferred because it is more reactive than 7-Cl-Trp (Corr et al, 2017). Fermentative production of 7-Cl-Trp has recently been established using recombinant Corynebacterium glutamicum (Veldmann et al, 2019)
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