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 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.

Impact of CRISPR interference on strain development in biotechnology

“…The model organism C. glutamicum is widely used in biotechnological industry, since it is a workhorse able to produce various compounds with high production titers [106–117], especially the amino acids l ‐glutamate [118–121] and l ‐lysine [122–127]. Globally, amino acids from C. glutamicum are produced in million ton scale [109].…”

Recent Advances in Synthetic Application and Engineering of Halogenases

“…Based on these results, Veldmann et al . subsequently addressed the fermentative production of 7‐Br‐Trp . Utilizing the engineered C. glutamicum strain enabled upscaling to a reaction volume of 2 L in a batch and fed‐batch mode resulting in significantly higher product titers of 7‐Br‐Trp compared to 7‐Cl‐Trp (1.2 g L −1 vs. 0.1 g L −1 ).…”

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

“…[215] Whereas a higher titer of brominated tryptophan was fermented using a bromide tolerant recombinant Corynebacterium glutamicum strain that carried tryptophan Fl-Hals. [240] In phenylpropanoid acid-based pathways, the reconstruction of nonnative enzyme could introduce pathway intermediates such as cinnamic and coumaric acids that are toxic to the cell growth. [241] The low catalytic efficiency of down-stream pathway enzymes often seems to be an issue with many early stage halogenation metabolic pathways.…”