Co-production of 7-chloro-tryptophan and indole pyruvic acid based on an efficient FAD/FADH2 regeneration system

Efficient FAD/FADH(2) regeneration is vital for enzymatic biocatalysis and metabolic pathway optimization. Here, we constructed an efficient and simple FAD/FADH(2) regeneration system through a combination of l-amino acid deaminase (l-AAD) and halogenase (CombiAADHa), which was applied for catalyzing the conversion of an l-amino acid to halide and an & alpha;-keto acid. For cell-free biotransformation, the optimal activity ratio of l-AAD and halogenase was set between 1:50 and 1:60. Within 6 h, 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) were synthesized in the selected mono-amino acid system. For whole-cell biotransformation, 7-Cl-Trp and IPA synthesis was enhanced by 15% (from 96 to 110 mg/L) and 12% (from 115 to 129 mg/L), respectively, through expression fine-tuning and the strengthening of FAD/FADH(2) supply. Finally, ultrasound treatment was applied to improve membrane permeability and adjust the activity ratio, resulting in 1.6-and 1.4-fold higher 7-Cl-Trp and IPA yields. The products were then purified. This system could also be applied to the synthesis of other halides and & alpha;-keto acids.

Automated summary

Efficient FAD/FADH(2) regeneration was achieved through a combination of l-amino acid deaminase (l-AAD) and halogenase (CombiAADHa). The system successfully catalyzed the conversion of l-amino acid to halide and α-keto acid, yielding 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) within 6 hours. The use of ultrasound treatment further improved the yields by 1.6- and 1.4-fold for 7-Cl-Trp and IPA, respectively.