Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum

Abstract Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum , resulting in a TrpD A162D mutation that exhibits feedback-resistant against l -tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpD A162D in C. glutamicum TP851 strain with two copies of trpD A162D in the genome led to the production of 3.1 g/L l -tryptophan in flask culture. Subsequent step for l -tryptophan chlorination into 7-chloro- l -tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli , Pseudomonas fluorescens , or Lechevalieria aerocolonigenes yielded higher production of 7-chloro- l -tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro- l -tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens . Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the l -tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica , fre from E. coli , and prnB from S. plymuthica , APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum .