Production of Heterodimeric Diketopiperazines Employing a Mycobacterium-Based Whole-Cell Biocatalysis System

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L^-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NAS_S1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.