Production of Heterodimeric Diketopiperazines Employing a <i>Mycobacterium</i>-Based Whole-Cell Biocatalysis System

Abstract

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 <i>Escherichia coli</i> and difficult to operate in <i>Streptomyces</i>. Through validation of the functions of two pairs <i>Mycobacterium smegmatis</i> sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that <i>M. smegmatis</i> is much more efficient, robust, and cleaner in metabolites background than the regularly used <i>E. coli</i> or <i>Streptomyces</i> systems. The <i>M. smegmatis</i>-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 <i>Actinomycetes</i>.