Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids

Pavlina Dubois,Isabelle Correia,Isabelle Jacques,Olivier Lequin,Robert Thai,Fabien Le Chevalier,Pascal Belin,Nicolas Canu,Mireille Moutiez,Steven Dubois,Muriel Gondry

doi:10.1038/s41598-019-45519-y

Abstract

Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2–26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.

Highlights

Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors
This approach could be used to increase the diversity of prenylated indole DKP alkaloids, provided that non-ribosomal peptide synthetases (NRPSs) that synthesise different Trp-containing CDPs can be co-expressed with PTs
cyclodipeptide synthases (CDPSs) genes were cloned in pIJ196 under the control of the T5 promoter followed by two lacO operator sequences

Summary

Introduction

Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. 15 DMATS superfamily PTs are active on Trp-containing CDPs and most have been extensively biochemically characterised in vitro[12] They catalyse prenylation in either a regular (reg) or reverse (rev) mode, depending on the allylic carbon atom involved in attachment to the indole ring (Fig. 1b). Combinatorial engineering was implemented in Aspergillus using the co-expression of one NRPS gene from Neosartorya fischeri, ftmPS, with one of three different DMATS superfamily PT genes, CdpC2PT from N. fischeri, CdpNPT from A. fumigatus, or CdpC3PT from N. fischeri[15] This approach could be used to increase the diversity of prenylated indole DKP alkaloids, provided that NRPSs that synthesise different Trp-containing CDPs can be co-expressed with PTs

Methods

Results

Conclusion