Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors

Abstract

Bacteria are a source of a large number of secondary metabolites with several biomedical and biotechnological applications. In recent years, there has been tremendous progress in the development of novel synthetic biology approaches both to increase the production rate of secondary metabolites of interest in native producers and to mine and reconstruct novel biosynthetic gene clusters in heterologous hosts. Here, we present the recent advances toward the engineering of novel microbial biosensors to detect the synthesis of secondary metabolites in bacteria and in the development of synthetic promoters and expression systems aiming at the construction of microbial cell factories for the production of these compounds. We place special focus on the potential of Gram-negative bacteria as a source of biosynthetic gene clusters and hosts for pathway assembly, on the construction and characterization of novel promoters for native hosts, and on the use of computer-aided design of novel pathways and expression systems for secondary metabolite production. Finally, we discuss some of the potentials and limitations of the approaches that are currently being developed and we highlight new directions that could be addressed in the field.

Figure 1

Main strategies to engineer novel control systems for SM production/discovery. As a central approach, several works have focused on the development of novel transcriptional control modules such as those based on the strong T7 RNA polymerase/T7 promoter. On a different perspective, natural or engineered small metabolite-responsive regulators have been used to control gene expression in response to a ligand of interest. More sophisticated approaches have focused on combining engineered ligand-binding and DNA-binding domains to create new expression devices. An entirely novel approach has been the usage of VHH antibody domains to couple ligand recognition to gene expression elements. Additionally, gene expression modulation using modified CRISPR/Cas9 modules is becoming more frequent every day. Finally, posttranslational regulation of protein production has been addressed through either the engineering of novel ligand-specific riboswitches or temperature responsive regulatory elements (thermometers). A full description of these main cases is presented in the text. GOI: gene of interest; O: operator, a cis-regulatory element; T7P: T7 promoter; RBS: ribosome binding site.

Figure 2

Metagenomics has become an important tool to improve and expand NP discovery from DNA retrieved from the microbial community inhabitants of environmental samples. Novel genetic parts (structural genes, regulators, terminators, peptide signals, transporters, etc.) are provided, which, when rewired, allow the creation of novel complex bioactive molecules.

Figure 3

Chemical diversity of NPs identified from Burkholderia. The main groups of molecules belonging to NRP and PK are shown.