Chassis optimization as a cornerstone for the application of synthetic biology based strategies in microbial secondary metabolism

Tiago Beites,Marta V Mendes

doi:10.3389/fmicb.2015.00906

Abstract

The increased number of bacterial genome sequencing projects has generated over the last years a large reservoir of genomic information. In silico analysis of this genomic data has renewed the interest in bacterial bioprospecting for bioactive compounds by unveiling novel biosynthetic gene clusters of unknown or uncharacterized metabolites. However, only a small fraction of those metabolites is produced under laboratory-controlled conditions; the remaining clusters represent a pool of novel metabolites that are waiting to be “awaken”. Activation of the biosynthetic gene clusters that present reduced or no expression (known as cryptic or silent clusters) by heterologous expression has emerged as a strategy for the identification and production of novel bioactive molecules. Synthetic biology, with engineering principles at its core, provides an excellent framework for the development of efficient heterologous systems for the expression of biosynthetic gene clusters. However, a common problem in its application is the host-interference problem, i.e., the unpredictable interactions between the device and the host that can hamper the desired output. Although an effort has been made to develop orthogonal devices, the most proficient way to overcome the host-interference problem is through genome simplification. In this review we present an overview on the strategies and tools used in the development of hosts/chassis for the heterologous expression of specialized metabolites biosynthetic gene clusters. Finally, we introduce the concept of specialized host as the next step of development of expression hosts.

Highlights

Over the last century, specialized metabolites derived from microbial secondary metabolic pathways have been used by the pharmaceutical industry as a source of lead compounds to feed the drug discovery pipeline
Multiple reports on genome reduction of industrial relevant organisms show a maximum reduction level below 25% of the total genome: E. coli, 15.3% (Posfai et al, 2006); B. subtillis, 20.7% (Morimoto et al, 2008); S. avermitilis, 16.9–18.6% (Komatsu et al, 2010) and Corynebacterium glutamicum 22% (Unthan et al, 2015). These low reduction percentages highlight how we are still very far from knowing how the networks that compose life are wired. In spite of these limitations, the application of directed mutagenesis to genome streamlining rendered some strains with interesting properties for biotechnological applications such as Streptomycesderived strains, where genome reduction allowed the heterologous expression and production of small molecules (Table 1)
The streamlining project of S. coelicolor M145 strain (derivative of S. coelicolor A3(2) that lacks the two natural plasmids SCP1 and SCP2) was not as drastic as the S. avermitilis project, since it only targeted the native secondary metabolite biosynthetic gene clusters (Gomez-Escribano and Bibb, 2011). In this case the biosynthetic gene clusters of the secondary metabolites majorly produced by this strain were sequentially deleted by homologous double-recombination, generating a plethora of streamlined strains characterized by a low percentage of genome reduction (2%)

Summary

Introduction

Over the last century, specialized metabolites derived from microbial secondary metabolic pathways have been used by the pharmaceutical industry as a source of lead compounds to feed the drug discovery pipeline. These low reduction percentages highlight how we are still very far from knowing how the networks that compose life are wired In spite of these limitations, the application of directed mutagenesis to genome streamlining rendered some strains with interesting properties for biotechnological applications such as Streptomycesderived strains, where genome reduction allowed the heterologous expression and production of small molecules (Table 1). The usage of random mutagenesis and isolation of mutants with interesting phenotypes is at the core of strain development in the pharmaceutical industry This means that genome streamlining has been practiced for decades with different purposes than obtaining a suitable chassis for expressing synthetic pathways. The generated strains presented advantages for the heterologous expression of biosynthetic gene clusters, presumably due to the lack of endogenous metabolic pathways that would compete for cell resources and to a decrease in genome instability. NRP, non-ribosomal peptide; PK, polyketide; NP, no production detected

38.5 NP – Cryptic 185 NP – Cryptic

Findings

Conclusion and Future Perspectives