The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms

Abstract

Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs). However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

Keywords: bioactive gene clusters; cryptic; heterologous expression; metagenomics; non-ribosomal peptide synthases; polyketide synthases; silent.

Figure 1

The decline in new chemical entities (NCE) development has continued in spite of an exponential increase in biosynthetic gene clusters (BGC) discovery. There has been more than a 50% reduction in the number of NCEs being approved by the FDA which can be categorized as natural products or derived from such. This has occurred at the same time that an explosion of papers citing “biosynthetic gene clusters” has emerged in the literature. Notwithstanding the lag time in drug development, serious bottlenecks exist in translating the bioactive potential from these microbial systems.

Figure 2

The integration of classical culture based and molecular biology based approaches for the elicitation of natural products from silent BGCs. A major challenge to the biodiscovery community, the cooperation of biological and chemical expertise is driving integrative technology developments that will unlock the silent potential of the cryptic gene clusters. Synthetic biology and combinatorial chemistry approaches further underpin the potential diversity of natural product that can be achieved.

Figure 3

LysR regulation and cryptic clusters. (A) LysR transcriptional regulators are comprised of two domains, a DNA binding HTH domain typically found at the N-terminal, and a co-inducer or signal binding domain; (B) As with several other families of transcriptional regulators, LTTRs are signal responsive, present either in an inactive or sometimes repressive state on the promoters of biosynthetic genes in the absence of an environmental cue. Upon addition of the appropriate signal or co-inducer, conformational changes occur at the promoter eliciting activation of the target gene(s); (C) The frequency of genes encoding LTTR proteins is significantly higher among the genomes of families known to include well characterized bioactive producing organisms. The specificity with which these proteins regulate gene expression and their inactivity in the absence of an activating signal make them likely candidates for control of cryptic biosynthetic gene expression.