Next generation sequencing has revealed the existence of tens of thousands of unknown natural product biosynthesis pathways. Our detailed understanding of the biosynthesis of secondary metabolism has facilitated bioinformatics analysis, which has suggested that these pathways might encode numerous novel chemical entities with strong biological activities. Streptomyces populate a highly competitive ecological niche, which they co-inhabit with innumerable other bacteria and fungi. The soil environment is scarce in energy, minerals and is subjected to strong fluctuation in temperature and water content. An accumulating body of evidence has demonstrated that Streptomyces have a plethora of advanced sensory systems for detection of the changing conditions and are able to fine tune their metabolic pathways in response. Genome sequencing projects have indicated that these bacteria typically harbour more than 20 biosynthetic gene clusters (BGCs) for production of secondary metabolites, but only 2-4 compounds can usually be detected under standard monoculture conditions in the laboratory. Most of these pathways remain dormant and require some kind of an environmental signal for activation. The key question in the field is how can this potential be harnessed for the discovery of improved bioactive compounds?

We are interested in developing methodology for awakening these cryptic metabolic pathways using synthetic biology. In particular, our focus has been in a method called Reported Guided Mutant Selection, where we link expression of the targeted pathway to the survival of the bacterial strain under selective pressure. We identify key promoter sequences from the gene cluster and clone these in front of a reporter system that allows for monitoring of transcription levels from the promoter and provides resistance to antibiotics. We then generate a library of millions of mutant strains with the hope that the target pathway has become active in some of these. If this has occurred, only positive mutants are able to survive in a medium supplemented with antibiotics. Single mutants are then isolated and their production profiles are compared to those of the wild type to identify novel compounds. Finally, the structure of the metabolite is elucidated using natural products chemistry.


FIGURE Reporter Guided Mutant Selection.


An alternative method for activation of cryptic gene clusters is to search for small molecular elicitors, such as other antibiotics, that might solicit the desired metabolic response. High-throughput screening of strains using chemical libraries and varying culture conditions are used to search for the activator molecule. Finally, the silent gene clusters may be cloned in a heterologous host and artificial promoter sequences may be utilized to drive the expression of the biosynthetic genes. This typically requires advanced molecular biology methods to manipulate the large (up to 50 kb DNA) gene clusters in E. coli, followed by transfer to the Streptomyces expression host.



In addition to activation of naturally occurring gene clusters, new chemical entities can also be made using metabolic engineering. In a process reminiscent to the natural evolution, we are able to rapidly generate new gene combinations that have not previously existed in Nature by combining material from two or more pathways. In case of anthracycline antibiotics, we have engineered strains that function as chasses for production of desired core carbon skeletons. New genes from related pathways may then be cloned into these strains individually and in different combinations using the engineering principles of synthetic biology. We have chosen the BioBricks approach to rapidly assemble 2-4 gene tailoring pathways from synthetic oligonucleotides.  Our work on the BIOCHEMISTY and BIOSYNTHESIS of these metabolites has provided us with a library of dozens of well-characterized gene products that we are able to use to modify previously existing anthracycline scaffolds. The compounds are then tested for their biological activities at our collaborators laboratories.


Updated: 12 May 2018