In the search for new drugs and drug leads, cyanobacteria hold great promise. Cyanobacteria are gram- negative photosynthetic organisms that produce a wide range of bioactive secondary metabolites, including compounds with anticancer, antibacterial, antimalarial and antiviral properties. However, because of the low isolated yield from natural collections, alternate sources of the bioactive compounds of interest must be developed. Chemical synthesis has been used, but some compounds have proved refractory to this method. Microbial fermentation is economical, but cyanobacteria are typically slow growing, not genetically tractable, and typically contaminated with bacteria that are difficult to remove. All of these factors have discouraged use of cyanobacteria as compound production factories, pointing instead towards a host organism capable of heterologous expression. The ideal heterologous host would be faster-growing, well-understood, and able to produce the desired bioactive compounds, using cyanobacterial gene clusters. Since these gene clusters are not completely expressed in traditional hosts (likely due to the lack of promoter recognition), the ideal host would also be capable of recognizing cyanobacterial promoters, thus allowing access to the tremendous chemical diversity of cyanobacteria. For this study, we will use a range of procedures to optimize Anabaena 7120 as a heterologous host, creating tools and a workflow for the production of anticancer compounds derived from cyanobacteria. We have chosen to identify and express the gene clusters responsible for the biosynthesis of three published anticancer compounds and one compound that could shed light on previously unidentified mammalian biology (including humans) that are produced by three cyanobacterial strains. Though present in laboratory culture, the strains are impure, in part due to the adherence of contaminating bacteria to the outer saccharide sheath. Using a technique we have recently developed, we will separate cyanobacterial cells from contaminating bacteria. Non- axenic laboratory cultures of anticancer compound-producing cyanobacteria will be purified from contaminant bacteria with fluorescence activated cell sorting for DNA extraction and genome sequencing. The genomes of the purified cyanobacteria will then be analyzed to identify the gene clusters involved in production of the desired compounds. In concurrent work, we will develop genetic tools for use in Anabaena, including a new replicative plasmid, defined transcriptionally neutral sites in the genome, and vectors for site-specific insertion of large DNA fragments. To validate the genetic constructs and gene cluster identification, the research will converge to produce the desired anticancer compounds in the Anabaena heterologous system using the genetic tools created. This research will allow us to study how these anticancer compounds work, produce sufficient quantities for biological characterization, and investigate the biosynthetic logic that cyanobacteria use to assemble these compounds.
Cyanobacteria make a wide variety of chemically unique bioactive metabolites (anticancer to antiviral) but the isolated yield is typically low and the bacteria are slow growing and sometimes recalcitrant to laboratory culture. We have developed a workflow that allows cyanobacterial genomes to be quickly sequenced, the gene clusters responsible for bioactive compound production identified and moved to an easier to cultivate microbial strain. This will allow cyanobacterial compounds to be accessed in quantities necessary for biological characterization allowing advances in cancer biology and drug lead development that are currently not possible.
| Wells, Kaitlyn N; Videau, Patrick; Nelson, Dylan et al. (2018) The influence of sigma factors and ribosomal recognition elements on heterologous expression of cyanobacterial gene clusters in Escherichia coli. FEMS Microbiol Lett 365: |
