Microbial Genomics and Biosynthetic Engineering

Genomic screening of marine cyanobacteria for the detection of targeted secondary metabolite biosynthetic genes has been achieved through fosmid DNA libraries construction using HMW metagenomic DNA isolated from environmental cyanobacterial samples. Conserved enzymatic steps shared by diverse biosynthetic gene clusters are used as starting point for the design of degenerate primers from the corresponding biosynthetic genes. Cyanobacteria-derived fosmid DNA library pools are screened by PCR and single hit clones are recovered through few rounds of dilution/PCR screening. Using this approach we isolated hybrid NRPS/PKS gene clusters encoding for various natural products from phylogenetically diverse cyanobacteria. In parallel, we sequence (meta)genomes using next generation sequencing followed by genome mining.

In order to address the ‘supply problem’ we want to heterologously express the recovered gene clusters in gifted, laboratory friendly and genetically amenable microbial producers. Streptomyces sp. are prolific producers of secondary metabolites providing almost two thirds of the antibiotics currently in clinical use and can be further optimized to express cyanobacterial metabolites. Although genetic refactoring of individual gene clusters can result in overproduction of the specific end product, we want to adapt a more generalized approach where global regulators of secondary metabolism are being targeted in strain improvement strategies allowing the use of the same engineered hosts for the heterologous expression of diverse biosynthetic clusters.

One powerful way to screen metagenomic DNA libraries especially for the discovery of completely novel molecules is the function-based screening that selects only for expressible genes that they confer a phenotypic trait to the host. This selection strategy does not require any prior sequence information and therefore it is free of the associated homology bias allowing the high-throughput recovery of active novel clones. We have developed an interspecies conjugation protocol utilizing the high integration efficiency of jBT1 integrase for the en masse transfer of marine-derived metagenomic DNA libraries into our panel of optimized heterologous hosts that will allow the expression and characterization of novel bioactive compounds.