Members of the Actinobacteria phylum (especially those from the Streptomyces genus) are some of the most fruitful sources of pharmaceuticals. Discovery of bioactive secondary metabolites from these species has moved from a largely ?grind and find? strategy (using chemical screens and bioactivity-guided fractionation) to a more targeted ?genome mining? one (using modern sequencing and algorithms). However, frequently promising biosynthetic gene clusters from these species are found in unculturable or genetically intractable organisms. Additionally, because of our greater understanding of the ?rules? of biosynthesis, reprogramming biosynthetic pathways through synthetic biology is of considerable interest. Due to a number of issues with folding,expression,andprecursorsupply,theworkhorsechassisofsyntheticbiology,E.coliandS.cerevisiae, havebeenshowntobeimperfectforexpressingproteinsofactinomycetalorigin.However,thedevelopmentof Streptomyces as a chassis for synthetic biology endeavors has been hindered by a number of features including:aslowgrowthrate,ahighGCgenome(whichcomplicatesgeneticmanipulations),andamyceliating phenotypeinliquidmedia.Thisproposalseekstocapitalizeontherecentworktodevelopgenetictoolsforthis class of bacteria (including orthogonal integration vectors;? characterization of promoters, ribosomal binding sites, and expression systems;? and application of the CRISPR/Cas9 genome editing platform) to improve a strain that naturally has attractive growth characteristics relative to other Streptomyces strains, Streptomyces venezuelae ATCC 10712. Specifically, this work is focused on issues related to heterologous expression of and metabolite formation by bacterial megasynthase enzymes that form ?thiotemplated? natural products, polyketides (generated by polyketide synthases, PKSs) and non-ribosomal peptides (generated by non- ribosomalpeptidesynthetases,NRPS).Toaddressthischallenge,severalapproacheswillbeemployed.First, using the CRISPR/Cas9 genome editing platform, a genome minimized version of Streptomyces venzueale ATCC 10712 will be generated to reduce background expression and improve growth characteristics. Then, various sites on the chromosome will be probed to determine where the level of transcription (and thus translation)arethehighest(firstwiththefluorescentprotein,mCherry,thenwithanNRPS-PKShybridnatural product biosynthetic pathway). Finally, the bottlenecks in posttranslational modification and precursor supply will be evaluated and addressed through metabolic engineering. The modifications to Streptomyces venezueale ATCC 10712 will undoubtedly accelerate discovery and optimization through providing a better chassisorganismtoproducenaturalandengineeredmetabolitescaffolds.
To engineer natural product analogs and uncover new secondary metabolites from unculturable and/or genetically intractable bacteria, heterologous hosts (or ?chassis?) are required. Thisproposalpresentsastrategythatutilizesgenomeeditingandmetabolicengineeringtotransform a promising strain, Streptomyces venezuale ATCC 10712, into a more widely applicable host for natural product pathways. This work capitalizes on new genetic tools that have been developed in recent years (such as the CRISPR/Cas9 system) to accelerate drug discovery and development of biologicallyactivescaffolds.
