Executive Summary : | Indolocarbazoles (ICZs), a subtype of bisindole alkaloids, are potential anti-cancer drugs of commercial interest. Staurosporine and Rebeccamycin were the first ICZs antibiotics shown to have potential anti-tumor activity as inhibitors of protein kinase and topoisomerase I [1]. Rebeccamycin is a conventional topoisomerase I poison capable of stabilizing DNA- topoisomerase I covalent complexes, thereby promoting secondary DNA double strand breaks due to the collision of the replication fork with the cleavage complex [2]. Several rebeccamycin analogues, which target DNA topoisomerase I or II, have already entered clinical trials as anticancer drugs [3]. It also shows antibacterial activity against several Gram-positive bacteria, including Staphylococcus aureus and Streptococcus faecalis. Rebeccamycin, is a halogenated natural product of the indolocarbazole family, and is produced by Saccharothrix aerocolonigenes ATCC39243[2]. There is a considerable effort placed on the design and synthesis of new rebeccamycin analogs and derivatives with enhanced antitumor activity. The cloning and sequencing of the complete gene cluster encoding Rebeccamycin biosynthesis from Saccharothrix aerocolonigenes ATCC39243 has been reported [2] and the identified set of genes has been shown to be necessary and sufficient for rebeccamycin production by heterologous expression in Streptomyces albus host strain.
The main goal of this proposal is to design and develop a modular framework for metabolically engineering of Escherichia coli to produce Rebeccamycin using the systems/synthetic biology paradigm. The work plan involves development of an integrative strategy to address the challenge of trade off between growth and product for strain development by genome engineering in Escherichia coli for Rebeccamycin. A synthetic biology approach of gene sequence optimization for transcription, translation rates of the multigene operon will be combined with systems biology techniques to successfully design in a modular fashion a strain portfolio for Rebeccamycin. The approach is to start with the host cell metabolic network reconstruction and incorporate the Rebeccamycin pathway. The genome-scale metabolic reconstruction will be translated into a stoichiometric model for mathematical analysis to identify and develop prospective strain designs. Constraints based analysis will predict knock-outs, gene additions, promoter and enzyme activity manipulations, optimal media components, oxygen uptake, growth rate/yield and redox balance. The resultant candidate strain designs determined in silico, will be implemented in the host strain for testing. A systems biology approach integrating high throughput OMICS data collection, computational analysis and evolutionary engineering will be used to finetune the strain for optimal production. Such integrated approaches will allow us to engineer an strain portfolio for rebeccamycin production scalable to other indolecarbazole drug molecules. |