Investigating and Developing Strategies for the Expression of Biosynthetic Genes In Vitro and In Vivo

Natural products have historically served as a rich source for a wide range of useful applications such aspesticides, veterinary agents, therapeutics, and bioproducts. To discover new natural products, manipulate them for analog generation, and harness the potential of these bioactive compounds for synthetic biology, it is necessary to develop robust methods for the expression of biosynthetic genes. A broad range of clinically useful natural products originate fromactinomycetes, especially those from the genus Streptomyces, which have been recognized as one of the predominant sources of microbial bioactive natural products. Actinobacteria are known for their large genomes, Guanine-Cytosine rich, and complex secondary metabolism. Some of these secondary metabolite pathways are composed of multienzyme proteins termed “megasynthases” which produce a wealth of clinically important natural product compounds (e.g., penicillin, daptomycin, and vancomycin antibiotics). The three projects in this dissertation investigate and develop new strategies to express megasynthase originating from actinomycetes in vivo and in vitro.

Often, the native or engineered pathways must be moved into a suitable surrogate, especially when the native host organism is not genetically tractable. Heterologous hosts must be genetically manipulatable and generally contain both sufficient fluxes of metabolic precursor as well as an appropriate environment for protein folding and expression. The first project of this dissertation reveals the effects of refactoring biosynthetic megasynthases using Escherichia coli as a heterologous host to express a non-ribosomal peptide synthase originating from Streptomyces.

Microbial metabolic engineering has focused on creating “cell factories'' that can synthesize valuable metabolites from readily available substrates. Cell-free synthetic biology is emerging as an important complementary approach because it is highly desirable to express protein on a more rapid timescale and does not rely upon the genetic tractability of a strain thus improving the throughput of design-build-test-learn (DBTL) cycles. The second project focuses on investigating and optimizing the lysate-based expression for megasynthase proteins.

The development of cell-free systems can then be used for prototyping these complex pathways to accelerate efforts towards engineered biosynthesis of these pathways. Take advantage of that, the third project uses a lysate-base cell-free system platform to profile expression strategies for a megasynthase protein.