The proposed research seeks to combine techniques of metabolic engineering with those of directed enzyme evolution (termed 'molecular pathway breeding') for the protection of useful novel chemical compounds and materials in recombinant cells. This application seek so to expand this approach to the biosynthesis of yet other, even more complex novel chemical compounds-porphyrins-demanding the development of new screening and analytical tools. Porphyrins are a fascinating, structurally complex compound class of enormous significance and potential for a variety of applications in e.g. medicine, chemistry or material sciences. Mimicking natural breeding processes through gene assembly and in vitro evolution of only a few functionally diverse available microbial porphyrin biosynthetic genes will generate new pathways for unnatural porphyrins. The strategies and methods developed in this project can be readily applied to other metabolic pathways and provide new engineering tools for the discovery and production, in recombinant cells, of a kaleidoscope of diverse natural and non-natural compounds for a variety of applications. In particular, the developed high-throughput (HT) screening methods for the identification of synthesized small molecules in large cell libraries will be highly useful for other combinatorial approaches as well. The biosynthetically produced new porphyrin structures with various reactive functional groups will make valuable scaffolds for chemical modifications for the synthesis of additional porphyrin structures.
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