FWF - TransAuto - Transcriptional auto-reulation of synthetic enzyme cascades

Nature has evolved efficient metabolicsystemsinvolvingmultiple enzymes arranged in pathways or cascadestoprovide robust cellular functions such as growth and reproduction.A key feature of natural pathways isthat they are finely regulated by proteinscalled transcription factors (TFs).TFs bind small molecules from the environment (e.g., nutrients) or sense metabolites inside the cell. Upon binding, TFs can specifically target genomic DNA, which switches on, adapts, and coordinates theproductionof enzymesactive in related metabolic pathwaysto appropriately respond to the stimulus.This project aims at adding this plasticity to artificial pathway designs by implementing customized TFs to regulate the induction and coordinated production of pathway enzymes depending on the presence of cascade-related molecules (e.g., substrates, intermediates, and potential byproducts). Therefore, proof-of-concept cascades consisting ofenzymes from different microorganisms (i.e., different metabolic systems) are introduced, together with pathway-specific TFs,into Escherichia coli, one of the most commonlyused microbialhostsin biotechnology.Customization of TFs follows complementary strategies: (1) screening of known TFs for binding of nonnative but structurally related compounds, (2) expanding binding specificities of known TFs by protein engineering, which is usually applied to engineer enzymes rather than TFs, and (3) identification of novel TFs by screening ofsolute-binding protein (SBP) libraries.Similar to TFs, SBPs bind nutrients and small molecules and transport them inside host cells. The innovation of this research proposal lies in physically linking the binding of pathway-related compounds by SBPs to their localization on microbial genomes. Subsequently, bioinformatic tools are used to analyze the genetic neighborhoods to discover new TFs. This is feasible as SBPs regularly colocalize on microbial chromosomes with their related metabolic pathways and, importantly, the TFs regulating them.Proof-of-concept cascades transform primary alcohol substrates viaaldehyde intermediates into phenylacetylcarbinols, which are precursors for important pharmaceuticals to treat a variety of diseases including hypotension, obesity, or chronic asthma.Implementation of customized TFs enables the timedproductionof all cascade enzymesand dynamic adaption in the amounts needed to efficiently produce the desired phenylacetylcarbinols.This has advantages over current pathway designs that produce enzymes in an all-or-nothingfashion, which burdens host cells and, in turn, can negatively influence synthetic cascade performanceand product yields.Completion ofthis projectrequiresmethodsfrom differentdisciplines includingsynthetic biology, systems biocatalysis, protein engineering, and bioinformatics. Thisinterdisciplinarycharacter is essentialto integrate pathway-specific TFs toauto-regulatethe proof-of-concept cascades. Since this unprecedentedconceptcan be applied toother artificial pathways, this work not only offersecological alternativestotraditional synthetic routes for the production of pharmaceuticals and other chemicalsemployed by the industry, but to auto-regulate and optimize synthetic enzyme cascadessimilar to how nature does it