Transcriptional auto-regulation of synthetic enzyme cascades

Nature has evolved efficient metabolic systems involving multiple enzymes arranged in pathways or cascades to provide robust cellular functions such as growth and reproduction. A key feature of natural pathways is that they are finely regulated by proteins called 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 the production of enzymes active in related metabolic pathways to 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 of enzymes from different microorganisms (i.e., different metabolic systems) are introduced, together with pathway-specific TFs, into Escherichia coli, one of the most commonly used microbial hosts in 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 of solute-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 via aldehyde 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 timed production of all cascade enzymes and 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-nothing fashion, which burdens host cells and, in turn, can negatively influence synthetic cascade performance and product yields. Completion of this project requires methods from different disciplines including synthetic biology, systems biocatalysis, protein engineering, and bioinformatics. This interdisciplinary character is essential to integrate pathway-specific TFs to auto-regulate the proof-of-concept cascades. Since this unprecedented concept can be applied to other artificial pathways, this work not only offers ecological alternatives to traditional synthetic routes for the production of pharmaceuticals and other chemicals employed by the industry, but to auto-regulate and optimize synthetic enzyme cascades similar to how nature does it.