A main objective of synthetic biology is to make the process of designing genetically-encoded biological systems more systematic, predictable, robust, scalable, and efficient. The examples of genetic systems in the field vary widely in terms of operating hosts, compositional approaches, and network complexity, ranging from a simple genetic switch to search-and-destroy systems. While significant advances in synthesis capabilities support the potential for the implementation of pathway- and genome-scale programs, several design challenges currently restrict the scale of systems that can be reasonably designed and implemented. Synthetic biology offers much promise in developing systems to address challenges faced in manufacturing, the environment and sustainability, and health and medicine, but the realization of this potential is currently limited by the diversity of available parts and effective design frameworks. As researchers make progress in bridging this design gap, advances in the field hint at ever more diverse applications for biological systems.

Summary For synthetic biology applications, protein-based transcriptional genetic controllers are limited in terms of orthogonality, modularity, and portability. Although ribozyme-based switches can address these issues, their current two-stage architectures and limited dynamic range hinder their broader incorporation into systems-level genetic controllers. Here, we address these challenges by implementing an RNA-protein hybrid controller with a three-stage architecture that introduces a transcription-based amplifier between an RNA sensor and a protein actuator. To facilitate the construction of these more complex circuits, we use a model-guided strategy to efficiently match the activities of stages. The presence of the amplifier enabled the three-stage controller to have up to 200-fold higher gene expression than its two-stage counterpart and made it possible to implement higher-order controllers, such as multilayer Boolean logic and feedback systems. The modularity inherent in the three-stage architecture along with the sensing flexibility of RNA devices presents a generalizable framework for designing and building sophisticated genetic control systems.

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.

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