C-GEM is working to solve a “Holy Grail” level problem in modern science: the inability to synthesize chemically diverse polymers with protein-like sequence definition. Such ‘sequence-defined chemical polymers’ have virtually limitless potential for information storage, anti-counterfeiting, drug delivery, remediation, even drug discovery, but strategies to prepare them are barely in their infancy.
The C-GEM strategy is to synthesize sequence-defined chemical polymers using the ribosome, nature’s protein synthesizing machine. Repurposing the ribosome in this way demands experiment and computation, requires innovation in chemistry, biology, and engineering, and has already revealed deep secrets about the evolution and function of the translational apparatus. C-GEM scientists are discovering tRNAs and protein factors that deliver new chemical monomers, non-natural ribosomes that support novel bond-forming reactions, engineered organisms that produce never-before-made polymers at scale, and designed catalysts that add even greater chemical diversity.
Highlights from Phase I
New reactions from an old catalyst
Here we report that wild type E. coli ribosomes accept and elongate pre-charged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.
Genetic code expansion at the N-terminus
Here we show that one can initiate translation with aromatic non‐canonical amino acids (ncAAs) using a chimeric and orthogonal initiator tRNA (itRNATy2) that is a substrate for M. jannaschii TyrRS. itRNATy2 initiates translation in vivo with aromatic ncAAs bearing diverse sidechains. Translational efficiency was enhanced by deleting redundant copies of tRNAfMet from the genome. The end result was a system capable of introducing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable new synthetic biology tool and demonstrates the versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
A closer look at an engineered ribosome
Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome–P7A7-evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.
Working together, better
The Center for Genetically Encoded Materials (C-GEM) is an NSF Phase I Center for Chemical Innovation that comprises six laboratories spread across three university campuses. Our success as a multi-institution research team demanded the development of a software infrastructure, GEM-NET, that allows all C-GEM members to work together seamlessly—as though everyone was in the same room. GEM-NET was designed to support both science and communication by integrating task management, scheduling, data sharing, and collaborative document and code editing with frictionless internal and public communication; it also maintains security over data and internal communications. In this Article, we document the design and implementation of GEM-NET: our objectives and motivating goals, how each component contributes to these goals, and the lessons learned throughout development. We also share open source code for several custom applications and document how GEM-NET can benefit users in multiple fields and teams that are both small and large. We anticipate that this knowledge will guide other multi-institution teams, regardless of discipline, to plan their software infrastructure and utilize it as swiftly and smoothly as possible.