Date of Award
Fall 2021
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Isaacs, Farren
Abstract
There has been significant progress in the field of synthetic biology to develop modular tools with which to achieve greater ability to probe, manipulate and predict biological phenomena. Genetic code expansion aims to engineer genomes and the translation apparatus to enable site-specific incorporation of nonstandard amino acids (nsAAs) into proteins. However, the ability of living systems to incorporate multiple instances of nsAAs or even abiological monomers that are not L-α-amino acids into biopolymers is severely limited. Improved aminoacyl tRNA synthetase (aaRS) and tRNA pairs that are orthogonal to the native translation system, and new genomically recoded organisms (GROs) with open codons have been developed to enable multi-site incorporation of nsAAs into proteins. In order to allow for the translation of biopolymers consisting of synthetic monomers that are not L-α-amino acids, the peptidyl transferase center (PTC) of the ribosome must be evolved. Here, I describe efforts to address these challenges with the goal of expanding nature’s ability to expand the chemical diversity of genetically encoded polymers produced by the ribosome of living systems. Specifically, we sought to develop new approaches to produce protein biopolymers with an expanded chemical alphabet.Several technologies have converged to realize this vision, including orthogonal tethered ribosomes (oRiboT) that are insulated from the cell’s native translational system, in vitro translation systems, genome editing methods that can make multiplexed edits in precise positions across the genome, and GROs with open codons allowing more efficient multi-site incorporation of nsAAs into proteins in vivo. We commenced this work by developing genome editing methods to generate vast combinatorial diversity for evolution of orthogonal tethered ribosomes in vivo. As there are seven ribosomal operons in E. coli that share sequence with oRiboT, we needed to develop a genome editing method that could specifically target one out of many repetitive genetic elements in a genome. In this method, which we call filtered editing, we embed group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduced intron-containing ribosomes into the E. coli genome and performed targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering (MAGE). Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. We then developed a strategy to evolve peptidyl transferase center mutants of orthogonal tethered ribosomes that can accommodate non-L--amino acids for polymerization of synthetic biopolymers. Our integrated strategy combines genomically recoded Escherichia coli strains, a library of orthogonal tethered ribosome variants, and iterative rounds of positive and negative selections. As a model, we applied our strategy to isolate mutant ribosomes able to accommodate an extended backbone monomer, β-O-methyl-tyrosine (β3OmY). Using selected orthogonal tethered ribosome variants, we demonstrate multi-site incorporation of β3OmY into polypeptides in vitro, which were not accommodated by the natural ribosome. To more rationally guide our efforts to engineer the ribosome’s vast sequence space for expanded function, we sought to develop computational methods to study functional relationships between bases in the PTC of oRiboT ribosomes. The fitness landscape of a gene maps the sequence to function relationship across its sequence space and can aid in optimizing its molecular evolution for improved activity. I present a method that relies on the function of three orthogonal genes to empirically assay each mutant of a degenerate library of oRiboT ribosomes and computationally reconstruct the fitness landscape of oRiboT. Using this method, we determine the flexibility to mutation of bases across the PTC of oRiboT, as well as their epistatic interactions. This work complements previous understanding of the functional role of nucleotides in the PTC, and expands it to allow for an understanding of how additional mutations can compensate for fitness costs to ribosome function, allowing the production of functional ribosomes that significantly differ from WT ribosome sequence. I also discuss work to expand our ability to introduce multiple distinct nsAAs into protein biomaterials in a GRO at open codons. This approach involved the construction of a modular multi-plasmid system to encode multiple instances of two nsAAs at the UAG and UGA codons, respectively, into protein biopolymers in vivo. We systematically characterized the strain fitness, and nsAA incorporation by fluorescence as well as LC-MS measurements. Together, this work establishes the technical foundation to simultaneously encode two distinct synthetic monomers into novel biopolymers in a recodes strain of E. coli containing two open codons. In total, the work discussed in this dissertation describes early efforts for engineering the central dogma for the biosynthesis of sequence-defined biopolymers and expansion of the cell’s chemical repertoire. Applications of this research have broad significance in basic and applied research, and for the development of new protein biomaterials, therapeutics, environmental remediation, and sustainable manufacturing.
Recommended Citation
Radford, Felix, "Engineering the genetic code and the ribosome to expand the chemical palette of living systems" (2021). Yale Graduate School of Arts and Sciences Dissertations. 764.
https://elischolar.library.yale.edu/gsas_dissertations/764
