Yane-Shih Wang

Upgrading protein synthesis for synthetic biology

Genetic code expansion for synthesis of proteins containing noncanonical amino acids is a rapidly growing field in synthetic biology. Creating optimal orthogonal translation systems will require re-engineering central components of the protein synthesis machinery on the basis of a solid mechanistic biochemical understanding of the synthetic process.

A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum.

Together with tRNA(CUA)(Pyl), a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large para substituents into superfolder green fluorescent protein at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pairs.

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

A genetically encoded photocaged Nε-methyl-L-lysine

A photocaged N(epsilon)-methyl-L-lysine has been genetically incorporated into proteins at amber codon positions in Escherichia coli using an evolved pyrrolysyl-tRNA synthetase-pylT pair. Its genetic incorporation and following photolysis to recover N(epsilon)-methyl-L-lysine at physiological pH provide a convenient method for the biosynthesis of proteins with monomethylated lysines at specific sites.

The de novo engineering of pyrrolysyl-tRNA synthetase for genetic incorporation of L-phenylalanine and its derivatives

Using evolved pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli.

Genetic incorporation of twelve meta-substituted phenylalanine derivatives using a single pyrrolysyl-tRNA synthetase mutant.

When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Significance Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. We found that a series of PylRS variants that were initially selected to be specific for the posttranslational modification Nε-acetyl-l-Lys displayed polyspecificity [i.e., activity with a broad range of noncanonical amino acid (ncAA) substrates]. Our structural and biochemical data indicate that the engineered tRNA synthetases can accommodate ncAA substrates in multiple binding modes. The data further suggest that in vivo selections do not produce optimally specific tRNA synthetases and that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Exploring the Substrate Range of Wild-Type Aminoacyl- tRNA Synthetases

We tested the substrate range of four wild‐type E. coli aminoacyl‐tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). Although these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also show that E. coli tryptophanyl‐tRNA synthetase (TrpRS) and tyrosyl‐tRNA synthetase have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge in the aim of incorporating multiple different amino acids site‐specifically into proteins.

The genetic incorporation of thirteen novel non-canonical amino acids

Thirteen novel non-canonical amino acids were synthesized and tested for suppression of an amber codon using a mutant pyrrolysyl-tRNA synthetase-tRNA(Pyl)(CUA) pair. Suppression was observed with varied efficiencies. One non-canonical amino acid in particular contains an azide that can be applied for site-selective protein labeling.

Genetic Incorporation of Seven ortho-Substituted Phenylalanine Derivatives

Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.