Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase

Abstract

Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as N ε-((((E)-cyclooct-2-en-1-yl)­oxy)­carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of M. mazei PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCO*Lys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.

Naturally Occurring tRNAs With Non-canonical Structures

“…Ultimately, Dh PylSc binds to tRNA Pyl through contacts with the acceptor and D-stem, and has no direct contact with the anticodon stem, variable loop, or T-stem (Nozawa et al, 2009). The PylRS-tRNA Pyl pair in the seventh-order methanogen M. alvus has recently been explored as an additional tool for genetic code expansion with advantages over its previously studied counterparts (Meineke et al, 2018;Willis and Chin, 2018;Yamaguchi et al, 2018;Beránek et al, 2019;Dunkelmann et al, 2020;Seki et al, 2020). Ma tRNA Pyl has many unusual features that distinguish it from canonical tRNAs as well as previously characterized tRNA Pyl (Figure 8C).…”

Genetic Incorporation of ϵ‐N‐Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl‐tRNA Synthetase

“…The smaller size of MaPylRS makes it more amenable to delivery via viral vectors with limited capacity and easier to construct gene libraries for directed evolution. MaPylRS is also more stable and has higher expression level in E. coli cells [28] . Transplantation of mutations identified in the active sites of MmPylRS or MbPylRS into the MaPylRS often lead to improved ncAA incorporation efficiency in E. coli [29,30] .…”

“…Transplantation of mutations identified in the active sites of MmPylRS or MbPylRS into the MaPylRS often lead to improved ncAA incorporation efficiency in E. coli [29,30] . MaPylRS protein can also be concentrated several fold higher without aggregation in vitro , which greatly facilitates cell‐free genetic code expansion as well [28] …”

“…We therefore decided to use MaPylRS as the parental synthetase to engineer BzK‐specific mutants. On the basis of the crystal structure of MaPylRS (Figure 1b), [28] we randomized Tyr126, Met129, and Val168 in the active site and fixed His227 to Ile and Tyr228 to Pro. The H227I/Y228P mutations are at the second layer of the amino acid‐binding pocket and have been shown to improve ncAA incorporation efficiency in E. coli [28] .…”

Expanding the Genetic Code for Neuronal Studies

“…UAAs can be incorporated in vitro by different synthetic, semisynthetic and biosynthetic methods, such as native chemical ligation, inteinmediated protein ligation, protein trans-splicing, cell-free and flexible in vitro translation. [5] Genetic code expansion (GCE) enables the incorporation of UAAs into proteins in bacteria, yeast, cell lines, multicellular organisms, and human hematopoietic stem cells. [4c,d,6] This affords us the opportunity to modify and visualise dynamic biological processes with unprecedented submolecular precision.…”