Upgrading protein synthesis for synthetic biology

Abstract

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.

Figure 1. Engineering efficient OT Ss

Protein synthesis requires amino acid (AA)-tRNAs as building blocks for ribosomal protein synthesis. An amino acid is ligated to the tRNA by a dedicated AARS. The product (AA-tRNA) is then delivered by an elongation factor (for example, EF-Tu) to the ribosome, where the anticodon of the tRNA matches the triplet codon of the mRNA. Protein synthesis using an expanded genetic code is shown with an ncAA incorporated by PylRS, tRNA^Pyl and EF-Tu at the UAG codon. For efficient ncAA incorporation, all three components of the above OTS should be optimized. First, an efficient and specific AARS needs to be developed for each ncAA (AA*). It will be necessary to combine the power of new methods to engineer and select mutant libraries. High-throughput screening of amino acid chemical libraries, biochemical analysis and structural determination are important factors in developing new OTSs. This cycle may need to be repeated several times to eventually produce AARS-ncAA pairs that match the efficiency and specificity of natural AARSs. Second, the synthetic tRNA carrying the desired ncAA must be orthogonal (that is, not recognized by endogenous AARSs) in each organism. Third, EF-Tu requires mutations to accommodate ncAAs with negative charges or bulky side chains. For certain ncAAs, even the ribosome may need to be engineered to improve ncAA incorporation in protein.

Figure 2. Chemical diversity of amino acids in the standard and expanded genetic codes

Current genetically encoded ncAAs are mainly derivatives of lysine and phenylalanine. Future efforts are needed to develop a wide variety of orthogonal systems to expand the genetic code with a much larger pool of ncAAs including diverse functional groups. The tree relates the canonical amino acids according to the JTT similarity matrix. Amino acid similarity reflects the substitution frequency of one amino acid for another in standardized sets of multiple sequence alignments.

Figure 3. Reassignment of CUN codons in yeast mitochondria provides insight into sense codon recoding

The ancestor mitochondria contain a tRNA^Leu with a UAG anticodon that pairs with CUN codons. Both the CUN codons and tRNA^Leu _UAG were lost during evolution. A duplicated copy of tRNA^His then evolved to decode CUN. Although this new tRNA is no longer recognized by histidyl-tRNA synthetase (HisRS), it becomes a substrate for the coevolved threonyl-tRNA synthetase (MST1). With the emergence of the orthogonal MST1-tRNA^Thr _UAG pair, CUN codons reappeared in the mitochondrial genome to complete the codon reassignment event. This naturally evolved system could serve as a model for sense codon recoding.