This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Available online at www.sciencedirect.com Recent advances in genetic code engineering in Escherichia coli Michael Georg Hoesl and Nediljko Budisa The expansion of the genetic code is gradually becoming a modifications (PTMs). These reactions are selectively core discipline in Synthetic Biology. It offers the best possible and timely coordinated chemistries performed by dedi- platform for the transfer of numerous chemical reactions and cated enzymes and enzymatic complexes, usually in processes from the chemical synthetic laboratory into the specialized cell compartments. biochemistry of living cells. The incorporation of biologically occurring or chemically synthesized non-canonical amino Certainly, one of the main goals of Synthetic Biology is acids into recombinant proteins and even proteomes via to generate new and emergent biological functions in reprogrammed protein translation is in the heart of these streamlined cells which are equipped with ‘tailor-made efforts. Orthogonal pairs consisting of aminoacyl-tRNA biochemical production lines’. However, it is extremely synthetase and its cognate tRNA proved to be a general difficult to mimic nature’s complex machineries such as tool for the assignment of certain codons of the genetic code the PTM-apparatus. Thus, we usually highjack and/or with a maximum degree of chemical liberty. Here, we divert cellular systems such as protein translation to gain highlight recent developments that should provide a solid additional chemical diversity. To achieve this goal, we basis for the development of generalist tools enabling a need to find a way for efficient cellular uptake, meta- controlled variation of chemical composition in proteins bolic stability and translational activity (i.e. incorpora- and even proteomes. This will take place in the frame of tion) of useful non-canonical amino acids (ncAAs) which a greatly expanded genetic code with emancipated are usually chemically synthesized. Furthermore, we codons liberated from the current function or with totally need to (re)assign coding units (i.e. codons) in the new coding units. genetic code to accommodate ncAAs into target Address proteins. Technische Universita¨ t Berlin (Berlin Institute of Technology), Department of Chemistry, Biocatalysis Group, Franklinstraße 29, 10587 Here, we briefly sketch the most important developments Berlin, Germany in the field in the last three years. For further and more comprehensive information we refer to other recently Corresponding author: Budisa, Nediljko ([email protected]) published reviews which give an excellent overview of the methods, the incorporated ncAAs, and their diverse Current Opinion in Biotechnology 2012, 23:751–757 applications [1 ,2 ,3,4 ,5 ]. This review comes from a themed issue on Tissue, cell and pathway engineering General in vivo incorporation strategies Edited by Hal Alper and Wilfried Weber ncAAs for protein engineering can be divided into two For a complete overview see the Issue and the Editorial groups: first, amino acids which are isostructural to cAAs and therefore recognized by the endogenous host cell Available online 9th January 2012 machinery, and second, amino acids which are orthogonal 0958-1669/$ – see front matter, # 2012 Elsevier Ltd. All rights to the host cell system because they do not participate in reserved. conventional translation. To exploit the beneficial fea- http://dx.doi.org/10.1016/j.copbio.2011.12.027 tures of both ncAA groups for protein engineering, two distinct in vivo approaches are available for their co- translational incorporation (see Figure 1). Introduction Nature builds up proteins with the 20 canonical amino For isostructural ncAAs, residue-specific replacement of acids (cAAs) encoded by the 61 sense codons. However, cAAs is performed with the supplementation-based these 20 side-chain functionalities are obviously not suf- incorporation method (SPI) using auxotrophic host strains ficient for proteins to cover all the chemical diversity [6–8]. In contrast, orthogonal ncAAs are added to the amino necessary to maintain many vital biological functions in acid repertoire by site-specific incorporation in response to both unicellular and multicellular organisms. Evolution stop or quadruplet codons (stop codon suppression, SCS) invented two strategies to increase the side-chain inven- using orthogonal aminoacyl-tRNA synthetase:tRNA pairs tory: a small fraction of proteins is co-translationally (o-pairs) [9,10]. Both approaches have particular advan- equipped with special proteinogenic amino acids such tages and pitfalls, depending on the specific biological as selenocysteine (Sec) and pyrrolysine (Pyl) by reassign- questions and problems being addressed. Biological func- ment of termination codons. However, the major tions based on collective effects of many residues (e.g. classes of chemical modifications that contribute to the activity [11], stability [12,13], or conformational prefer- protein structure/function diversity are post-translational ences [14]) are particularly well studied and tailored by www.sciencedirect.com Current Opinion in Biotechnology 2012, 23:751–757 Author's personal copy 752 Tissue, cell and pathway engineering Figure 1 (a) Normal aminoacylation natural correctly tRNA charged aaRS tRNA canonical amino acid (cAA) natural aaRS natural protein (b) Supplementation based incorporation method (SPI) natural mis-acylated tRNA tRNA aaRS isostructural non-canonical natural amino acid (ncAA) aaRS protein variant (c) Stop codon suppressions approaches (SCS) mutated suppressor natural tRNA tRNA aaRS aaRS orthogonal ncAA natural mutated aaRS aaRS protein variant heterologous orthogonal charged aaRS:tRNA pair (o-pair) suppressor tRNA Current Opinion in Biotechnology Aminoacylation with canonical and non-canonical amino acids for protein translation. (a) In the natural scenario, tRNA aminoacylation is catalyzed by the corresponding aminoacyl-tRNA synthetase (aaRS) responsible for charging the tRNA with the cognate amino acids. (b) The supplementation- based incorporation method (SPI) exploits the natural substrate tolerance of the endogenous host aaRSs by using auxotrophic host strains. This allows the simultaneous exchange of many residues in a target protein by sense-codon reassignment [57]. The substrates for this procedure are non- canonical amino acids (ncAAs) which are isostructural to their canonical counterparts (i.e. atomic mutation concept [58]). (c) Stop codon suppression methodologies (SCS) are nominally site-specific and make use of a heterologous orthogonal aaRS:tRNA pair (o-pair) to incorporate an orthogonal amino acid in response to a stop or quadruplet codon. At this level, orthogonality is defined by a lack of cross-reactivity between the o-pair (including the ncAA) and the endogenous host synthetases, amino acids and tRNAs. Orthogonality of the o-pair is based on species-specific differences in tRNA recognition by the aaRS. The o-pair has to be evolved to enable the participation of a desired orthogonal ncAA in ribosome mediated protein synthesis (reviewed in [1 ]). SPI. In contrast, the site-specific SCS approach enables the (AzF) [20] for site-specific coupling reactions are certainly punctual dissection and modification of proteins (e.g. the most popular ones. [15,16]). Now we are witnessing the spreading utilization of pyr- Methanocaldococcus jannaschii TyrRS versus rolysyl-tRNA synthetase (PylRS) and its cognate suppres- Methanosarcinaceae PylRSs sor tRNA pylT from Methanosarcinaceae species in the Since the introduction of orthogonality as a tool for whole field. In the native context, this ‘natural o-pair’ protein engineering by Furter [9], various o-pairs were enables cells to incorporate the mostly aliphatic Pyl into developed (see [1 ] and [17] for comprehensive lists). target proteins in response to the amber (UAG) stop Nevertheless, until recently the most frequently used codon. In contrast to mjTyrRS, however, PylRSs already o-pair was based on the TyrRS from Methanocaldococcus naturally display a broad substrate tolerance towards jannaschii (mjTyrRS). Altogether, the incorporation of orthogonal ncAAs [21]. Within a very short time, a large around 40 different ncAAs, mainly aromatic analogs of number of different PylRS variants for incorporation of Phe and Tyr, was reported [1 ]. Among these, 4-ben- highly valuable aliphatic ncAAs were developed (see zoylphenylalanine (Bpa) for crosslinking [18] or 4-acetyl- Figure 2 and [4 ]). Remarkably, the evolved Pyl o-pair phenylalanine (ActF) [19] and 4-azidophenylalanine systems show a very high flexibility with respect to amino Current Opinion in Biotechnology 2012, 23:751–757 www.sciencedirect.com Author's personal copy Expanded genetic code Hoesl and Budisa 753 Figure 2 (a) Isostructural ncAAs F F N3 HN HN COOH HO O HO O H2N