Michael G. Hoesl

Recent advances in genetic code engineering in Escherichia coli.

The expansion of the genetic code is gradually becoming a core discipline in Synthetic Biology. It offers the best possible platform for the transfer of numerous chemical reactions and processes from the chemical synthetic laboratory into the biochemistry of living cells. The incorporation of biologically occurring or chemically synthesized non-canonical amino acids into recombinant proteins and even proteomes via reprogrammed protein translation is in the heart of these efforts. Orthogonal pairs consisting of aminoacyl-tRNA synthetase and its cognate tRNA proved to be a general tool for the assignment of certain codons of the genetic code with a maximum degree of chemical liberty. Here, we highlight recent developments that should provide a solid basis for the development of generalist tools enabling a controlled variation of chemical composition in proteins and even proteomes. This will take place in the frame of a greatly expanded genetic code with emancipated codons liberated from the current function or with totally new coding units.

In Vivo Incorporation of Multiple Noncanonical Amino Acids into Proteins

Expansion of the standard genetic code enables the design of recombinant proteins with novel and unusual properties. Traditionally, such proteins have contained only a single type of noncanonical amino acid (NCAA) in their amino acid sequence. However, recently reported initial efforts demonstrate that it is possible with suppression-based methods to translate two chemically distinct NCAAs into a single recombinant protein by combining the suppression of different termination codons and nontriplet coding units (such as quadruplets). The possibility of expanding the code with various NCAAs simultaneously further increases the toolkit for the generation of multifunctionalized proteins.

Lipase Congeners Designed by Genetic Code Engineering

Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Azatryptophans endow proteins with intrinsic blue fluorescence.

Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible (Aza)Indoles (Inds)/(Aza)Tryptophans (Trp) as optical probes represent almost ideal isosteric substitutes for natural Trp in cellular proteins. To overcome the limits of the traditionally used (7-Aza)Ind/(7-Aza)Trp, we substituted the single Trp residue in human annexin A5 (anxA5) by (4-Aza)Trp and (5-Aza)Trp in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable on routine UV irradiations. We identified (4-Aza)Ind as a superior optical probe due to its pronounced Stokes shift of ≈130 nm, its significantly higher quantum yield (QY) in aqueous buffers and its enhanced quenching resistance. Intracellular metabolic transformation of (4-Aza)Ind into (4-Aza)Trp coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.

Chemical Evolution of a Bacterial Proteome

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.

Expanding and Engineering the Genetic Code in a Single Expression Experiment

Various new biological properties of proteins can emerge from the presence of noncanonical amino acids (NCAAs) at multiple positions in the protein sequence. In this context, “genetic code engineering” is based on in vivo sense codon reassignment by the supplementation-based incorporation method (SPI). By exploiting the substrate tolerance of various cellular systems (uptake, metabolism, and translation apparatus), different NCAAs were successfully translated into target protein sequences in a residue-specific manner. Proteins produced in this way can also be called variants, alloproteins, or congeners because they originate from the same gene sequence, but contain a small fraction of amino acids exchanged by related NCAAs. Many translationally active NCAAs are isostructural side-chain analogues of existing canonical amino acids and often have chemical diversity that is not available in the standard genetic code. Accordingly, “genetic code engineering” follows an evolutionary logic because the disruption of the resulting protein structure should be minimized by the modest alterations in the amino acid side chains. Nonetheless, common academic criticism refers to the absence of “site-directedness” in the SPI approach. To circumvent this, heterologous orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs have been developed for in vivo co-translational position-specific incorporation of NCAAs (“genetic code expansion”) by stop codon suppression (SCS). However, the SCS approaches are only well suited for a few amino acid insertions because the increase of in-frame stop/nontriplet codons in the mRNA decreases the efficiency of its translation. To date, the suppression of only three stop codons in one mRNA sequence is reported in vivo. More importantly, “code expansion” is generally not applicable to isostructural NCAAs because it relies on the use of “orthogonal” aaRSs developed by guided-evolution approaches. These methods are generally not capable of generating enzymes that discriminate efficiently between subtle structural variations. Examples include exchanges at the level of single atoms or small functional groups such as -H/-F, -H/-CH3, -S-/-CH2-, -CH = /-N = or -H/-OH (“atomic mutations”). In nature, canonical amino acids of similar shape (Val/Thr), molecular volume (Ile/Val) or very similar functional groups (Ser/Thr) are normally recognized by enzymes that have an additional editing domain beside the catalytic domain (“double sieve”). The above-mentioned small modifications can usually only be introduced post-translationally by means of orthogonal pairs (“code expansion”), that is, after cotranslational chemical masking with bulky groups such as onitrobenzyl. By taking into account the limitations and advantages of both the SPI and SCS approach, it would be highly desirable to combine these in a single in vivo expression experiment. In this way, SCS should allow us to add NCAAs at permissive sites in a position-specific manner, whereas SPI would bring a multiple residue-specific incorporation of isostructural NCAAs in response to sense codons. In this study, we followed this line of argument and combined the residue-specific substitutions of aliphatic (Met) and endocyclic (Pro) analogues with the site-directed incorporation of the aromatic photo-crosslinking amino acid p-benzoyl-phenylalanine (Bpa) as outlined in Figure 1. The Bpa system was chosen because it proved to be a well-performing orthogonal pair without background incorporation of canonical amino acids. As a model protein, we first chose the enhanced green fluorescent protein (EGFP) because its refolding properties with cis-4-fluoroproline ((4S-F)Pro) are well known. In addition, the position-specific insertion of Bpa has been proved to work with satisfying yields at the permissive sequence position 150 of Aequorea victoria GFPs (avGFPs). To expand the number of model proteins for these experiments, we also tested the cysteine-free barstar from Bacillus amyloliquefaciens (y-b*), which is normally used for proteinfolding studies and the industrially relevant lipase from Thermoanaerobacter thermohydrosulfuricus (TTL) (Figure 1). In both proteins, we performed a search for permissive positions amenable for efficient in-frame stop codon read-through. For parallel incorporation of Bpa and (4S-F)Pro into EGFP (Figure 2 A, lane 4), a pQE80L vector containing a C-terminally His-tagged EGFP with an amber stop codon (UAG) at N150 was used in combination with pSup-BpaRS-6TRN. This plasmid contains an engineered mjTyrRS (BpaRS) along with six copies of tRNA CUA capable of recognizing and incorporating Bpa in response to UAG. Expression was performed in the Pro auxotrophic strain CAG18515 by growing the cells in New Minimal Medium (NMM) with a limited Pro concentration until depletion at an OD600 between 0.6–0.8. Subsequently, 1 mm of Bpa and 0.5 mm of (4S-F)Pro were added to the expression culture, and target protein expression was induced (4 h at 30 8C). Additional expression experiments were performed for production of the mutant EGFP(N150Bpa) (Figure 2 A, lane 3) and a control experiment with 1 mm of Tyr instead of Bpa (Figure 2 A, lane c). The expression of parent EGFP (Figure 2 A, lane 1) and its congener EGFP[(4S-F)Pro] (Figure 2 A, lane 2) was performed [a] M. G. Hoesl , Prof. Dr. N. Budisa Max Planck Institute of Biochemistry, Molecular Biotechnology Am Klopferspitz 18, 82152 Martinsried (Germany) Fax: (+ 49) 89-8578-3557 [b] M. G. Hoesl , Prof. Dr. N. Budisa Berlin Institute of Technology, Department of Chemistry, Biocatalysis Group Franklinstrasse 29, 10587 Berlin (Germany) Fax: (+ 49) 30-314-28279 E-mail : budisa@biocat.tu-berlin.de Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000586.Expanding and engineering the code simultaneously: This concept was experimentally realized in a single in vivo expression experiment whereby residue-specific, sense codon reassignments Met→Nle/Pro→(4S-F)Pro (code engineering) were combined with position-specific STOP→Bpa read-through by an amber suppressor tRNA (code expansion).

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

In vivo expression of colored proteins without post‐translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell‐permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red‐shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2‐, 4‐, 5‐, 6‐, and 7‐azaindole) 4‐azaindole has exhibited the largest Stokes shift (∼130 nm) in steady‐state fluorescence measurements. It is also highly biocompatible and as 4‐azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono‐ and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4‐azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole‐containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo‐ or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady‐state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coli Strain Produces a New-to-nature Bioactive Lantibiotic Containing Thienopyrrole-Alanine.

Genetic code engineering that enables reassignment of genetic codons to non-canonical amino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesized peptides and proteins with functions not commonly found in Nature. Here we report the expression of a ribosomally synthesized and post-translationally modified peptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan (Trp) residue replaced by the ncAA L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) which does not sustain cell growth in the culture. We have demonstrated that cellular toxicity of [3,2]Tpa for the production of the new-to-nature bioactive congener of lichenicidin in the host Escherichia coli can be alleviated by using an evolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but also uses it as a proteome-wide synthetic building block. This work underscores the feasibility of the biocontainment concept and establishes a general framework for design and large scale production of RiPPs with evolutionarily adapted host strains.

Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein

Aequorea victoria green fluorescent protein and its widely used mutants enhanced green fluorescent protein and enhanced cyan fluorescent protein (ECFP) are ideal target proteins to study protein folding. The spectral signals of their chromophores are directly correlated with the folding status of the surrounding protein matrix. Previous studies revealed that tryptophan at position 57 (Trp57) plays a crucial role for the green fluorescent proteins structural and functional integrity. To precisely dissect its role in ECFP folding, we performed its substitution with the isosteric analogs 4‐azatryptophan [(4‐Aza)Trp] and 7‐azatryptophan [(7‐Aza)Trp]. Although Trp is moderately hydrophobic, these isosteric analogs are hydrophilic, which makes them an almost ideal tool to study the role of Trp57 in ECFP folding. We achieved high‐level expression of both (4‐Aza)Trp‐ECFP and (7‐Aza)Trp‐ECFP. However, great portions (70–90%) of protein samples were insoluble and did not contain a maturated chromophore. All attempts to refold the insoluble protein fractions failed. Nevertheless, low amounts of fully labeled, soluble, chromophore containing fractions with altered spectral features were also isolated and identified. The most probable reason for the high yield of misfolding is the introduction of strong hydrophilicity at position 57 which strongly interferes with productive and efficient folding of ECFP. In addition, the results support a strong correlation between translational kinetics of non‐canonical amino acids in the ribosome and in vivo folding of the related modified protein sequence. Copyright