Our Troika: what are we particularly interested in?

Our research focuses on studies and engineering of the genetic code using three canonical amino acids ("troika"):

• Proline (Pro, P)
• Methionine (Met, M)
• Tryptophan (Trp, W).

as targets for replacements (as well as the code amino acid repertoire expansions and reductions) with their analogs, surrogates, natural and synthetic counterparts with chemically changed side chains (Met, Trp) and protein backbone itself (Pro).

We perform mainly in vivo translations with various analogs or chemically similar structures of our "troika" in combination with their chemical or metabolic syntheses (transformations) using all available tools and approaches (including orthogonalized translation). We are also very interested to use their chemical analogs to delivery elements not used by nature (e.g. fluorine, tellurium and boron) into the biochemistry of life.

You will find more about proline, tryptophan and methionine in the Resources section, where all canonical members of the amino acid repertoire of the universal genetic code are presented in a comprehensible way.

Note that Proline (codons: CCU, CCA, CCG, and CCC) is considered to be a "primordial" amino acid and an integral part of the very early ‘GC code’. It has critical importance for protein folding and functions, in particular in repetitive oligopeptide motifs and processes that involve cis/trans isomerization.

On the other hand, both Met (AUG) and Trp (UGG) are considered as a "late addition" to the extant ‘GCAU code’ with special chemical functions in proteins. They are the least occurring amino acid in the proteome with low abundance in proteins (~1%) but highest metabolic synthesis cost (especially Trp) of all canonical amino acids.

Our main achievements with “Troika”:

Why code engineering?

In the extant living beings, the set of 20 canonical amino acids prescribed by the universal genetic code does not span all dimensions of chemical variability necessary for diverse cellular processes advanced (e.g. metazoan) cellular forms.

Keep always in mind: only a few proteins have a final covalent structure which is a simple accurate translation of mRNA. Thus, the release of proteins from a ribosome is usually not the last chemical step in their functional maturation to become e.g. structural components of the cell or to serve as functional biochemical machines.

The majority of the proteins reach maturity and converts into active forms, either co-translationally or by post-translational processing. Indeed, evolution invented these two strategies to increase amino acid side-chain inventory. First, a small fraction of proteins is co-translationally equipped with special proteinogenic amino acids such as selenocysteine (Sec) and pyrrolysine (Pyl) by reassignment of termination codons. The second and major class of chemical modifications that contribute to the protein structure/ function diversity are post-translational modifications (PTMs). These reactions are selectively and timely coordinated events performed by dedicated enzymes or enzymatic complexes, usually in a specialized cell compartment.

Why then code engineering? Indeed, we have PTMs that encompass all the dimensions of chemical variability necessary for different cellular processes and functions.

Nonetheless, there are at least three good reasons (all good things go by three) to use code engineering.

1. PTM machinery is highly complex (compartmentalization!)
2. It is difficult to achieve large quantities of homogeneous product
3. The recognition features for specific PTM are normally easily destroyed in laboratory settings

It is extremely difficult to mimic nature's complex machines or processes such as the PTM. Therefore, we will require genetic code engineering as long as research in microfluidic devices and systems provides us with suitably miniaturized devices or micro-devices. For example, efficiently closed membrane or semipermeable orthogonal compartments such as vesicles might act as containers (or even protocells!) with encapsulated biochemical processes.

Academic application and potentials

Over the past 20 years, Ned teams have worked with various groups on joint projects. Many of these collaborations were very successful. Thereby, non-canonical amino acids proven to be valuable and useful tools for various research fields.

• Drug Design – code engineering as a route to diversify small bioactive molecules
• Biomaterials – new-to-nature congeneric peptides, proteins & complex scaffolds
• Bioorthogonal conjugations and chemistries – custom-made in vivo chemistries
• Structural Biology – noninvasive markers for 3D structures determination
• Fluorine biochemistry – fluorine as element of life and tool for NMR spectroscopy
• Spectroscopic probes in proteins – electron/proton pathways, signal transduction
• Photobiology – chromophore design (photophysics & optogenetics)
• Biophysics – protein folding, stability & energy dissipation pathways
• Biocatalysis – biocatalytic processes in cellular systems & enzymatic cascades
• Metabolic Engineering – reprograming intracellular amino acid syntheses
• Directed evolution – engineering & selection of enzymes and bacterial strains

Xenobiology, Synthetic Biology and related Technologies

Latest at the end of the 20th century it become clear that engineering of e.g. different microbial strains for fermentative production is much more efficient than classical chemical engineering. For example, Danielli predicted that “the production of sequence-determined polymers rather than the random polymers…” will create a “degree of sophistication…. far beyond thought”. In 1971 he said: “I am convinced that so many people – even biologists – aren’t aware that biology is moving from an age of analysis into an age of synthesis” [New Scientist, 1971, pp. 124].

While Danielli in 1970th was a voice in the wilderness, nowadays is well established that the main goal of Synthetic Biology (SB) is the creation of biodiversity applicable for biotechnological needs. In contrast, Xenobiology (XB) aims to expand the framework of natural chemistries with the non-natural building blocks in living cells to accomplish artificial biodiversity.

The molecules, molecular complexes and processes along the flow of genetic information ("central dogma") are particularly attractive targets for such engineering. For example, the development of alternative nucleic acids (xenonucleic acids, XNAs) based upon new base pairs, sugars and backbones. Alternatively, estranging the genetic code from its current form via systematic introduction of non-canonical amino acids should enable the development of bio-containment mechanisms in synthetic cells potentially endowing them with a ‘genetic firewall’ i.e. orthogonality which prevents genetic information transfer to natural systems.

In general, XB is an attempt to produce non-natural molecules (xenobiotic materials) using chemically modified organisms that may be endowed with a genetic firewall. In this context, processed products derived from microbes (e.g. E. coli, B. subtilis, S. cerevisiae) are particularly attractive. Such microbes are generally very suitable ‘workhorses’ for redesigning, recoding, re-inventing natural processes within short timescales.

According to Phillipe Marliere both Xenobiology and Synthetic biology have following industrial goals:

• Produce chemicals, materials and energy vectors from renewable resources.
• Design, construct and evolve microbes with novel metabolic cores and coding pathways.
• Propagate synthetic eco-systems and food-chains.
• Diversify modes of element cycling and fixation (CHNOPS + Fluorine + Silicon + Boron)
• Promote evolutionary bifurcations, prevent genetic pollution (Biocontainment, Genetic firewall).

New to-nature xenobiotic materials of future will be elaborated through combination of bio-inspired modules that will greatly surpass existing technologies with potentially high value for the society as a whole.