Tackling Achilles’ Heel in Synthetic Biology: Pairing Intracellular Synthesis of Noncanonical Amino Acids with Genetic‐Code Expansion to Foster Biotechnological Applications

Abstract

For the last two decades, synthetic biologists have been able to unlock and expand the genetic code, generating proteins with unique properties through the incorporation of noncanonical amino acids (ncAAs). These evolved biomaterials have shown great potential for applications in industrial biocatalysis, therapeutics, bioremediation, bioconjugation, and other areas. Our ability to continue developing such technologies depends on having relatively easy access to ncAAs. However, the synthesis of enantiomerically pure ncAAs in practical quantitates for large‐scale processes remains a challenge. Biocatalytic ncAA production has emerged as an excellent alternative to traditional organic synthesis in terms of cost, enantioselectivity, and sustainability. Moreover, biocatalytic synthesis offers the opportunity of coupling the intracellular generation of ncAAs with genetic‐code expansion to overcome the limitations of an external supply of amino acid. In this minireview, we examine some of the most relevant achievements of this approach and its implications for improving technological applications derived from synthetic biology.

An Engineered Escherichia coli Strain with Synthetic Metabolism for in‐Cell Production of Translationally Active Methionine Derivatives

“…Recently, though, it has been argued that the limited access to reprogrammed protein translation to metabolically produced ncAAs is an Achilles' heel for the whole field, and hinders its broader application in biotechnology. [59] Current technologies for the expression of custom-made proteins and peptides require ready-synthesized unnatural amino acids. In most cases, their chemical or chemo-enzymatic syntheses are complicated and expensive.…”

“…Chemical diversity gained in this way will allow for a dramatic increase in the scope of protein biosynthesis, and enable the expression of peptide‐based biomaterials having properties not found in nature. Recently, though, it has been argued that the limited access to reprogrammed protein translation to metabolically produced ncAAs is an Achilles′ heel for the whole field, and hinders its broader application in biotechnology [59] . Current technologies for the expression of custom‐made proteins and peptides require ready‐synthesized unnatural amino acids.…”

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

“…Lastly, scientists in the twenty-first century are not restricted to designing enzyme pathways containing only the standard 20 amino acids. Over 200 different non-canonical amino acids have been incorporated into proteins through altering the genetic code machinery (Biava, 2020). Recent advances are reviewed elsewhere (Arranz-Gibert et al, 2019;Biava, 2020;Ros et al, 2020); key challenges include ensuring the orthogonality of the introduced aminoacyl tRNA synthetase/tRNA pair to the native system, the efficiency of incorporation, and providing a source of the amino acid either intracellularly through metabolic engineering or through uptake from the growth media.…”

Intersecting Xenobiology and Neometabolism To Bring Novel Chemistries to Life

“…[141] As indicated above, FAAs should be externally added to the culture to force their incorporation into the proteome, which is considered a disadvantage in terms of cost and sustainability. [142] Deciphering regulatory details of the 4-fluorothreonine biosynthetic pathway in S. cattleya could be a major breakthrough to overcome this limitation, but this is not the only route towards in vivo production of FAA by neometabolism. In this sense, the use of tyrosine phenol-lyases is remarkable because of its simplicity.…”