Abstract
In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re‐engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans‐sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio‐orthogonal compounds. Our reprogrammed E. coli strain is capable of the in‐cell production of l‐azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology‐based production.
Highlights
The incorporation of natural or synthetic noncanonical amino acid (ncAA) into proteins during translation is a very attractive approach to expand the scope of ribosomal protein synthesis beyond the 20 canonical amino acids, generating novel structural and functional protein diversity
In our enzymatic reaction scheme, O-acetyl homoserine sulfhydrylase (OAHSS) from C. glutamicum will react with the unnatural substrate sodium azide (NaN3), instead of sulfide, leading to Aha production in place of methionine (Scheme 1, green branch)
When using the selective pressure incorporation method (SPI) method for the production of ncAA-substituted proteins, the required amount of noncanonical amino acid added to the culture medium varies from 0.5 to mM
Summary
Christian Johannes Schipp+,[a] Ying Ma+,[b] Ammar Al-Shameri,[c] Federico D’Alessio,[d]. It has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies For this reason, extensive genetic code re-engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. Extensive genetic code re-engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors.
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