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

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.

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

“…Autonomous biosynthesis of ncAAs and their concurrent incorporation into enzyme of interest in vivo could significantly reduce the production cost and permeability issues ( Pagar et al, 2021 ). Using enzymatic and metabolic pathways, some ncAAs like p -amino-phenylalanine, 5-hydroxytryptophan, l -phosphothreonine, l -dihydroxyphenylalanine, fluorotyrosine, and S -allylcysteine were biosynthesized in E. coli and concurrently incorporated into target proteins ( Ma et al, 2014 ; Exner et al, 2017 ; Kim et al, 2018 ; Won et al, 2019a ; Nojoumi et al, 2019 ; Schipp et al, 2020 ). The production of an increasing number of ncAAs in engineered cells will be aided by advances in biochemistry, molecular biology, and synthetic biology.…”

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

“…Similarly, Nojoumi and co-workers reported the biosynthesis of S -allyl- l -homocysteine from allyl mercaptan, coupled with incorporating the ncAA into proteins by SPI. Di Salvo and collaborators in a series of papers − could show how the “hijacking” of methionine biosynthesis of E. coli by introducing acetylated homoserine (two genes for direct sulfhydration from Corynebacterium ) can be used for the in situ production of different ncAAs. TPL-catalyzed biosynthesis and concurrent incorporation of L-DOPA via SCS and SPI and fluorotyrosines via SPI have also recently been achieved.…”

Functionalization of Oxide‐Free Silicon Surfaces for Biosensing Applications

“…However, also by applying recently developed protocols of metabolic pathway engineering in bacteria, unnatural azide-functionalized amino acids can be inserted at specific positions with high precision. [113,114] This scalable method is a convenient way to functionalize proteins and allows covalent immobilization of biomolecules at a specific position without side reactions. This technology was further developed so that L-azidohomoalanine can be directly produced from sodium azide and is efficiently and specifically incorporated into a recombinant model protein.…”