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

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.

Powering Artificial Enzymatic Cascades with Electrical Energy.

We have developed a scalable platform that employs electrolysis for an in vitro synthetic enzymatic cascade in a continuous flow reactor. Both H2 and O2 were produced by electrolysis and transferred through a gas-permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H2 and O2 served as electron mediators for the biocatalysts. We demonstrate the production of methylated N-heterocycles from diamines with up to 99 % product formation as well as excellent regioselective labeling with stable isotopes. Our platform can be applied for a broad panel of oxidoreductases to exploit electrical energy for the synthesis of fine chemicals.

Hydrogenase-based oxidative biocatalysis without oxygen.

Biocatalysis-based synthesis can provide a sustainable and clean platform for producing chemicals. Many oxidative biocatalytic routes require the cofactor NAD+ as an electron acceptor. To date, NADH oxidase (NOX) remains the most widely applied system for NAD+ regeneration. However, its dependence on O2 implies various technical challenges in terms of O2 supply, solubility, and mass transfer. Here, we present the suitability of a NAD+ regeneration system in vitro based on H2 evolution. The efficiency of the hydrogenase-based system is demonstrated by integrating it into a multi-enzymatic cascade to produce ketoacids from sugars. The total NAD+ recycled using the hydrogenase system outperforms NOX in all different setups reaching up to 44,000 mol per mol enzyme. This system proves to be scalable and superior to NOX in terms of technical simplicity, flexibility, and total output. Furthermore, the system produces only green H2 as a by-product even in the presence of O2.

H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions.

The soluble hydrogenase from Ralstonia eutropha provides an atom efficient regeneration system for reduced flavin cofactors using H2 as an electron source. We demonstrated this system for highly selective ene-reductase-catalyzed C[double bond, length as m-dash]C-double bond reductions and monooxygenase-catalyzed epoxidation. Reactions were expanded to aerobic conditions to supply H2O2 for peroxygenase-catalyzed hydroxylations.

Correction: H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions.

Correction for 'H2 as a fuel for flavin- and H2O2-dependent biocatalytic reactions' by Ammar Al-Shameri et al., Chem. Commun., 2020, DOI: 10.1039/d0cc03229h.