Whole-cell biocatalysis

Genetic incorporation of noncanonical amino acids (ncAAs) harbouring catalytic side chains into proteins allows the creation of enzymes able to catalyse reactions that have no equivalent in nature. Here, we present for the first time the use of the ncAA 3-aminotyrosine (aY) as catalytic residue in a designer enzyme for iminium activation catalysis. Incorporation of aY into protein scaffold LmrR gave rise to an artificial Friedel-Crafts (FC) alkylase exhibiting complementary enantioselectivity to a previous FC-alkylase design using p-aminophenylalanine as catalytic residue in the same protein. The new FC-alkylase was optimized by directed evolution to afford a quadruple mutant that showed increased activity and excellent enantioselectivity (up to 95% ee). X-ray crystal structures of the parent and evolved designer enzymes suggest that the introduced mutations cause a narrowing of the active site and a reorientation of the catalytic -NH2 group. Furthermore, the evolved FC-alkylase was applied in whole-cell catalysis, facilitated by the straightforward incorporation of aY. Our work demonstrates that aY is a valuable addition to the biochemists toolbox for creating artificial enzymes. 

Combining a natural decarboxylase and an artificial metathase, a microbial cell factory is created that enables the synthesis of cycloalkenes from fatty diacids in a whole-cell hybrid biocatalytic cascade process.

We report in vivo biocatalytic cascade reactions comprising a combination of canonical enzyme-catalysed reactions with an artificial-enzyme-catalysed new-to-nature reaction. The artificial enzyme contains a genetically encoded unnatural catalytic residue, which catalyses the formation of a hydrazone product from biosynthetically produced benzaldehydes in E. coli. 

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

The approach of combining enzymatic and transition‐metal catalysis has been focused almost exclusively on using purified, isolated enzymes. The use of whole‐cell biocatalysis, instead of isolated enzymes, with transition‐metal catalysis, however, has been investigated only sparsely, to date. Herein we present the development of two transition‐metal catalyzed reactions used to derivatize styrene obtained from whole‐cell biosynthesis. Using a biocompatible ruthenium cross‐metathesis catalyst up to 1.5 mM stilbene could be obtained in the presence of E. coli , which simultaneously produced styrene. Using palladium catalysts and arylboronic acids, titers of up to 1 mM of several stilbene derivatives were obtained. These two transition‐metal catalyzed reactions are valuable additions to the toolbox of combined whole‐cell biocatalysis and transition‐metal catalysis, offering the possibility to supplement biosynthetic pathways with the chemical versatility of abiological transition metal catalysis.