Combining an artificial metathase with a fatty acid decarboxylase in a whole cell for cycloalkene synthesis

Artificial metalloenzymes (ArMs) offer powerful tools to catalyse new-to-nature reactions. Accordingly, ArMs offer great potential to complement natural enzymes in concurrent cascade reactions. For in cellulo applications, however, the abundance of thiols in the cytosol of aerobic organisms limits the use of ArMs that rely on precious-metal cofactors. To overcome this limitation, compartmentalization of ArMs either in the periplasm or on the surface of Escherichia coli has shown promise. Here we report on our efforts to combine a surface-displayed artificial metathase with UndB, an inner-membrane decarboxylase. The resulting concurrent cascade converts medium-chain dicarboxylates to cyclic alkenes. To optimize the cascade, we evolved both enzymes independently and fine-tuned their expression levels. Compared with the wild-type whole-cell enzyme cascade, the yield of the engineered strain was improved by >75-fold for the conversion of sebacic acid to cyclohexene. Combining natural and artificial metalloenzymes thus offers a promising strategy for whole-cell biocatalysis and synthetic biology to catalyse new-to-nature concurrent cascade reactions.