Abstract

Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. colimetabolites into ​4-methyl-pentanol (​4MP) via ​coenzyme A (​CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for ​4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a ​carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from ​glucose demonstrates selective carbon chain extension and acid reduction with ​4MP constituting 81% (90±7 mg l−1) of the observed alcohol products. The highest observed ​4MP titre is 192±23 mg l−1. These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.