Cell-Free Biosynthesis of ω-Hydroxy Acids Boosted by a Synergistic Combination of Alcohol Dehydrogenases

The activity orchestration of an unprecedented cell-free enzyme system with self-sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into omega-hydroxy acids at the expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell-free system reached up to 100% conversion using 100 mM of linear C(s )diols and performed the desymmetrization of prochiral branched diols into the corresponding omega-hydroxy acids with an exquisite enantioselectivity (ee 99%). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5-hydroxypetanoic acid. Finally, the cell-free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as omega-hydroxy acids.

Automated summary

In this study, a cell-free enzyme system with self-sufficient cofactor recycling was used for efficient transformation of aliphatic diols into omega-hydroxy acids. Two compatible alcohol dehydrogenases were selected as specialist biocatalysts to maximize the efficiency of the biosynthetic route. The cell-free system achieved high conversion rates and exhibited excellent enantioselectivity. It also demonstrated superior sustainability compared to traditional metal catalysts and whole cells. Moreover, the system allowed for enzyme reutilization.