Cell-free biosynthesis of ω-hydroxy acids boosted by a synergistic combination of alcohol dehydrogenases

15 November 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

The activity orchestration of an unprecedented cell-free enzyme system with self-sufficient cofactor recycling enables the step-wise transformation of aliphatic diols into -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 C5 diols, and performed the dessymetrization of prochiral branched diols into the corresponding -hydroxy acids with an exquisite enantioselectivity (ee > 99%). Green metrics demostrate a superior sustanability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5-hydroxy petanoic 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 -hydroxy acids.

Supplementary materials

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Supporting Information
Description
Supporting information contains NMR, HPLC, GC-MS data, tables with enzyme activities, reaction courses for cascade catalyzed with different multi-enzyme systems using different activity ratios.
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