Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases.

Christoph Mähler,Franziska Kratzl,Kathrin Castiglione,Dirk Weuster-Botz,Christian Burger

doi:10.3390/molecules24142550

Christoph Mähler, Franziska Kratzl + Show 3 more

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https://doi.org/10.3390/molecules24142550

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Abstract

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts’ activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.

Highlights

Biocatalysts may represent a green and sustainable alternative to chemical catalysts. They can be used under mild conditions and are biodegradable [1]
An additional T7 promotor as well as a ribosome binding site were inserted for an improved co-expression of the genes encoding for NostocER1 and the formate dehydrogenase (FDH) from
Mycobacterium vaccae, which was used for cosubstrate regeneration [19]

Summary

Introduction

Biocatalysts may represent a green and sustainable alternative to chemical catalysts. The application of wild type biocatalysts for industrial purposes is often not cost-effective, due to low activities and space-time yields [1,3]. This can be circumvented by the usage of recombinant microorganisms and optimized enzymes [3]. The asymmetric reduction of C=C double bonds is a widely employed reaction, since up to two stereogenic centers can be generated [4] In this context, ene reductases (ERs) from the old yellow enzyme family (OYE, EC 1.6.99.1) represent highly interesting biocatalysts for anti-specific hydrogenations of activated alkenes [5]. Large-scale production processes with OYEs are cost-intensive and only applicable when products of high value are produced, e.g., active pharmaceutical compounds

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