Engineering of a borneol dehydrogenase from P. putida for the enzymatic resolution of camphor

Benjamin Begander,Michael Hofer,Volker Sieber,Robert Kourist,Julia Diener

doi:10.1007/s00253-021-11239-5

Abstract

Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (−)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (−)-camphor are limited, and the oxidation of precious (−)-borneol would be too costly. Hence, synthesis of (−)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (−)-camphor with an ee of > 99%.Key points• Characterization of a novel borneol dehydrogenase (BDH) from P. putida.• Development of enantioselective BDH variants for the reduction of camphor.• Enzymatic kinetic resolution of camphor with borneol dehydrogenase.Graphical abstract

Highlights

Terpenes are hydrocarbons classified by the number of isoprene units (C5H8), ranging from hemiterpenes to polyterpenes, andAppl Microbiol Biotechnol (2021) 105:3159–3167Selescu et al 2013; Sherkheli et al 2013); a simple and efficient approach for the separation of camphor racemate is highly desirable
The best enzyme variant was used for the catalytic separation of a camphor racemate (Fig. 1)
Point mutations are well known for influencing protein solubility (Smialowski et al 2007; Tian et al 2010; Klesmith et al 2017), which might be the reason for the good solubility of borneol dehydrogenase (BDH) from Pseudomonas sp

Summary

Introduction

Terpenes are hydrocarbons classified by the number of isoprene units (C5H8), ranging from hemiterpenes (one isoprene unit) to polyterpenes (more than eight isoprene units), andAppl Microbiol Biotechnol (2021) 105:3159–3167Selescu et al 2013; Sherkheli et al 2013); a simple and efficient approach for the separation of camphor racemate is highly desirable. No camphor-specific alcohol dehydrogenase is available; several borneol dehydrogenases (BDH) are known. The most active BDH enzymes to date are not from plant but have a bacterial origin and are the non-stereoselective borneol dehydrogenases from Pseudomonas sp. Besides BDH enzymes, only a tropinone reductase from Cochlearia officialis showed activity in oxidation of (−)-borneol as well as reduction of (+)-camphor (Reinhardt et al 2014). None of these enzymes has shown activity or selectivity in the reduction of (+/−)-camphor that would suffice for the synthesis of optically pure (−)-camphor. The best enzyme variant was used for the catalytic separation of a camphor racemate (Fig. 1)

Methods

Results

Conclusion