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  • In this study we have developed and investigated three NADPH


    In this study we have developed and investigated three NADPH-regenerating fusion partners with two different enzyme systems: a BVMO and an ADH. With the exception of one fusion construct (StGDH with LbADH), all fusion L-685,458 resulted in good soluble expression as well as fully functional as self-sufficient biocatalysts. The fusion biocatalysts displayed tolerance to various organic cosolvents, including DES. From the different fusion partners, FDH was the most tolerant to organic solvents, fully converting cyclohexanone for the BVMO fusion at 15% v/v MTBE, and acetophenone for the ADH fusion even at 40% v/v MTBE. We also demonstrate that the fusion enzymes can be used as enantioselective biocatalysts for a large variety of reactions by retaining almost all catalytic characteristics of the native non-fused enzymes. We demonstrated the applicability of three different NADPH regenerating fusions for these two enzyme systems, which broadens the choice of regenerating enzymes for future applications of these systems.
    Experimental section
    Acknowledgements The research for this work has received funding from the European Union (EU) project ROBOX (grant agreement n°635734) under EU’s Horizon 2020 Program Research and Innovation actions H2020-LEIT BIO-2014-1. The views and opinions expressed in this article are only those of the authors and do not necessarily reflect those of the European Union Research Agency. The European Union is not liable for any use that may be made L-685,458 of the information contained herein. Financial support from the Spanish Ministry of Economy and Competitiveness (MEC, Projects CTQ2013-44153-P and CTQ2016-75752-R) is gratefully acknowledged. Á.M.-I thanks MEC for a predoctoral fellowship inside the FPI program.
    Introduction Sterols are an abundant source of steroids in nature and a large variety of microorganisms are able to transform them, either partially, or completely to carbon dioxide and water. One such sterol is cholesterol (1 in Fig. 1). Its complex chemical structure requires the concerted action of a large number of enzymes to completely degrade it. The occurrence of genes coding for cholesterol-degrading enzymes in several bacterial and fungal genome sequences [1], indicates that cholesterol degradation pathways may be active in a variety of microorganisms. A typical bacterial cholesterol degradation pathway is presented in Fig. 1. Generally, the pathway is supposed to start with the oxidation or dehydrogenation of cholesterol (1) to 5-cholesten-3-one (59; Fig. 2), followed by isomerization to 4-cholesten-3-one (2). Under aerobic conditions, this transformation is catalyzed by oxygen-dependent bifunctional cholesterol oxidases/isomerases or 3β-hydroxysteroid dehydrogenases/isomerases [1,2,3,4], but under anaerobic conditions anoxic bifunctional cholesterol dehydrogenase/isomerase enzymes take care of the conversion [5,6]. Under aerobic conditions, the degradation of the eight-carbon aliphatic side chain of cholesterol is initiated with the hydroxylation of the C26 or C27 atom by the cytochrome P450 monooxygenase Cyp125 [7,8] or Cyp142 [9], followed by oxidation of the hydroxyl group to a carboxylate by the same enzyme. The resulting C26- or C27-carboxylate intermediate is subsequently activated as its coenzyme A (CoA) derivative by an ATP-dependent steroid-CoA ligase [10,11]. The release of the side chain has been elucidated biochemically to proceed through three cycles of a process similar to the β-oxidation of fatty acids, yielding the nineteen-carbon steroid core intermediate, e.g. 4-androstene-3,17-dione (AD; 8), by releasing successively propionic acid, acetic acid, and another propionic acid [12,13]. Under anaerobic conditions, bacteria use a similar route to degrade the side chain [14]. However, the degradation is initiated by hydroxylation of 4-cholesten-3-one (2) at C25, instead of at C26 or C27, to yield 25-hydroxy-4-cholesten-3-one (16), by an oxygen-independent hydroxylase using a water molecule as the oxygen donor [5,6], and subsequent isomerization to form 27-hydroxy-4-cholesten-3-one (3) [14]. The degradation of the steroid nucleus is primed with the introduction of the 1(2)-double bond into the steroid ring system (see below). The 1(2)-unsaturated intermediate then follows either the 9,10-seco pathway for aerobic degradation (magenta arrows in Fig. 1) or the 2,3-seco pathway for anaerobic degradation (blue arrows in Fig. 1). More detailed information on microbial cholesterol degradation can be found elsewhere [1,2,3].