Two Biotechnological Approaches to the Preparative Synthesis of Natural Dihydrocoumarin

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the enzyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not exceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared recombinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield.

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

Cascading Old Yellow Enzyme, Alcohol Dehydrogenase and Glucose Dehydrogenase for Selective Reduction of (E/Z)-Citral to (S)-Citronellol

Citronellol is a kind of unsaturated alcohol with rose-like smell and its (S)-enantiomer serves as an important intermediate for organic synthesis of (-)-cis-rose oxide. Chemical methods are commonly used for the synthesis of citronellol and its (S)-enantiomer, which suffers from severe reaction conditions and poor selectivity. Here, the first one-pot double reduction of (E/Z)-citral to (S)-citronellol was achieved in a multi-enzymatic cascade system: N-ethylmaleimide reductase from Providencia stuartii (NemR-PS) was selected to catalyze the selective reduction of (E/Z)-citral to (S)-citronellal, alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) performed the further reduction of (S)-citronellal to (S)-citronellol, meanwhile a variant of glucose dehydrogenase from Bacillus megaterium (BmGDHM6), together with glucose, drove efficient NADPH regeneration. The Escherichia coli strain co-expressing NemR-PS, YsADH, and BmGDHM6 was successfully constructed and used as the whole-cell catalyst. Various factors were investigated for achieving high conversion and reducing the accumulation of the intermediate (S)-citronellal and by-products. 0.4 mM NADP+ was essential for maintaining high catalytic activity, while the feeding of the cells expressing BmGDHM6 effectively eliminated the intermediate and by-products and shortened the reaction time. Under optimized conditions, the bio-transformation of 400 mM citral caused nearly complete conversion (>99.5%) to enantio-pure (S)-citronellol within 36 h, demonstrating promise for industrial application.

Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

<a>2-Arylpropionic acid </a><a>derivatives</a>, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of<a> 2-arylacrylic acid</a> derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of <a>2-arylacrylic acid derivatives</a> by either ERED wild-types or variants resulted solely in nonpharmacological (<i>R</i>)-enantiomers as the products. <a></a><a>Here, </a>we present the engineering of <i>Saccharomyces pastorianus</i> old yellow enzyme 1 (OYE1) into (<i>S</i>)-stereoselective enzymes, which afford pharmacologically active (<i>S</i>)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-<i>S</i> model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (<i>S</i>)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (<i>S</i>)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (<i>S</i>)-derivatives including (<i>S</i>)-ibuprofen and (<i>S</i>)-naproxen methyl esters with up to >99% <i>ee</i> values. <a>Moreover, the key residues and mutation strategy obtained from OYE1 </a>could be further transferred to OYE 2.6 (from <i>Pichia stipitis</i>) and KnOYE1 (from <i>Kazachstania naganishii</i>) to create the (<i>S</i>)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (<i>S</i>)-profens.