Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstr. 15, D-85748 Garching, Germany.
Institute of Bioprocess Engineering, Friedrich-Alexander-University Erlangen-Nürnberg, Paul-Gordan-Str. 3, D-91052 Erlangen, Germany.
Molecules. 2019 Jul 12;24(14):2550. doi: 10.3390/molecules24142550.
(2,5)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of ()-carvone. ( ) cells overexpressing an ene reductase from 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 cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM ()-carvone. 287 mM (2,5)-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.
(2,5)-二氢蒈烯是一种工业应用的构建块，可以通过对（）-蒈烯的位点选择性和立体选择性 C=C 键生物还原来合成。（）细胞过表达来自 sp. PCC7120 的烯还原酶（NostocER1），并结合共底物再生系统，被证明是该反应非常有效的生物催化剂。然而，生物催化剂的工业适用性与其催化剂的活性密切相关。由于细胞内 NADH 的浓度比 NADPH 的浓度高约 20 倍，我们生产了 细胞，其中优先接受 NADPH 的 NostocER1 被三种不同的 NADH 接受 NostocER1 突变体取代。这些全细胞生物催化剂在 0.7 l 规模的分批搅拌釜反应器中用于还原 300 mM（）-蒈烯。在 5 h 内形成了 287 mM 的（2,5）-二氢蒈烯，非对映过量值为 95.4%，收率为 95.6%。因此，通过使用 NADH 接受酶，全细胞生物催化剂得到了显著改善，与文献相比，初始产物形成速率提高了 2.1 倍，时空产率提高了 1.8 倍。
