Assil-Companioni Leen, Büchsenschütz Hanna C, Solymosi Dániel, Dyczmons-Nowaczyk Nina G, Bauer Kristin K F, Wallner Silvia, Macheroux Peter, Allahverdiyeva Yagut, Nowaczyk Marc M, Kourist Robert
Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
ACS Catal. 2020 Oct 16;10(20):11864-11877. doi: 10.1021/acscatal.0c02601. Epub 2020 Sep 4.
Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h L was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.
重组蓝藻中的光驱动生物催化提供了高度原子高效的辅因子再生光合作用，从而缓解了与牺牲性共底物相关的限制。然而，尽管光生物催化剂具有显著的比活性，但在中高细胞密度下的自我遮光限制了异源酶促反应的时空产率。此外，将人工电子受体有效整合到蓝藻电子途径的严格调控网络中可能极具挑战性。在这里，我们使用依赖NADPH的烯还原酶YqjM对2-甲基马来酰亚胺进行C=C键还原作为光依赖生物转化的模型反应。时间分辨NADPH荧光光谱法可以直接监测细胞内YqjM的活性，并揭示不同基因构建体之间NADPH稳态水平和氧化动力学的差异。这种效应与全细胞的比活性相关，全细胞的转化率>99%。通过使黄素二铁蛋白（Flv1/Flv3）失活，进一步将电子导向异源YqjM，在中等细胞密度下比活性提高了2倍，从而阐明了通过去除天然竞争性电子受体来加速光驱动生物转化的可能性。在最佳情况下，初始产物形成速率达到18.3 mmol h L，可在4小时内将60 mM底物溶液完全转化。
