Wissner Julian L, Ludwig Julian, Escobedo-Hinojosa Wendy, Hauer Bernhard
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
MethodsX. 2020 Nov 13;7:101143. doi: 10.1016/j.mex.2020.101143. eCollection 2020.
-Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of -dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant , harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the catalyzed degradation of -dihydrodiendiols into catechols dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant platforms pursuing the production of -dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in BW25113, catalyzing the degradation of -dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a -dihydrodiendiol of choice.•Novel screening strategy for BW25113 dehydrogenase knock-outs, incapable of degrading -dihydrodiendiols was validated for -1,2-dihydrocatechol as substrate•Corresponding knock-outs can be used for recombinant production of -dihydrodiendiols•Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD).
-二氢二醇是有价值的化合物，在有机化学中作为手性合成子有多种应用。生成-二氢二醇的生物技术路线涉及芳香族化合物的二羟基化反应，由 Rieske 非血红素铁双加氧酶催化。迄今为止，文献中可以找到许多携带此类双加氧酶的重组体的例子。然而，仅有少数出版物涉及-二氢二醇被脱氢酶催化降解为儿茶酚的情况。识别并消除这种脱氢酶催化的降解对于建立旨在生产-二氢二醇的增强型重组平台至关重要。在此，我们提供了一种快速简便的策略，用于鉴定大肠杆菌 BW25113 中催化-二氢二醇降解为儿茶酚的混杂醇脱氢酶。该方法基于对脱氢酶缺陷型 KEIO 菌株的筛选，考察它们降解所选-二氢二醇的能力。•针对以-1,2-二氢儿茶酚为底物，验证了大肠杆菌 BW25113 脱氢酶敲除体不能降解-二氢二醇的新型筛选策略•相应的敲除体可用于重组生产-二氢二醇•基于配备二极管阵列检测器的液相色谱法（HPLC-DAD）进行简单分析。
