Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
J Ind Microbiol Biotechnol. 2020 May;47(4-5):395-402. doi: 10.1007/s10295-020-02274-8. Epub 2020 Apr 18.
The biodesulfurization 4S pathway can specifically desulfurize an aromatic S heterocyclic compound (which is difficult to desulfurize by hydrodesulfurization) and maintain the integrity of its combustion value. The four Dsz enzymes in the pathway convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). DszC is the first enzyme in the 4S pathway and is subject to feedback inhibition and substrate inhibition. This study is the first attempt to further modify the DszC mutant AKWC to improve its tolerance to DBT. Alanine scanning was performed on the dimeric surface of the DszC mutant AKWC, and the HBP yield of the BAD (AKWCP413A) strain was increased compared to the BAD (AKWC) strain. Site-directed saturation mutagenesis was performed on the 413th amino acid of AKWC, and the substrate inhibition parameter K value of the mutant AKWCPI was 5.6 times higher than that of AKWC. When the DBT concentration was 0.25 mM, the HBP production of the recombinant strain overexpressing AKWCPI was increased by approximately 1.4-fold compared to the BL21(DE3)/BADC*+C* strain. The protein engineering of DszC further improved the substrate tolerance after overcoming the feedback inhibition, which provided a reference for the analysis of the inhibition mechanism of DszC substrate. Overexpression of DszC-beneficial mutants also greatly improved the efficiency of desulfurization.
生物脱硫 4S 途径可以特异性脱硫芳族 S 杂环化合物（其难以通过加氢脱硫脱硫）并保持其燃烧值的完整性。该途径中的四种 Dsz 酶将模型化合物二苯并噻吩（DBT）转化为无硫化合物 2-羟基联苯（HBP）。DszC 是 4S 途径中的第一酶，受反馈抑制和底物抑制的影响。本研究首次尝试进一步修饰 DszC 突变体 AKWC，以提高其对 DBT 的耐受性。对 DszC 突变体 AKWC 的二聚体表面进行丙氨酸扫描，与 BAD（AKWC）菌株相比，BAD（AKWCP413A）菌株的 HBP 产量增加。对 AKWC 的第 413 位氨基酸进行定点饱和突变，突变体 AKWCPI 的底物抑制参数 K 值比 AKWC 高 5.6 倍。当 DBT 浓度为 0.25mM 时，与 BL21(DE3)/BADC*+C*菌株相比，过表达 AKWCPI 的重组菌株的 HBP 产量增加了约 1.4 倍。克服反馈抑制后，DszC 的蛋白质工程进一步提高了底物耐受性，为分析 DszC 底物抑制机制提供了参考。DszC 有益突变体的过表达也大大提高了脱硫效率。
