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 KI 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.

中文翻译：

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通过丙氨酸扫描和定点饱和诱变相结合提高DszC的底物耐受性。

生物脱硫4S途径可以特异性地使芳族S杂环化合物脱硫（很难通过加氢脱硫来脱硫），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途径中的第一个酶，受到反馈抑制和底物抑制。该研究是进一步修饰DszC突变体AKWC以提高其对DBT的耐受性的首次尝试。在DszC突变体AKWC的二聚体表面上进行了丙氨酸扫描，与BAD（AKWC）菌株相比，BAD（AKWCP413A）菌株的HBP产量增加。对AKWC的第413个氨基酸进行了定点饱和诱变，突变体AKWCPI的底物抑制参数KI值是AKWC的5.6倍。当DBT浓度为0.25 mM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。DszC的蛋白质工程克服了反馈抑制作用后，进一步提高了底物的耐受性，为分析DszC底物的抑制机理提供了参考。DszC有益突变体的过表达也大大提高了脱硫效率。与BL21（DE3）/ BADC * + C *菌株相比，是4倍。DszC的蛋白质工程克服了反馈抑制作用后，进一步提高了底物的耐受性，为分析DszC底物的抑制机理提供了参考。DszC有益突变体的过表达也大大提高了脱硫效率。与BL21（DE3）/ BADC * + C *菌株相比，是4倍。DszC的蛋白质工程克服了反馈抑制作用后，进一步提高了底物的耐受性，为分析DszC底物的抑制机理提供了参考。DszC有益突变体的过表达也大大提高了脱硫效率。