Protein engineering of a sulfotransferase and a monooxygenase for the synthesis of high-value chemicals
Ji, Yu; Schwaneberg, Ulrich (Thesis advisor); Elling, Lothar (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2020
The push for sustainability (less waste and less resource use) is a global issue related to environmental, economic, and social concerns. Enzymes as biodegradable catalysts play an important role in the development of sustainable technologies. Enzymatic catalysis, a safer process compared to chemical catalysis occurs usually with high catalytic efficiency and specificity under mild reaction conditions using less hazardous and less toxic chemicals. In this thesis, two different classes of enzymes (transferases and oxidoreductases) were engineered for the detoxification of phenolic compounds and production of biodegradable surfactants, respectively. Catechols (extensively used in the chemical industry) are abundantly found in the environment and cause human health problems, e.g., vasoconstriction, renal tube degeneration, liver malfunction, and neurodegeneration. Sulfation is an important way for detoxification of xenobiotics and endobiotics including catechols. Metabolites such as catechol sulfates act in our body as important antioxidants and often have anti-inflammatory properties. Moreover, they are used as biomarkers to monitor diseases such as Parkinson’s disease, cardiovascular diseases, urinary tract infections, candidemia, and various forms of cancer. Because of their high biological relevance, there is an increasing interest in the synthesis of catechol sulfates. Enzymatic conversion of catechols using aryl sulfotransferase is a synthetically attractive, environmentally-friendly, and sustainable route for regio-/chemoselective sulfation of catechols. A two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Optimization yielded a coefficient of variation below 15 % for the p-NPS-4-AAP screening system. The validation of the established screening system was accomplished by directed aryl sulfotransferase evolution toward 3-chlorocatechol, which yielded the beneficial variant ASTB-M5 (V430A) and showed up to 2.4-fold increased turnover number and a significantly improved sulfate stoichiometry of ASTB-M5 (from 29 % to 58 %). The reengineering of loop12 and loop13 of aryl sulfotransferase B (ASTB) was performed in order to improve the sulfate transfer efficiency of six catechols. The obtained ASTB variants were generated in a KnowVolution campaign using the random mutagenesis method SeSaM and the multi-site saturation method OmniChange. The catalytic activity (kcat) and catalytic efficiency (kcat/KM) of the final variant ASTB-OM2 (Q191Y/Y218W/L225V) were improved for all six investigated catechols when compared to ASTB-WT (e.g., 13.6-fold improvement of kcat for 3-bromocatechol). HPLC-MS analysis confirmed the improved sulfate stoichiometry of ASTB-OM2 with a transfer efficiency of up to 94 % for 3-methylcatechol in comparison to 24 % for ASTB-WT. A molecular understanding of the improved sulfation activity of ASTB-OM2 was achieved through molecular docking studies and electron effects of catechol substituents were analyzed by the Hammett plot. The final variant ASTB-OM2 was also applied for the sulfation of important pharmaceutical compounds. Cetyl-trimethylammonium bromide (CTAB) is a widely used cationic surfactant in nature. Apart from compound expenses, the key parameters for usage of cationic surfactants in the industry are biodegradability and surfactant solubility. CTAB biodegradation requires hydroxylation in the first step, which is rate-limiting and crucial for solubility in water. Nowadays bolaform surfactants (such as hydroxylated CTAB), consist of two hydrophilic groups that are separated by a hydrophobic spacer chain, attracted the interest of industry and academia due to their reduced micelle size and excellent solubilization properties. Enzymatic hydroxylation of CTAB by monooxygenase P450 BM3 is a sustainable route for biodegradable bolaform surfactant hydroxylated CTAB synthesis. The OmniChange multi-site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity toward the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188). In total, 1740 clones from the P450 BM3 OmniChange library were screened with the NADPH depletion assay and H2O2 detection assay. Several improved P450 BM3 variants were identified and finally, four were kinetically characterized with respect to CTAB hydroxylation based on both performance and coupling efficiency. The P450 BM3 variant P3A8 (R47E/Y51M/F87V/L188E) displayed an initial activity (64.9 ± 4.8 s-1, 13.5-fold increased activity compared to wild-type P450 BM3) which is in the range of the specific activity for its natural fatty acid substrate (palmitic acid, 32-122 s-1) and it showed high coupling efficiency (92.5 %), while wild-type P450 BM3 displayed a very low coupling efficiency (0.5 %). HPLC-MS/MS detection confirmed that P3A8 and P2E7 (R47D/Y51L/F87V/L188A) form 13 and 35 times more 2-hydroxylated CTAB than P450 BM3. In addition, di-hydroxylated CTAB products were detected for all four investigated P450 BM3 variants (up to a yield of 77 %; P3A8).
- Department of Biology 
- Chair of Biotechnology