Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH
Abstract
Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error-prone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (kcat/Km) for 2,6-dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (∼105 M–1 s–1). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-β-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme–hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.
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- Ruihua Zhang, Chenyi Li, Jian Wang, Yajun Yan. Microbial Ligninolysis: Toward a Bottom-Up Approach for Lignin Upgrading. Biochemistry 2019, 58 (11) , 1501-1510. https://doi.org/10.1021/acs.biochem.8b00920
- Elena Fernández-Fueyo, Irene Davó-Siguero, David Almendral, Dolores Linde, Maria Camilla Baratto, Rebecca Pogni, Antonio Romero, Victor Guallar, Angel T. Martínez. Description of a Non-Canonical Mn(II)-Oxidation Site in Peroxidases. ACS Catalysis 2018, 8 (9) , 8386-8395. https://doi.org/10.1021/acscatal.8b02306
- Giang-Son Nguyen, Anna Sofia Lewin, Francesca Di Bartolomeo, Alexander Wentzel. Recent Advances in Enzymatic Conversion of Lignin to Value Added Products. 2021,,, 439-471. https://doi.org/10.1007/978-3-030-58315-6_14
- Stefan Hofbauer, Vera Pfanzagl, Hanna Michlits, Daniel Schmidt, Christian Obinger, Paul G. Furtmüller. Understanding molecular enzymology of porphyrin-binding α + β barrel proteins - One fold, multiple functions. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2021, 1869 (1) , 140536. https://doi.org/10.1016/j.bbapap.2020.140536
- Célica Cagide, Susana Castro-Sowinski. Technological and biochemical features of lignin-degrading enzymes: a brief review. Environmental Sustainability 2020, 3 (4) , 371-389. https://doi.org/10.1007/s42398-020-00140-y
- Xiaopeng Wang, Lu Lin, Jizhong Zhou. Links among extracellular enzymes, lignin degradation and cell growth establish the models to identify marine lignin‐utilizing bacteria. Environmental Microbiology 2020, 9 https://doi.org/10.1111/1462-2920.15289
- A.O. Falade, T.C. Ekundayo. Emerging biotechnological potentials of DyP‐type peroxidases in remediation of lignin wastes and phenolic pollutants: a global assessment (2007–2019). Letters in Applied Microbiology 2020, 13 https://doi.org/10.1111/lam.13392
- Vivek Chauhan, Shamsher S. kanwar. Impact of Industrial Dyes on the Environment and Bacterial Peroxidase Isolated from Bacillus sp. BTS-P5 as a Possible Solution. Current Biotechnology 2020, 9 (1) , 45-56. https://doi.org/10.2174/2211550109666200303110926
- L. E. Khmelevtsova, I. S. Sazykin, T. N. Azhogina, M. A. Sazykina. Prokaryotic Peroxidases and Their Application in Biotechnology (Review). Applied Biochemistry and Microbiology 2020, 56 (4) , 373-380. https://doi.org/10.1134/S0003683820030059
- Catarina Barbosa, Célia M. Silveira, Diogo Silva, Vânia Brissos, Peter Hildebrandt, Lígia O. Martins, Smilja Todorovic. Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing. Biosensors and Bioelectronics 2020, 153 , 112055. https://doi.org/10.1016/j.bios.2020.112055
- Timothy D.H. Bugg, James J. Williamson, Goran M.M. Rashid. Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass. Current Opinion in Chemical Biology 2020, 55 , 26-33. https://doi.org/10.1016/j.cbpa.2019.11.007
- Muhammad Bilal, Hafiz M. N. Iqbal. Ligninolytic Enzymes Mediated Ligninolysis: An Untapped Biocatalytic Potential to Deconstruct Lignocellulosic Molecules in a Sustainable Manner. Catalysis Letters 2020, 150 (2) , 524-543. https://doi.org/10.1007/s10562-019-03096-9
- Abdulrahman H. A. Alessa, Kang Lan Tee, David Gonzalez-Perez, Hossam E. M. Omar Ali, Caroline A. Evans, Alex Trevaskis, Jian-He Xu, Tuck Seng Wong. Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY). Bioresources and Bioprocessing 2019, 6 (1) https://doi.org/10.1186/s40643-019-0255-7
- Siseon Lee, Minsik Kang, Jung-Hoon Bae, Jung-Hoon Sohn, Bong Hyun Sung. Bacterial Valorization of Lignin: Strains, Enzymes, Conversion Pathways, Biosensors, and Perspectives. Frontiers in Bioengineering and Biotechnology 2019, 7 https://doi.org/10.3389/fbioe.2019.00209
- Can Liu, Hong Yuan, Fei Liao, Chuan-Wan Wei, Ke-Jie Du, Shu-Qin Gao, Xiangshi Tan, Ying-Wu Lin. Unique Tyr-heme double cross-links in F43Y/T67R myoglobin: an artificial enzyme with a peroxidase activity comparable to that of native peroxidases. Chemical Communications 2019, 55 (46) , 6610-6613. https://doi.org/10.1039/C9CC02714A
- Chonlong Chio, Mohini Sain, Wensheng Qin. Lignin utilization: A review of lignin depolymerization from various aspects. Renewable and Sustainable Energy Reviews 2019, 107 , 232-249. https://doi.org/10.1016/j.rser.2019.03.008
- Rahman Rahman Pour, Austine Ehibhatiomhan, Yuling Huang, Ben Ashley, Goran M. Rashid, Sharon Mendel-Williams, Timothy D.H. Bugg. Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates. Enzyme and Microbial Technology 2019, 123 , 21-29. https://doi.org/10.1016/j.enzmictec.2019.01.002
- Naofumi Kamimura, Shingo Sakamoto, Nobutaka Mitsuda, Eiji Masai, Shinya Kajita. Advances in microbial lignin degradation and its applications. Current Opinion in Biotechnology 2019, 56 , 179-186. https://doi.org/10.1016/j.copbio.2018.11.011
- Chenxian Yang, Fangfang Yue, Yanlong Cui, Yuanmei Xu, Yuanyuan Shan, Bianfang Liu, Yuan Zhou, Xin Lü. Biodegradation of lignin by Pseudomonas sp. Q18 and the characterization of a novel bacterial DyP-type peroxidase. Journal of Industrial Microbiology & Biotechnology 2018, 45 (10) , 913-927. https://doi.org/10.1007/s10295-018-2064-y
- Rupam Sarma, Md. Islam, Mark Running, Dibakar Bhattacharyya. Multienzyme Immobilized Polymeric Membrane Reactor for the Transformation of a Lignin Model Compound. Polymers 2018, 10 (4) , 463. https://doi.org/10.3390/polym10040463
- Lei Wang, Yongmei Chen, Shuangyan Liu, Haomin Jiang, Linan Wang, Yanzhi Sun, Pingyu Wan. Study on the cleavage of alkyl-O-aryl bonds by in situ generated hydroxyl radicals on an ORR cathode. RSC Advances 2017, 7 (81) , 51419-51425. https://doi.org/10.1039/C7RA11236J