1. Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion
- Author
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Guimin Cheng, Hongbing Sun, Qian Wang, Jinxing Yang, Jing Qiao, Cheng Zhong, Tao Cai, and Yu Wang
- Subjects
Formolase ,Dihydroxyacetone ,C1 bioconversion ,Carbon fixation ,Synthetic methylotrophy ,Technology ,Chemical technology ,TP1-1185 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde or C3 dihydroxyacetone (DHA). FLS lays the foundation for several artificial carbon fixation and valorization pathways, such as the artificial starch anabolic pathway. However, the application of FLS is limited by its low catalytic activity and product promiscuity. Findings FLS, designed and engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected as a candidate for modification. To evaluate its catalytic activity, 25 residues located within an 8 Å distance from the active center were screened using single-point saturation mutagenesis. A screening approach based on the color reaction of the DHA product was applied to identify the desired FLS variants. After screening approximately 5,000 variants (approximately 200 transformants per site), several amino acid sites that were not identified by directed evolution were found to improve DHA formation. The serine-to-phenylalanine substitution at position 236 improved the activity towards DHA formation by 7.6-fold. Molecular dynamics simulations suggested that the mutation increased local hydrophobicity at the active site, predisposing the cofactor-C2 intermediate to nucleophilic attack by the third formaldehyde molecule for subsequent DHA generation. Conclusions This study provides improved FLS variants and valuable information into the influence of residues adjacent to the active center affecting catalytic efficiency, which can guide the rational engineering or directed evolution of FLS to optimize its performance in artificial carbon fixation and valorization.
- Published
- 2024
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