Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion

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.

Keywords

Formolase / Dihydroxyacetone / C1 bioconversion / Carbon fixation / Synthetic methylotrophy

References

[1] Cai T, Sun H, Qiao J, Zhu L, Zhang F, Zhang J, Tang Z, Wei X, Yang J, Yuan Q, Wang W, Yang X, Chu H, Wang Q, You C, Ma H, Sun Y, Li Y, Li C, Jiang H, Wang Q, Ma Y. Cell-free chemoenzymatic starch synthesis from carbon dioxide. Science, 2021, 373: 1523-1527.

[2] Case DA, Aktulga HM, Belfon K, Ben-Shalom I, Brozell SR, Cerutti DS, Cheatham TE, Cruzeiro VWD, Darden TA, Duke RE. Amber 2021, 2021, San Francisco: University of California.

[3] Chen J, Wang Y, Zheng P, Sun J. Engineering synthetic auxotrophs for growth-coupled directed protein evolution. Trends Biotechnol, 2022, 40: 773-776.

[4] Darden T, York D. An N· log (N) method for Ewald sums in large systems. J Chem Phys, 1993, 98: 10089-10092.

[5] Guan A, Hou Y, Yang R, Qin J. Enzyme engineering for functional lipids synthesis: recent advance and perspective. Bioresour Bioprocess, 2024, 11: 1.

[6] Hann EC, Overa S, Harland-Dunaway M, Narvaez AF, Le DN, Orozco-Cárdenas ML, Jiao F, Jinkerson RE. A hybrid inorganic–biological artificial photosynthesis system for energy-efficient food production. Nat Food, 2022, 3: 461-471.

[7] Hu G, Li Z, Ma D, Ye C, Zhang L, Gao C, Liu L, Chen X. Light-driven CO₂ sequestration in Escherichia coli to achieve theoretical yield of chemicals. Nat Catal, 2021, 4: 395-406.

[8] Jakalian A, Jack DB, Bayly CI. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation. J Comput Chem, 2002, 23: 1623-1641.

[9] Larini L, Mannella R, Leporini D. Langevin stabilization of molecular-dynamics simulations of polymers by means of quasisymplectic algorithms. J Chem Phys, 2007, 126: 104101.

[10] Li T, Tang Z, Wei H, Tan Z, Liu P, Li J, Zheng Y, Lin J, Liu W, Jiang H, Liu H, Zhu L, Ma Y. Totally atom-economical synthesis of lactic acid from formaldehyde: combined bio-carboligation and chemo-rearrangement without the isolation of intermediates. Green Chem, 2020, 22: 6809-6814.

[11] Liu Z, Shi S, Ji Y, Wang K, Tan T, Nielsen J. Opportunities of CO₂-based biorefineries for production of fuels and chemicals. Green Carbon, 2023, 1: 75-84.

[12] Lu X, Liu Y, Yang Y, Wang S, Wang Q, Wang X, Yan Z, Cheng J, Liu C, Yang X, Luo H, Yang S, Gou J, Ye L, Lu L, Zhang Z, Guo Y, Nie Y, Lin J, Li S, Tian C, Cai T, Zhuo B, Ma H, Wang W, Ma Y, Liu Y, Li Y, Jiang H. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nat Commun, 2019, 10: 1378.

[13] Qian J, Fan L, Yang J, Feng J, Gao N, Cheng G, Pu W, Zhou W, Cai T, Li S, Zheng P, Sun J, Wang D, Wang Y. Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde. Synth Syst Biotechnol, 2023, 8: 386-395.

[14] Qiao Y, Ma W, Zhang S, Guo F, Liu K, Jiang Y, Wang Y, Xin F, Zhang W, Jiang M. Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: advances, challenge and perspectives. Synth Syst Biotechnol, 2023, 8: 578-583.

[15] Ryckaert J-P, Ciccotti G, Berendsen HJ. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys, 1977, 23: 327-341.

[16] Siegel JB, Smith AL, Poust S, Wargacki AJ, Bar-Even A, Louw C, Shen BW, Eiben CB, Tran HM, Noor E, Gallaher JL, Bale J, Yoshikuni Y, Gelb MH, Keasling JD, Stoddard BL, Lidstrom ME, Baker D. Computational protein design enables a novel one-carbon assimilation pathway. Proc Natl Acad Sci U S A, 2015, 112: 3704-3709.

[17] Tan C, Xu P, Tao F. Carbon-negative synthetic biology: challenges and emerging trends of cyanobacterial technology. Trends Biotechnol, 2022, 40: 1488-1502.

[18] Wang J, Wang W, Kollman PA, Case DA. Antechamber: an accessory software package for molecular mechanical calculations. J Am Chem Soc, 2001, 222: 2001.

[19] Wang Y, Tao F, Ni J, Li C, Xu P. Production of C3 platform chemicals from CO₂ by genetically engineered cyanobacteria. Green Chem, 2015, 17: 3100-3110.

[20] Wang X, Wang Y, Liu J, Li Q, Zhang Z, Zheng P, Lu F, Sun J. Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway. Bioresour Bioprocess, 2017, 4: 41.

[21] Wang Y, Fan L, Tuyishime P, Zheng P, Sun J. Synthetic methylotrophy: a practical solution for methanol-based biomanufacturing. Trends Biotechnol, 2020, 38: 650-666.

[22] Wu S, Bornscheuer U T. A chemoenzymatic cascade with the potential to feed the world and allow humans to live in space. Eng Microbiol, 2022, 2: 100006.

[23] Yang J, Sun S, Men Y, Zeng Y, Zhu Y, Sun Y, Ma Y. Transformation of formaldehyde into functional sugars via multi-enzyme stepwise cascade catalysis. Catal Sci Technol, 2017, 7: 3459-3463.

[24] Yang J, Song W, Cai T, Wang Y, Zhang X, Wang W, Chen P, Zeng Y, Li C, Sun Y, Ma Y. De novo artificial synthesis of hexoses from carbon dioxide. Sci Bull, 2023, 68: 2370-2381.

[25] Yang J, Fan L, Cheng G, Cai T, Sun J, Zheng P, Li S, Wang Y. Engineering of cofactor preference and catalytic activity of methanol dehydrogenase by growth-coupled directed evolution. Green Carbon, 2024

[26] Zhang Z, Wang Y, Zheng P, Sun J. Promoting lignin valorization by coping with toxic C1 byproducts. Trends Biotechnol, 2021, 39: 331-335.

[27] Zhang J, Liu D, Liu Y, Chu H, Bai J, Cheng J, Zhao H, Fu S, Liu H, Fu Y, Ma Y, Jiang H. Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide. Green Chem, 2023, 25: 3247-3255.

[28] Zheng T, Zhang M, Wu L, Guo S, Liu X, Zhao J, Xue W, Li J, Liu C, Li X, Jiang Q, Bao J, Zeng J, Yu T, Xia C. Upcycling CO₂ into energy-rich long-chain compounds via electrochemical and metabolic engineering. Nat Catal, 2022, 5: 388-396.

Funding

National Natural Science Foundation of China(32222004)

Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110201)