Enzyme engineering for functional lipids synthesis: recent advance and perspective

Guan, Ailin; Hou, Yue; Yang, Run; Qin, Jiufu

doi:10.1186/s40643-023-00723-7

Cited by 3 publications

(1 citation statement)

References 87 publications

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“…Directed evolution is a powerful strategy for obtaining desired enzyme variants based on random mutation and activity screening (Chen et al 2022 ; Guan et al 2024 ). However, considering that the theoretical library size of randomly mutated FLS is extremely large (528 20 =2.8 × 10 54 ), it is impractical to cover every possible mutation during library construction and screening.…”

Section: Resultsmentioning

confidence: 99%

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

Cheng,

Sun,

Wang

et al. 2024

Bioresour. Bioprocess.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Cheng,

Sun,

Wang

et al. 2024

Bioresour. Bioprocess.

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Recent insight into the advances and prospects of microbial lipases and their potential applications in industry

Eskandari,

Leow,

Rahman

et al. 2024

Int Microbiol

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Simultaneous high-throughput particle-bacteria separation and solution exchange via in-plane and out-of-plane parallelization of microfluidic centrifuges

Norouzy,

Zabihihesari,

Rezai

2024

Biomicrofluidics

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Inertial microfluidic devices have gained attention for point-of-need (PoN) sample preparation. Yet, devices capable of simultaneous particle-bacteria solution exchange and separation are low in throughput, hindering their applicability to PoN settings. This paper introduces a microfluidic centrifuge for high-throughput solution exchange and separation of microparticles, addressing the need for processing large sample volumes at elevated flow rates. The device integrates Dean flow recirculation and inertial focusing of microparticles within 24 curved microchannels assembled in a three-layer configuration via in-plane and out-of-plane parallelization. We studied solution exchange and particle migration using singleplex and duplex samples across devices with varying curve numbers (2-curve, 8-curve, and 24-curve). Processing 5 and 10 μm microparticles at flow rates up to 16.8 ml/min achieved a solution exchange efficiency of 96.69%. In singleplex solutions, 10 and 5 μm particles selectively migrated to inner and outer outlets, demonstrating separation efficiencies of 99.7% and 90.3%, respectively. With duplex samples, sample purity was measured to be 93.4% and 98.6% for 10 and 5 μm particles collected from the inner and the outer outlets, respectively. Application of our device in biological assays was shown by performing duplex experiments where 10 μm particles were isolated from Salmonella bacterial suspension with purity of 97.8% while increasing the state-of-the-art particle solution exchange and separation throughput by 16 folds. This parallelization enabled desirable combinations of high throughput, low-cost, and scalability, without compromising efficiency and purity, paving the way for sample preparation at the PoN in the future.

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Enzyme engineering for functional lipids synthesis: recent advance and perspective

Cited by 3 publications

References 87 publications

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

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

Recent insight into the advances and prospects of microbial lipases and their potential applications in industry

Simultaneous high-throughput particle-bacteria separation and solution exchange via in-plane and out-of-plane parallelization of microfluidic centrifuges

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