Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion DOI Creative Commons
Guimin Cheng, Hongbing Sun, Qian Wang

et al.

Bioresources and Bioprocessing, Journal Year: 2024, Volume and Issue: 11(1)

Published: May 12, 2024

Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde C3 dihydroxyacetone (DHA). FLS lays foundation for several artificial carbon fixation and valorization pathways, such as starch anabolic pathway. However, application limited by its low catalytic activity product promiscuity. FLS, engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected candidate modification. To evaluate activity, 25 residues located within an 8 Å distance active center were screened using single-point saturation mutagenesis. A screening approach color reaction DHA applied to identify desired variants. After approximately 5,000 variants (approximately 200 transformants per site), amino acid sites not identified directed evolution found improve formation. The serine-to-phenylalanine substitution at position 236 improved towards formation 7.6-fold. Molecular dynamics simulations suggested mutation increased local hydrophobicity site, predisposing cofactor-C2 intermediate nucleophilic attack third molecule subsequent generation. This study provides valuable information influence adjacent affecting efficiency, which can guide rational engineering optimize performance in valorization.

Language: Английский

Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering of Clostridium ljungdahlii DOI Creative Commons
Junzhe Zhang, Yuzhen Li,

Zhi-Ning Xi

et al.

Applied and Environmental Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: March 26, 2025

ABSTRACT Clostridium ljungdahlii , as a model acetogen strain, represents novel platform for biotechnological production CO 2 fixation. The genome of C. harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions these GRM identified inducible promoters, P choline1 was induced by choline, while 1,2-PD 1,2-PD. Subsequently, highly expressed tightly controlled were applied improve CRISPR/Cas9 editing tools. Specifically, used develop efficient knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency multiple genes, including pyrE pduS aor2 eutT . On other hand, cas9 integrated downstream into genome. efficiently mediated introducing plasmids containing gRNA cassette along relevant homology arms. exemplified construction Δbdh::pdc where 2,3-butanediol dehydrogenase replaced pyruvate decarboxylase from Zymomonas mobilis 3-HB Syn KI artificial 3-hydroxybutyric acid synthesis pathway inserted highlights effectiveness convenience systems, thereby enriching CRISPR/Cas toolkit acetogens. IMPORTANCE A genetic constitutive promoter has been developed precise However, its hindered toxicity resulting expression large plasmids, leading low overall success rate. Inducible allow transcription target genes be switched off presence or absence inducers, have broad range applications. In this study, we identify promoters apply them enhance improved tools facilitate high efficiency, potentially playing significant advancing research metabolic engineering

Language: Английский

Citations

0
Hydrophobic microenvironment around atomic Fe sites for enhanced C–H bond oxidation of aromatic alkanes DOI
Zhenmei Zhang,

Jingwen Bai,

Wengang Liu

et al.

Science China Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 26, 2025

Language: Английский

Citations

0
Molecular Engineering and Utilization of Cyclohexanone Monooxygenase in the Cascade Biocatalytic Synthesis of ε-Caprolactone DOI

Yingjiao Zhang,

Jialong Zhang,

Shengliang Cai

et al.

ACS Sustainable Chemistry & Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: April 22, 2025

Language: Английский

Citations

0
Bacillus methanolicus: an emerging chassis for low-carbon biomanufacturing DOI
Xueting Yang, Zhaojuan Zheng, Yu Wang

et al.

Trends in biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: July 1, 2024

Language: Английский

Citations

2
Scanning the active center of formolase to identify key residues for enhanced C1 to C3 bioconversion DOI Creative Commons
Guimin Cheng, Hongbing Sun, Qian Wang

et al.

Bioresources and Bioprocessing, Journal Year: 2024, Volume and Issue: 11(1)

Published: May 12, 2024

Formolase (FLS) is a computationally designed enzyme that catalyzes the carboligation of two or three C1 formaldehyde molecules into C2 glycolaldehyde C3 dihydroxyacetone (DHA). FLS lays foundation for several artificial carbon fixation and valorization pathways, such as starch anabolic pathway. However, application limited by its low catalytic activity product promiscuity. FLS, engineered based on benzoylformate decarboxylase from Pseudomonas putida, was selected candidate modification. To evaluate activity, 25 residues located within an 8 Å distance active center were screened using single-point saturation mutagenesis. A screening approach color reaction DHA applied to identify desired variants. After approximately 5,000 variants (approximately 200 transformants per site), amino acid sites not identified directed evolution found improve formation. The serine-to-phenylalanine substitution at position 236 improved towards formation 7.6-fold. Molecular dynamics simulations suggested mutation increased local hydrophobicity site, predisposing cofactor-C2 intermediate nucleophilic attack third molecule subsequent generation. This study provides valuable information influence adjacent affecting efficiency, which can guide rational engineering optimize performance in valorization.

Language: Английский

Citations

1