Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering DOI Creative Commons
Yijie Deng,

Kai Etheridge,

Xinping Ran

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 20, 2024

ABSTRACT Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection screening. To address these challenges, we developed a growth-coupled continuous (GCCDE) approach, enabling automated efficient enzyme engineering. By linking activity to bacterial growth utilizing the MutaT7 system, GCCDE combines in vivo mutagenesis high-throughput of superior variants single process. validate this evolved thermostable CelB enhance β-galactosidase at lower temperatures while maintaining thermal stability. was coupled E. coli , allowing with improved metabolize lactose more efficiently promote faster minimal medium. Using culture achieved real-time over 10 9 per culture. Integrating vitro further increased genetic diversity, yielding significantly enhanced low-temperature compared wild type preserving thermostability. DNA sequencing identified key mutations likely responsible for substrate binding catalytic turnover. This approach broadly applicable optimizing diverse enzymes, demonstrating potential industrial research applications. IMPORTANCE Enzyme engineering aims develop enzymes or novel traits, but traditional methods are slow require repetitive manual steps. study presents faster, protein approach. We utilized an technique, tools, induce living bacteria established direct link between growth. A setup used enable better-performing variants. Bacteria grew selecting without intervention. method, engineered better performance high-temperature The adaptable many enzymes. It offers solution system enables simultaneously, showing power advance

Language: Английский

In vivo directed evolution of an ultra-fast RuBisCO from a semi-anaerobic environment imparts oxygen resistance DOI Creative Commons
Julie L. McDonald,

Nathan P. Shapiro,

Amanuella A. Mengiste

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 17, 2025

Abstract Carbon dioxide (CO 2 ) assimilation by the enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) underpins biomass accumulation in photosynthetic bacteria and eukaryotes. Despite its pivotal role, Rubisco has a slow carboxylation rate is competitively inhibited oxygen (O ). These traits impose limitations on efficiency, making compelling target for improvement. Interest Form II from Gallionellaceae bacteria, which comprise dimer or hexamer of large subunits, arises their nearly 5-fold higher than average enzyme. As well as having fast (25.8 s − 1 at 25 °C), we show that (GWS1B) extremely sensitive to O inhibition, consistent with evolution under semi-anaerobic environments. We therefore used novel vivo mutagenesis-mediated screening pipeline evolve GWS1B over six rounds oxygenic selection, identifying three catalytic point mutants improved ambient efficiency; Thr-29-Ala (T29A), Glu-40-Lys (E40K) Arg-337-Cys (R337C). Full kinetic characterization showed each substitution enhanced CO affinity conditions subduing affinity, leading 25% (E40K), 11% (T29A) 8% (R337C) enhancements efficiency °C. By contrast, near anaerobic natural environment , mutant was impaired ∼16%. findings demonstrate efficacy artificial directed access regions space Rubisco. Significance Given Rubisco’s crucial role carbon assimilation, addressing inhibition significant challenge. Utilizing one fastest known, yet also highly oxygen-sensitive, – applied Escherichia coli discover mutations specifically enhance oxygen, condition distinct Gallionellaceae’s environment. Our underscore potential unlock new capabilities Rubisco, implications both fundamental research practical agricultural applications.

Language: Английский

Citations

0
Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering DOI Creative Commons
Yijie Deng,

Kai Etheridge,

Xinping Ran

et al.

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

Published: March 27, 2025

ABSTRACT Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection screening. To address these challenges, we developed a growth-coupled continuous (GCCDE) approach, enabling automated efficient enzyme engineering. By linking activity to bacterial growth utilizing the MutaT7 system, GCCDE combines in vivo mutagenesis high-throughput of superior variants single process. validate this evolved thermostable CelB from Pyrococcus furiosus enhance its β-galactosidase at lower temperatures while maintaining thermal stability. was coupled E. coli , allowing with improved utilize lactose more efficiently promote faster minimal medium. Using culture achieved simultaneous real-time over 10⁹ per culture. Integrating vitro further increased genetic diversity, yielding significantly enhanced low-temperature compared wild type preserving thermostability. DNA sequencing identified key mutations likely responsible for substrate binding catalytic turnover. This approach broadly applicable optimizing diverse enzymes, demonstrating potential industrial research applications. IMPORTANCE Enzyme engineering aims develop enzymes or novel traits, but traditional methods are slow require repetitive manual steps. study presents faster, protein approach. We utilized an technique, tools, induce living bacteria established direct link between growth. A setup enables better-performing real time. Bacteria grew selecting without intervention. method, engineered better performance combining process, system bypasses cycles error-prone PCR, transformation, Our adaptable various providing solution

Language: Английский

Citations

0
T7 RNA Polymerase-Guided Base Editor for Accelerated Continuous Evolution in Bacillus subtilis DOI Creative Commons

Bin Wang,

Yaokang Wu, Xueqin Lv

et al.

Synthetic and Systems Biotechnology, Journal Year: 2025, Volume and Issue: 10(3), P. 876 - 886

Published: April 21, 2025

Language: Английский

Citations

0
Continuous Evolution of Protein through T7 RNA Polymerase-Guided Base Editing in Corynebacterium glutamicum DOI
Qing Wang,

Jiajia You,

Yichen Li

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 19, 2024

In vivo targeted mutagenesis technologies are the basis for continuous directed evolution of specific proteins. Here, an efficient system (CgMutaT7) gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers build CgMutaT7 system, which introduces mutations regions controlled by promoter. After a series optimizations, resulting (CgMutaT74) can increase mutant frequency target 1.12 × 104-fold, with low off-target frequency. Subsequently, high-throughput sequencing further revealed that CgMutaT74 performs uniform C → T transitions at least 1.8 kb DNA region. Finally, xylose isomerase successfully continuously evolved improve utilization, indicating has great potential applications protein function expression components.

Language: Английский

Citations

1
Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering DOI Creative Commons
Yijie Deng,

Kai Etheridge,

Xinping Ran

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 20, 2024

ABSTRACT Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection screening. To address these challenges, we developed a growth-coupled continuous (GCCDE) approach, enabling automated efficient enzyme engineering. By linking activity to bacterial growth utilizing the MutaT7 system, GCCDE combines in vivo mutagenesis high-throughput of superior variants single process. validate this evolved thermostable CelB enhance β-galactosidase at lower temperatures while maintaining thermal stability. was coupled E. coli , allowing with improved metabolize lactose more efficiently promote faster minimal medium. Using culture achieved real-time over 10 9 per culture. Integrating vitro further increased genetic diversity, yielding significantly enhanced low-temperature compared wild type preserving thermostability. DNA sequencing identified key mutations likely responsible for substrate binding catalytic turnover. This approach broadly applicable optimizing diverse enzymes, demonstrating potential industrial research applications. IMPORTANCE Enzyme engineering aims develop enzymes or novel traits, but traditional methods are slow require repetitive manual steps. study presents faster, protein approach. We utilized an technique, tools, induce living bacteria established direct link between growth. A setup used enable better-performing variants. Bacteria grew selecting without intervention. method, engineered better performance high-temperature The adaptable many enzymes. It offers solution system enables simultaneously, showing power advance

Language: Английский

Citations

0