TY  - JOUR
AU  - Rosenthal, R. G.
AU  - Diana Zhang, X.
AU  - Ilić Đurđić, Karla
AU  - Collins, J. J.
AU  - Weitz, D. A.
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6255
AB  - Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.
PB  - John Wiley and Sons Inc
T2  - Angewandte Chemie International Edition
T1  - Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics
VL  - 62
IS  - 24
SP  - e202303112
DO  - 10.1002/anie.202303112
ER  - 

@article{
author = "Rosenthal, R. G. and Diana Zhang, X. and Ilić Đurđić, Karla and Collins, J. J. and Weitz, D. A.",
year = "2023",
abstract = "Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.",
publisher = "John Wiley and Sons Inc",
journal = "Angewandte Chemie International Edition",
title = "Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics",
volume = "62",
number = "24",
pages = "e202303112",
doi = "10.1002/anie.202303112"
}

Rosenthal, R. G., Diana Zhang, X., Ilić Đurđić, K., Collins, J. J.,& Weitz, D. A.. (2023). Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics. in Angewandte Chemie International Edition
John Wiley and Sons Inc., 62(24), e202303112.
https://doi.org/10.1002/anie.202303112

Rosenthal RG, Diana Zhang X, Ilić Đurđić K, Collins JJ, Weitz DA. Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics. in Angewandte Chemie International Edition. 2023;62(24):e202303112.
doi:10.1002/anie.202303112 .

Rosenthal, R. G., Diana Zhang, X., Ilić Đurđić, Karla, Collins, J. J., Weitz, D. A., "Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics" in Angewandte Chemie International Edition, 62, no. 24 (2023):e202303112,
https://doi.org/10.1002/anie.202303112 . .