Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics

R G Rosenthal; X Diana Zhang; K Ilić Đurđić; J J Collins; D A Weitz

doi:10.1002/anie.202303112

Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics

Angew Chem Int Ed Engl. 2023 Jun 12;62(24):e202303112. doi: 10.1002/anie.202303112. Epub 2023 May 3.

Authors

R G Rosenthal^{1

2}, X Diana Zhang¹, K Ilić Đurđić^{1

3

4}, J J Collins^{2

3

5

6

7

8}, D A Weitz^{1

3

9}

Affiliations

¹ School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
² Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
³ Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA.
⁴ University of Belgrade-Faculty of Chemistry Studentski trg 12-16, 11000, Belgrade, Serbia.
⁵ Department of Biological Engineering, Massachusetts Institute of Technology 77 Massachusetts Ave, Cambridge, MA 02139, USA.
⁶ Synthetic Biology Center, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
⁷ Harvard-MIT Program in Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
⁸ Broad Institute of MIT and Harvard, 415 Main St, Cambridge, MA 02142, USA.
⁹ Department of Physics, Harvard University, Cambridge, MA 02138, USA.

PMID: 37019845
DOI: 10.1002/anie.202303112

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.

Keywords: CRISPR-Directed Evolution; Continuous Evolution; Droplet Microfluidics; Protein Engineering; Sustainable Chemistry.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Catalysis
Directed Molecular Evolution*
Gene Library
High-Throughput Screening Assays
Humans
Microfluidics*
Substrate Specificity