Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.
The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.
Département de Chimie, Université de Montréal, Montréal, QC, Canada.|Center for Green Chemistry and Catalysis (CGCC), Université de Montréal, Montréal, QC, Canada.|PROTEO, The Québec Network for Research on Protein Function, Engineering and Applications, Québec, QC, Canada.
Daniela
http://orcid.org/0000-0003-3756-3782
D
Quaglia
Center for Green Chemistry and Catalysis (CGCC), Université de Montréal, Montréal, QC, Canada.|PROTEO, The Québec Network for Research on Protein Function, Engineering and Applications, Québec, QC, Canada.|Département de Biochimie, Université de Montréal, Montréal, QC, Canada.
Maximilian C C J C
MC
Ebert
DSM Nutritional Products, 101 Research Drive, Dartmouth, NS, Canada.
Paul F
PF
Mugford
Département de Chimie, Université de Montréal, Montréal, QC, Canada.|Center for Green Chemistry and Catalysis (CGCC), Université de Montréal, Montréal, QC, Canada.|PROTEO, The Québec Network for Research on Protein Function, Engineering and Applications, Québec, QC, Canada.|Département de Biochimie, Université de Montréal, Montréal, QC, Canada.
Joelle N
JN
Pelletier
D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013329:Structure-Activity Relationship; D058615:Synthetic Biology / Q000379:methods
D004798:Enzymes