TY - JOUR
T1 - Synthetic RNA switches for yeast metabolic engineering
T2 - Screening recombinant enzyme libraries
AU - Michener, Joshua K.
AU - Smolke, Christina D.
N1 - Publisher Copyright:
© Springer Science+Business Media, LLC 2014.
PY - 2014
Y1 - 2014
N2 - Directed evolution is a powerful technique for increasing the activity of poorly active enzymes, for example when an enzyme is engineered to accept a new substrate or function in a new environment. Since enzyme activity greatly depends on the enzyme environment, screening should be performed under the same conditions as the ultimate application of the enzyme. When an enzyme will be used in live cells, RNA biosensors offer a powerful and fl exible method of linking the desired phenotype, production of a small molecule of interest, to an easily measured phenotype, such as fl uorescence. Here, we describe methods for screening enzyme libraries using an RNA biosensor, showing examples from the evolution of a P450 monooxygenase.
AB - Directed evolution is a powerful technique for increasing the activity of poorly active enzymes, for example when an enzyme is engineered to accept a new substrate or function in a new environment. Since enzyme activity greatly depends on the enzyme environment, screening should be performed under the same conditions as the ultimate application of the enzyme. When an enzyme will be used in live cells, RNA biosensors offer a powerful and fl exible method of linking the desired phenotype, production of a small molecule of interest, to an easily measured phenotype, such as fl uorescence. Here, we describe methods for screening enzyme libraries using an RNA biosensor, showing examples from the evolution of a P450 monooxygenase.
KW - Directed evolution
KW - High-throughput enzyme screening
KW - P450
KW - RNA switch
UR - http://www.scopus.com/inward/record.url?scp=84921424977&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-0563-8_7
DO - 10.1007/978-1-4939-0563-8_7
M3 - Article
C2 - 24744030
AN - SCOPUS:84921424977
SN - 1064-3745
VL - 1152
SP - 125
EP - 136
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -