posted on 2014-01-17, 00:00authored byGeorg Schendzielorz, Martin Dippong, Alexander Grünberger, Dietrich Kohlheyer, Ayako Yoshida, Stephan Binder, Chiharu Nishiyama, Makoto Nishiyama, Michael Bott, Lothar Eggeling
Enzymes
initiating the biosynthesis of cellular building blocks
are frequently inhibited by the end-product of the respective pathway.
Here we present an approach to rapidly generate sets of enzymes overriding
this control. It is based on the in vivo detection
of the desired end-product in single cells using a genetically encoded
sensor. The sensor transmits intracellular product concentrations
into a graded optical output, thus enabling ultrahigh-throughput screens
by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened
the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six
of the resulting N-acetyl-l-glutamate kinase
proteins were further developed and characterized and found to be
at least 20-fold less sensitive toward l-arginine inhibition
than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A
in C. glutamicumΔargR led
to the accumulation of 34 mM l-arginine in the culture medium.
We also screened mutant libraries of lysC-encoded
aspartate kinase and hisG-encoded ATP phosphoribosyltransferase.
We isolated 11 LysC muteins, enabling up to 45 mM l-lysine
accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in
vivo screening of enzyme libraries by using metabolite sensors
is extremely well suited to identify high-performance muteins required
for overproduction.