title: Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.
abstract: 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.
authors.affiliation: Institute of Bio- and Geosciences 1: Biotechnology, Forschungszentrum Jülich , D-52428 Jülich, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Schendzielorz
authors.forename: Martin
authors.initials: M
authors.lastname: Dippong
authors.forename: Alexander
authors.initials: A
authors.lastname: Grünberger
authors.forename: Dietrich
authors.initials: D
authors.lastname: Kohlheyer
authors.forename: Ayako
authors.initials: A
authors.lastname: Yoshida
authors.forename: Stephan
authors.initials: S
authors.lastname: Binder
authors.forename: Chiharu
authors.initials: C
authors.lastname: Nishiyama
authors.forename: Makoto
authors.initials: M
authors.lastname: Nishiyama
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
authors.forename: Lothar
authors.initials: L
authors.lastname: Eggeling
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001222:Aspartate Kinase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000378:metabolism*; D005434:Flow Cytometry; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006639:Histidine; D001120:Arginine; D001276:ATP Phosphoribosyltransferase; D017851:Phosphotransferases (Carboxyl Group Acceptor); D001222:Aspartate Kinase; C018721:acetylglutamate kinase; D008239:Lysine