title: Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production--Strategies for the elimination of feedback inhibition.
abstract: L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog β-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches.
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Kulis-Horn
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Persicke
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Electronic address: Joern.Kalinowski@CeBiTec.Uni-Bielefeld.DE.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D025461:Feedback, Physiological / Q000502:physiology*; D006639:Histidine / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000069456:Silent Mutation / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006639:Histidine; D001276:ATP Phosphoribosyltransferase
keywords: ATP-phosphoribosyl transferase; Chromosomal overexpression; Domain deletion; Feedback deregulation; Synonymous mutation; l-Histidine biosynthesis