title: Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.
abstract: Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Chen
authors.forename: Weiqian
authors.initials: W
authors.lastname: Meyer
authors.forename: Sugima
authors.initials: S
authors.lastname: Rappert
authors.forename: Jibin
authors.initials: J
authors.lastname: Sun
authors.forename: An-Ping
authors.initials: AP
authors.lastname: Zeng
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001222:Aspartate Kinase / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008239:Lysine / Q000096:biosynthesis*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011995:Recombination, Genetic
chemical_list: D050505:Mutant Proteins; D001222:Aspartate Kinase; D008239:Lysine