caa a22 4500 60550508X CHVBK 20210128100620.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00253-014-6109-5 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6109-5 The contest for precursors: channelling l -isoleucine synthesis in Corynebacterium glutamicum without byproduct formation [Elektronische Daten] [Michael Vogt, Karin Krumbach, Won-Gi Bang, Jan van Ooyen, Stephan Noack, Bianca Klein, Michael Bott, Lothar Eggeling] l-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of l-lysine and l-threonine, and four enzymes of l-isoleucine synthesis have an enlarged substrate specificity involved also in l-valine and l-leucine synthesis. As a consequence, constructing a strain specifically overproducing l-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on l-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53mM l-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94mM l-isoleucine with l-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109mM l-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188mol l-isoleucine (mol glucose)−1 which characterizes it as one of the best l-isoleucine producers available and which does not contain plasmids. Springer-Verlag Berlin Heidelberg, 2014 Genomic mutations nationallicence Activity control nationallicence Promoter mutations nationallicence Branched-chain amino acids nationallicence Flux directioning nationallicence Byproduct reduction nationallicence Enzyme quantification nationallicence Vogt Michael Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Krumbach Karin Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Bang Won-Gi Department of Agricultural Chemistry, College of Life and Environmental Sciences, Korea University, Seoul, Sungbook-gu, South Korea aut van Ooyen Jan Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Noack Stephan Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Klein Bianca Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Bott Michael Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Eggeling Lothar Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/2(2015-01-01), 791-800 0175-7598 99:2<791 2015 99 253 https://doi.org/10.1007/s00253-014-6109-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6109-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Vogt Michael Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Krumbach Karin Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Bang Won-Gi Department of Agricultural Chemistry, College of Life and Environmental Sciences, Korea University, Seoul, Sungbook-gu, South Korea aut NATIONALLICENCE 700 1- van Ooyen Jan Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Noack Stephan Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Klein Bianca Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Bott Michael Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 700 1- Eggeling Lothar Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/2(2015-01-01), 791-800 0175-7598 99:2<791 2015 99 253