AUTHOR=Krahn Irene , Bonder Daniel , Torregrosa-Barragán Lucía , Stoppel Dominik , Krause Jens P. , Rosenfeldt Natalie , Meiswinkel Tobias M. , Seibold Gerd M. , Wendisch Volker F. , Lindner Steffen N. TITLE=Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=9 YEAR=2021 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.669093 DOI=10.3389/fbioe.2021.669093 ISSN=2296-4185 ABSTRACT=

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.