title: Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.
abstract: The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction.
authors.affiliation: The National Food Institute, Technical University of Denmark, Kongens Lyngby 2800, Denmark.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: The National Food Institute, Technical University of Denmark, Kongens Lyngby 2800, Denmark.
authors.forename: Siu Hung Joshua
authors.initials: SHJ
authors.lastname: Chan
authors.affiliation: iAMB - Institute of Applied Microbiology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
authors.forename: Suresh
authors.initials: S
authors.lastname: Sudarsan
authors.affiliation: iAMB - Institute of Applied Microbiology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
authors.affiliation: The National Food Institute, Technical University of Denmark, Kongens Lyngby 2800, Denmark. Electronic address: perj@food.dtu.dk.
authors.forename: Peter Ruhdal
authors.initials: PR
authors.lastname: Jensen
authors.affiliation: The National Food Institute, Technical University of Denmark, Kongens Lyngby 2800, Denmark. Electronic address: chso@food.dtu.dk.
authors.forename: Christian
authors.initials: C
authors.lastname: Solem
mesh_terms: D001682:Biological Availability; D053898:Biosynthetic Pathways / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000502:physiology*; D005632:Fructose / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology; D018390:Gene Targeting / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008957:Models, Genetic; D009249:NADP / Q000096:biosynthesis* / Q000235:genetics*; D009876:Operon / Q000235:genetics*
chemical_list: D005632:Fructose; D009249:NADP
keywords: Convergent evolution; Corynebacterium glutamicum; Fructose operon; Hexose phosphates; NADPH; Transcriptomics