title: Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.
abstract: A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.
authors.affiliation: Degussa AG, Feed Additives, R&D Biotechnology, Kantstr. 2, D-33790 Halle, Westf., Germany. achim.marx@degussa.com
authors.forename: Achim
authors.initials: A
authors.lastname: Marx
authors.forename: Stephan
authors.initials: S
authors.lastname: Hans
authors.forename: Bettina
authors.initials: B
authors.lastname: Möckel
authors.forename: Brigitte
authors.initials: B
authors.lastname: Bathe
authors.forename: Albert A
authors.initials: AA
authors.lastname: de Graaf
authors.forename: Ashling C
authors.initials: AC
authors.lastname: McCormack
authors.forename: Cliona
authors.initials: C
authors.lastname: Stapleton
authors.forename: Kevin
authors.initials: K
authors.lastname: Burke
authors.forename: Michael
authors.initials: M
authors.lastname: O'Donohue
authors.forename: L K
authors.initials: LK
authors.lastname: Dunican
mesh_terms: D002455:Cell Division / Q000502:physiology; D003352:Corynebacterium / Q000166:cytology / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics* / Q000378:metabolism*; D008239:Lysine / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010101:Oxygen Consumption; D010641:Phenotype; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D005956:Glucose-6-Phosphate Isomerase; D008239:Lysine