Fermentative production of lysine by Corynebacterium glutamicum: transmembrane transport and metabolic flux analysis

2005 ◽  
Vol 40 (2) ◽  
pp. 499-508 ◽  
Author(s):  
Theodora Tryfona ◽  
Mark T. Bustard
Keyword(s):  
Corynebacterium Glutamicum ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Transmembrane Transport ◽  
Flux Analysis ◽  
Fermentative Production

scholarly journals Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose

2004 ◽  
Vol 70 (1) ◽  
pp. 229-239 ◽  
Author(s):  
Patrick Kiefer ◽  
Elmar Heinzle ◽  
Oskar Zelder ◽  
Christoph Wittmann
Keyword(s):  
Corynebacterium Glutamicum ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Gas Chromatography Mass Spectrometry ◽  
Overflow Metabolism ◽  
Flux Analysis ◽  
Substrate Uptake ◽  
Lysine Production ◽  
Fructose 1,6 Bisphosphatase

ABSTRACT A comprehensive approach to 13C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTSFructose) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTSMannose was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.

scholarly journals Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Its Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

2005 ◽  
Vol 71 (6) ◽  
pp. 3255-3268 ◽  
Author(s):  
Andrea T. Hüser ◽  
Christophe Chassagnole ◽  
Nic D. Lindley ◽  
Muriel Merkamm ◽  
Armel Guyonvarch ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Metabolic Flux Analysis ◽  
Carbon Flux ◽  
Rational Design ◽  
Metabolic Flux ◽  
De Novo ◽  
Batch Cultivation ◽  
Flux Analysis ◽  
Genome Wide ◽  
Production Strain

ABSTRACT A “second-generation” production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, dl-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

scholarly journals Study on roles of anaplerotic pathways in glutamate overproduction of Corynebacterium glutamicum by metabolic flux analysis

2007 ◽  
Vol 6 (1) ◽  
pp. 19 ◽  
Author(s):  
Tomokazu Shirai ◽  
Koki Fujimura ◽  
Chikara Furusawa ◽  
Keisuke Nagahisa ◽  
Suteaki Shioya ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis
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