title: Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis.
abstract: Corynebacterium glutamicum is known as a host species for amino acid production. This microorganism was recently noticed as a host that produces secreted proteins. In this study, we performed (13)C metabolic flux analysis ((13)C-MFA) on a recombinant C. glutamicum strain that secretes a heterologous transglutaminase (TGase) to improve TGase secretion. For the (13)C-MFA of a TGase-secreting C. glutamicum strain in batch cultivation, a (13)C-labeling experiment and measurement of mass isotopomer distributions of proteinogenic amino acids were performed, and metabolic fluxes were determined considering the changes in fractional (13)C-labeling of proteinogenic amino acids with respect to culture time. The TGase yield increased at the stationary phase but decreased toward its end. The results of (13)C-MFA revealed that the flux from glycolysis to the TCA cycle gradually increased during TGase secretion. We speculate that the NADH/NAD(+) ratio in the cells increases and that as a result, the specific glucose uptake rate decreases in the stationary phase because of the increased flux of the TCA cycle. Since it is expected that a decrease in the NADH/NAD(+) ratio would improve the TGase yield, we tried to enhance lactate production in a TGase-secreting C. glutamicum strain to decrease cellular NADH levels by increasing the pH level in the culture. The TGase yield increased in 1.4-fold by increasing the pH from 6.7 to 7.2, indicating that the TGase yield was successfully improved on the basis of the (13)C-MFA.
authors: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Motoki
authors: M
authors: Umakoshi
authors: Takashi
authors: T
authors: Hirasawa
authors: Chikara
authors: C
authors: Furusawa
authors: Yasuhiro
authors: Y
authors: Takenaka
authors: Yoshimi
authors: Y
authors: Kikuchi
authors: Hiroshi
authors: H
authors: Shimizu
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes / Q000378:metabolism; D002952:Citric Acid Cycle; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D007218:Industrial Microbiology; D008954:Models, Biological; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D011503:Transglutaminases; D005947:Glucose