Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids

Metabolites

Nobuyuki Okahashi¹  Shuichi Kajihata¹  Chikara Furusawa¹ 

[1] Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan; E-Mails:

关键词: metabolic flux analysis;    intracellular free amino acids;    Escherichia coli;    GC-MS;   

DOI  :  10.3390/metabo4020408

来源: mdpi

【 摘 要 】

¹³C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct ¹³C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal ¹³C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using ¹³C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the ¹³C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.