Metabolic flux analysis of Arthrobacter sp. CGMCC 3584 for cAMP production based on 13C tracer experiments and gas chromatography-mass spectrometry.
Arthrobacter sp. CGMCC 3584 are able to produce cAMP from glucose by the purine synthesis pathway via de novo or salvage biosynthesis. In order to gain an improved understanding of its metabolism, (13)C-labeling experiment and gas chromatography-mass spectrometry (GC-MS) analysis were employed to determine the metabolic network structure and estimate the intracellular fluxes. GC-MS analysis helps to reflect the activity of the intracellular pathways and reactions. The metabolic network mainly contains glycolytic and pentose phosphate pathways, the tricarboxylic acid cycle, and the inactive glyoxylate shunt. Hypoxanthine as a precursor of cAMP and sodium fluoride as an inhibitor of glycolysis were found to increase the cAMP production, as well as the flux through the PP pathway. The effects of adding hypoxanthine and sodium fluoride are discussed based on the enzyme assays and metabolic flux analysis. In conclusion, our results provide quantitative insights into how cells manipulate the metabolic network under different culture conditions and this may be of value in metabolic regulation for desirable production.
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China.
Huanqing
H
Niu
Yong
Y
Chen
Shiwei
S
Yao
Lixia
L
Liu
Chen
C
Yang
Bingbing
B
Li
Dong
D
Liu
Jingjing
J
Xie
Xiaochun
X
Chen
Jinglan
J
Wu
Hanjie
H
Ying
D001173:Arthrobacter / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002247:Carbon Isotopes / Q000737:chemistry; D002952:Citric Acid Cycle; D003470:Culture Media / Q000737:chemistry; D000242:Cyclic AMP / Q000096:biosynthesis* / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D019271:Hypoxanthine / Q000737:chemistry / Q000494:pharmacology; D010427:Pentose Phosphate Pathway; D012969:Sodium Fluoride / Q000737:chemistry / Q000494:pharmacology
D002247:Carbon Isotopes; D003470:Culture Media; D019271:Hypoxanthine; D012969:Sodium Fluoride; D000242:Cyclic AMP
(13)C tracer experiments; Arthrobacter sp. CGMCC 3584; Gas chromatography–mass spectrometry; Metabolic flux analysis