In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli.
The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E. coli and recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis.
Hong
Soon Ho
SH
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Park
Si Jae
SJ
Moon
Soo Yun
SY
Park
Jong Pil
JP
Lee
Sang Yup
SY
D000421:Alcaligenes / Q000235:genetics; D002952:Citric Acid Cycle; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005798:Genes, Bacterial; D005818:Genetic Engineering; D006885:Hydroxybutyrates / Q000378:metabolism*; D008660:Metabolism; D009154:Mutation; D016571:Neural Networks, Computer; D009876:Operon; D010957:Plasmids; D011091:Polyesters / Q000378:metabolism*; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D000094122:Polyhydroxybutyrates
D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates