In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli

• Published in: Biotechnology and bioengineering Vol. 83; no. 7; pp. 854 - 863
• Main Authors: Hong, Soon Ho, Park, Si Jae, Moon, Soo Yun, Park, Jong Pil, Lee, Sang Yup
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 30.09.2003; Wiley
• Subjects: 2-keto-3-deoxy-6-phosphogluconate aldolase; Alcaligenes - genetics; Biological and medical sciences; Biotechnology; Citric Acid Cycle; Entner-Doudoroff pathway; Escherichia coli; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetic Engineering; Genetic technics; Hydroxybutyrates - metabolism; metabolic flux analysis; Metabolism; Methods. Procedures. Technologies; Modification of gene expression level; Mutation; Neural Networks (Computer); Operon; Plasmids; poly(3-hydroxybutyrate); Polyesters - metabolism; Predictive Value of Tests; Recombinant Proteins - metabolism; Reproducibility of Results; Recombinant microorganism; metabolic flux analysis; Escherichia coli; Ester polymer; Butyrate(hydroxy)polymer; Metabolic pathway; Metabolism; 2-keto-3-deoxy-6-phosphogluconate aldolase; Entner-Doudoroff pathway; Production; poly(3-hydroxybutyrate); Bacteria; Metabolic engineering; Enterobacteriaceae
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10733

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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 854–863, 2003.