Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene.
The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).
Choi
Han-Pil
HP
School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
Lee
Young-Mi
YM
Yun
Cheol-Won
CW
Sung
Ha-Chin
HC
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000622:Aminolevulinic Acid / Q000378:metabolism*; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005798:Genes, Bacterial; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors; D012241:Rhodopseudomonas / Q000235:genetics*; D019802:Succinic Acid / Q000378:metabolism; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial; D000622:Aminolevulinic Acid; D019802:Succinic Acid; D000624:5-Aminolevulinate Synthetase; D000623:Porphobilinogen Synthase; D005998:Glycine