5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene

Journal Article · Tue Oct 01 04:00:00 EDT 1996 · Applied and Environmental Microbiology

OSTI ID:508348

Van Der Werf, M J ^[1]; Zeikus, J G ^[1]

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1. Michigan State Univ., East Lansing, MI (United States)

The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinate (ALA) synthase. This enzyme catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine-forming ALA. The R. sphaeroides hemA gene in the pUC18/19 vector system was transformed into Escherichia coli. The effects of both genetic and physiological factors on the expression of ALA synthase and the production of ALA were studied. ALA synthase activity levels were maximal when hemA had the same transcription direction as the lac promoter. The distance between the lac promoter and hemA affected the expression of ALA synthase on different growth substrates. The E. coli host strain used had an enormous effect on the ALA synthase activity level and on the production of ALA, with E. coli DH1 being best suited. The ALA synthase activity level was also dependent on the carbon source. Succinate, L-malate, fumarate, and L-aspartate gave the highest levels of ALA synthase activity, while the use of lactose as a carbon source resulted in a repression of ALA synthase. After growth on succinate, ALA synthase represented {approx}5% of total cellular protein. The ALA synthase activity level was also dependent on the pH of the medium, with maximal activity occurring at pH 6.5. ALA production by whole cells was limited by the availability of glycine, and the addition of 2 g of glycine per liter to the growth medium increased the production of ALA fivefold, to 2.25 mM. In recombinant E. coli extracts, up to 22 mM ALA was produced from succinate, glycine, and ATP. 58 refs., 4 figs., 7 tabs.

• DOE Contract Number: FG02-93ER20108
• OSTI ID: 508348
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 10 Vol. 62; ISSN AEMIDF; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
BIOCHEMISTRY
ESCHERICHIA COLI
GENES