5-Aminolevulinate production by Escherichia coli containing the Rhodobacter sphaeroides hemA gene.
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 approximately 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.
van der Werf
M J
MJ
Department of Biochemistry, Michigan State University, East Lansing, 48824, USA.
Zeikus
J G
JG
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism*; D001224:Aspartic Acid / Q000494:pharmacology; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007763:Lac Operon / Q000235:genetics; D007785:Lactose / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D003998:Dicarboxylic Acids; D001224:Aspartic Acid; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D000624:5-Aminolevulinate Synthetase; D007785:Lactose; D005998:Glycine