The 5-aminolevulinate (ALA) synthase gene (hemA) containing several codons rarely used by Escherichia coli was cloned from the genome of Rhodobacter sphaeroides and optimized in two strains of Escherichia coli: BL21(DE3) and Rosetta(DE3), which is a rare codon optimizer strain. The effects of initial isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, induction time, and temperature on enzyme activity were studied and compared for two strains. The results indicated that the ALA synthase expressed by Rosetta(DE3)/pET-28a(+)-hemA was higher than that by BL21 (DE3)/pET-28a(+)-hemA. The initial precursors, glycine and succinate, and initial glucose, which is an inhibitor for both ALA synthase and dehydratase, were observed to be the key factors affecting ALA production. ALA synthase activity was generally higher with Rosetta(DE3) than with BL21(DE3), so was ALA biosynthesis. Based on the optimal culture system using Rosetta(DE3), the yield of ALA achieved 3.8 g/l (29 mM) under the appropriate conditions in fermenter.