The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM D-glucose or D-Xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-Xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture System using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter. (C) 2008 Elsevier Ltd. All rights reserved.