Expression of 5-aminolevulinic acid synthase in recombinant Escherichia  coli

Abstract

The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinic acid synthase (EC.2.3.1.3), catalysing the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine to give 5-aminolevulinic acid. The gene was transformed into Escherichia coli K 12 using the pALA vector systems. Effects of host strains, vector plasmids, growth substrates and precursors on the expression and activity of 5-aminolevulinic acid synthase were studied. The E. coli host strain had an enormous effect on 5-aminolevulinic acid synthase activity and production of 5-aminolevulinic acid, with E. coli DH1 being best suited. RT-PCR of hemA mRNA indicated that the transcription of the hemA gene in the recombinant strain appeared to be higher than that of the wild-type strain. 5-Aminolevulinic acid synthase activity was maximal when hemA had the same transcription direction as the lac promoter. Distance between lac promoter and hemA affected the expression of 5-aminolevulinic acid synthase on different growth substrates. 5-Aminolevulinic acid synthase activities were also dependent on the carbon sources and precursors. L-Malate gave the highest activity of 5-aminolevulinic acid synthase, while lactose as a carbon source resulted in a repression of 5-aminolevulinic acid synthase. Production of 5-aminolevulinic acid by recombinants were limited by the availability of glycine, and repeated addition of glycine (20 mM) to the growth medium increased production of 5-aminolevulinic acid to 33.8 mM.

High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties

“…Recombinant bacteria containing the hemA gene from such organisms as Rhodobacter sphaeroides (Vanderwerf and Zeikus, 1996;Kiatpapan and Murooka, 2001;Xie et al, 2003b;Wang et al, 2006;Fu et al, 2007), Bradyrhizobium japonicum (Choi et al, 1999;Lee et al, 2004), Rhodopseudomonas palustris (Choi et al, 2008), and Agrobacterium radiobacter (Liu et al, 2005;Lin et al, 2009) were constructed for ALA production, achieving levels of 39 mmol/L (Xie et al, 2003a), 50 mmol/L (Fu et al, 2008), and 56 mmol/L (Lin et al, 2009), respectively.…”