Expression of a hemA Gene from Agrobacterium radiobacter in a Rare Codon Optimizing Escherichia coli for Improving 5-aminolevulinate Production

Abstract

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

Purification and functional characterization of thermostable 5-aminolevulinic acid synthases

“…5). The results were much lower than previously reported values of 50 mM (Fu et al 2010) and 56 mM (Lin et al 2009) obtained in controlled fermentors operating at optimized conditions. Another possible reason for the low values is that the three thermostable ALASs have high Km values for glycine (Table 1).…”

Metabolic engineering of Corynebacterium glutamicum for efficient production of 5‐aminolevulinic acid

“…Recombinant E. coli heterologously expressing ALAS from R. sphaeroides could accumulate 5‐ALA via C4 pathway, and the expression of ALAS in E. coli was further optimized to improve 5‐ALA production using factorial design (Liu et al, ). In addition, a rare codon optimizer strain was used to enhance ALAS activity, resulting in production of 6.56 g/L 5‐ALA in a 15 L fermenter (Fu et al, ). As succinyl‐CoA is a precursor for 5‐ALA, production was enhanced fourfold by expressing ALAS from R. sphaeroides ALAS in an E. coli strain engineered for succinate production (Kang et al, ).…”

Toward Heme: 5-Aminolevulinate Synthase and Initiation of Porphyrin Synthesis

“…83 Overexpression and purification of ALAS from various other sources followed, involving the use of different expression systems and different chromatographic techniques to obtain the protein in its pure form. [89][90][91] The ability to purify recombinant ALAS in sufficiently large quantities enabled detailed investigations of the structural and kinetic properties of the enzyme. [92][93][94][95][96][97] In fact, the understanding of the catalytic role of individual active site amino acid was largely dependent on the ability to purify murine ALAS2 in sufficiently large quantities required for detailed examinations of the biochemical and biophysical properties of the enzyme variants.…”