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Expression of a hemA Gene from Agrobacterium radiobacter in a Rare Codon Optimizing Escherichia coli for Improving 5-aminolevulinate Production
- Fu, Weiqi, Lin, Jianping, Cen, Peilin
- Applied biochemistry and biotechnology 2010 v.160 no.2 pp. 456-466
- gene expression, Agrobacterium tumefaciens, Escherichia coli, aminolevulinic acid, codons, genetic engineering, glycine (amino acid), succinic acid, glucose, microbial growth, batch fermentation, optimization, fermenters
- 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.