Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (P_rrnB or P_{rsp_7571}) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where P_rrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where P_rrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where P_rrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.