光合成細菌変異株による5-アミノレブリン酸の工業的生産

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The photosynthetic bacterium Rhodobacter sphaeroides accumulates 5-aminolevulinic acid(ALA), a precursor in tetrapyrrole biosynthesis, under light illumination and upon addition of levulinic acid as an inhibitor of ALA dehydratase. To generate an industrial strain that produces ALA in the absence of light, we sequentially mutated R.sphaeroides using N-methyl-N'-nitro-N-nitrosoguanidine(NTG). The mutant strain CR-286 was isolated as an ALA producer in the presence of yeast extract using low-melting-point agarose gel containing the ALA auxotroph Escherichia coli. Subsequent mutant strains were screened by cultivation in the absence of light and assayed for ALA by the Ehrlich reaction in a 96-well microtiter plate. The strain CR-386, derived from R.sphaeroides CR-286, was selected as a mutant that exhibited significant ALA accumulation. While CR-286 required light illumination for ALA production, CR-386 was able to accumulate 1.5 mM ALA in the presence of glucose, glycine, levulinic acid, and yeast extract with agitation in the absence of light. Another strain, CR-450, derived from CR-386, was further selected as a mutant that exhibited significant ALA accumulation but no accumulation of aminoacetone, an analogue of ALA. CR-450 accumulated 3.8 mM ALA under the same conditions. Similarly, the mutant strain CR-520, derived from CR-450, and CR-606, derived from CR-520, accumulated 8.1 mM and 11.2 mM ALA, respectively. In batch fermentation, strain CR-606 accumulated 20mM ALA over 18 h after the addition of glycine, levulinic acid, glucose, and yeast extract. Finally, the strain CR-720, derived from CR-606, was selected as a mutant that was stable and superior to strain CR-606 in ALA accumulation.
社団法人日本生物工学会の論文
2000-02-25

光合成細菌変異株による5-アミノレブリン酸の工業的生産

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