Genetically Engineered Rhodobacter Sphaeroides for the Overproduction of gama-aminolevulinic acid
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Genes for J3 and cc polypeptides of the lightharvesting I I complexes in Rhodobacter sphaeroides 2.4.1 are encodcd by the pucBA operon, and their expression is highly rcgulated by oxygen level and light intensity. 8-,Qminolcvulinic acid (ALA) in this bacterium is synthesized by ALA synthase isozymes encoded by hemA and hemT. Recombinant plasmids carrying either a pucB'-henlA transcriptional fusion (pAS608A) or a pucS-henlT translational fusion (pAS607A) have been constructed. Derivatives of Escherichia coli SASP19 (hemA- mutant), harboring either pAS608A or pAS607A. still required exogenous ALA supplementation. However, each of these functions could be expressed in R. sphaeroides AT1 (hemA- and hemTmutant of R. sphaeroides 2.4.1) such that AT1 could grow in the medium without exogenous ALA supplementation. Introduction of pAS608A into R. sphaeroides 2.4.1 resulted in the increase of ALA synthase activity under both aerobic and photosynthetic growth conditions, while the introduction of pAS607A resulted in the increase of ALA synthase only under aerobic conditions. The production of extracellular and intracellular ALA in R. sphaeroides 2.4.1 (pAS608A) or R. sphaeroides 2.4.1 (pAS607A) was not significantly different from that of the wild-type strain in either aerobic or photosynthetic growth conditions.