The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile were cloned by functional expression in These genes were not only expressed, but also osmoregulated in as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4.4 kb fragment revealed four major ORFs, which were designated and The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that codes for -2,4-diaminobutyric acid acetyltransferase, for -2,4-diaminobutyric acid transaminase and for -ectoine synthase. A DNA region upstream of was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.


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