There are two 5 '-flanking regions of bkt encoding beta-carotene ketolase in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a commercially valuable astaxanthin, with levels reaching up to 4% dry weight under environmental stress. In recent years, much effort has been devoted to understanding the molecular mechanisms regulating astaxanthin biosynthetic pathways. Beta-carotene ketolase (bkt), with control being exhibited at the transcription level, plays an important role in astaxanthin biosynthesis by H. pluvialis. Here we demonstrate the presence of two separate 5'-flanking regions [1.5 kilobase (kb) and 2 kb] of bkt (bkt1 and bkt2) that possess regulatory elements similar to those of known stress-responsive genes in plants. Results of 5'-deletion constructs and transient beta-galactosidase expression assays demonstrate that there may be positive regulatory elements governing expression in the shorter promoter at -1060/-900 from the 1.5 kb 5' region, and in the longer promoter at -1838/-1219 and at -1046/ -734 from the 2 kb 5' region relative to each homologous ATG start codon. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5 kb 5' untranslated region of bkt1 may function as a negative regulatory element to regulate its own promoter.

The unicellular green alga Haematococcus pluvialis accumulates a commercially valuable astaxanthin, with levels reaching up to 4% dry weight under environmental stress. In recent years, much effort has been devoted to understanding the molecular mechanisms regulating astaxanthin biosynthetic pathways. Beta-carotene ketolase (bkt), with control being exhibited at the transcription level, plays an important role in astaxanthin biosynthesis by H. pluvialis. Here we demonstrate the presence of two separate 5'-flanking regions [1.5 kilobase (kb) and 2 kb] of bkt (bkt1 and bkt2) that possess regulatory elements similar to those of known stress-responsive genes in plants. Results of 5'-deletion constructs and transient beta-galactosidase expression assays demonstrate that there may be positive regulatory elements governing expression in the shorter promoter at -1060/-900 from the 1.5 kb 5' region, and in the longer promoter at -1838/-1219 and at -1046/ -734 from the 2 kb 5' region relative to each homologous ATG start codon. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5 kb 5' untranslated region of bkt1 may function as a negative regulatory element to regulate its own promoter.