A β-carotene ketolase gene (bkt1) promoter regulated by sodium acetate and light in a model green microalga Chlamydomonas reinhardtii

Many microalgae could accumulate high level of astaxanthin, especially a green microalga Haematococcus pluvialis. β-carotene ketolase gene (bkt) is a key enzyme for astaxanthin biosynthesis in Haematococcus pluvialis, and astaxanthin accumulation could be induced effectively by abiotic stresses such as high concentration of sodium acetate and high light. In Haematococcus there are 3 bkt gene copies, among them, the transcription level of bkt1 was obviously correlated with high light and sodium acetate. Thus, the function of bkt1 promoter played a key role in understanding the regulatory mechanisms of astaxanthin biosynthesis. A TATA-box and cis-acting elements associated with high light and stress-related responses were detected in bkt1 promoter in previously studies. However, detailed information about cis-acting regulatory elements of this important promoter is still unavailable since a reliable genetic manipulation technique is not available yet. To identify the functions of cis-elements in bkt1 promoter, 8 constructs contained different length of bkt1 promoter were successfully introduced into a model microalga Chlamydomonas reinhardtii and different transformants were obtained with zeomycin resistant selection. Exposed to high light or sodium acetate, differentially increased ble transcripts and protein production in transformants indicated that these cis-elements respond to high light and sodium acetate. Moreover, we found that TATA-box in bkt1 promoter is essential for the promoter function and ble expression was correlated with cis-acting elements in bkt1 promoter. Our results shed lights on regulation mechanisms of the key genes from Haematococcus for astaxanthin biosynthesis in Chlamydomonas reinhardtii.