Preliminary Studies On Carotenoids Metabolic Engineering Of Two Eukaryotic Microalgae

Caroteniods have many important physiological functions in organism.Especially, astaxanthin has a high antioxidant activity, an anti-cancer activity, andsome other biological activities. Metabolic engineering of carotenoids has made greatprogress in Escherichia coli, yeast and plant. This paper aims at studying on thecarotenoids metabolic engineering of eukaryotic microalgae.β-carotene hydroxylase gene (crtR-B) was cloned from Haematococcus pluvialis,and transferred to the chloroplast genome of Chlamydomonas reinhardtii. HPLCassay showed that crtR-B transformants could rapidly synthesize xanthophylls(V+A+Z) in larger quantities than wild type upon transfer moderate adapted cells tohigh intensity light. The increase of carotenoids content may be due to the catalyticactivity of the additional β-carotene hydroxylase under high light.β-carotene ketolase gene (bkt) was cloned from H. pluvialis and then transferredto C. reinhardtii chloroplast genome. RT-PCR and RT-PCR Southern blot assaysshowed bkt could express at the transcriptional level in the C. reinhardtiitransformants. However, we couldn't detect any astaxanthin accumulation in thetransformants.Due to the high abundance quantities of β-carotene in Dunaliella salina cell, wethen turned to D. salina and optimized its genetic transformation system. 20 μg/ml ofBasta could inhibit 1.0×106/ml cells growth completely. Rupture-disc pressures of 450psi and bombardment distance of 6 cm was the optimal parameter for PDS1000/Hemicro-particle bombardment system transformation with the transient expression ofGUS reporter gene. The bar gene was introduced into the cells via the aboveestablished method. These results indicated that stable transformation system wassuccessfully established. Furthermore, phytoene synthase gene (psy) of D. salina wascloned including 459 bp 5′-flanking region.This is the first report on manipulation of carotenoids biosynthesis in C.reinhardtii using the metabolic engineering. With high light treatment, the amount ofxanthophylls increased significantly in the crtR-B transformants. The resultssuggested we successfully strengthen the carotenoids metabolic pathway.Micro-particle bombardment system transformation for D. salina was establishedusing selective marker bar gene and Basta screening. In addition, 5′-flanking regionof endogenetic psy was also cloned as a potential promoter. This research provided afoundation for further study to manipulate astaxanthin accumulation in D. salina.