Utilization Of Eukaryotic Astaxanthin Synthetases In The Development Of Tobacco Bioreactors

Astaxanthin is a keto-carotenoid with stronger antioxidant activity than vitamin E or coenzyme Q₁₀,and with pharmacological effects against cancer,cardiovascular disease,diabetes,and others.Extensive astaxanthin application in food,nutraceuticals,and cosmetic products has resulted in the broad market prospects of natural astaxanthin.But the approaches,which possess low efficiency and high cost for astaxanthin production with currently available resources,are far from meeting the rapidly growing market demand.The development of efficient astaxanthin bioreactors via tobacco is of great values to overcome this difficulty.But previously developed tobacco bioreactors for astaxanthin synthesis predominantly employed the modified microbial astaxanthin synthases.Whereas,the utilization of eukaryotic astaxanthin synthases in tobacco bioreactor is much limited.In order to determine the bioactivity of ectopically expressed eukaryotic astaxanthin synthases in tobacco,and to reveal the molecular basis for developing tobacco bioreactor for astaxanthin synthesis.In this study,we codon-optimized,and synthesized the eukaryotic astaxanthin synthases from Adonis aestivalis,Haematococcus pluvialis and Xanthophyllomyces dendrorhous respectively,and analyzed their astaxanthin synthetic bioactivities in tobacco using both transient and stable expression experiments.Eventually,transgenic tobacco plants stably producing astaxanthin were obtained.The main results are as follows:1.The transient expression experiments only showed that the bioactivities of astaxanthin synthases from A.aestivalis were higher than that from H.pluvialis and X.dendrorhous in tobacco,however,the stable expression experiments combined with molecular modification could distinguished thehe differential bioactivities of astaxanthin synthases from A.aestivalis,H.pluvialis and X.dendrorhous in tobacco.Therefore,the stable expression experiments are more sensitive and accurate than the transient expression experiments in determining the biological activities of astaxanthin synthases from different bio-resources in tobacco.2.By means of subcellular localization experiments and stable expression experiments,according to the localization of three eukaryotic astaxanthin synthases in tobacco cells,A.aestivalis-derived astaxanthin synthases could synthesize a large amount of astaxanthin in resistant callus and transgenic tobacco plants without molecular modification;H.pluvialis-derived astaxanthin synthases could synthesize a small amount of astaxanthin in resistant callus after molecular modification by addition of chloroplast-targeting peptide;X.dendrorhous-derived astaxanthin synthases could not synthesize astaxanthin even after the addition of chloroplast-targeting peptide.3.Subcellular localization experiments showed that the eukaryotic astaxanthin synthetase from different resources were located to differential organelles of tobacco cells.4.The results of stable expression experiments,RT-PCR and UPLC analysis of resistant callus showed that the A.aestivalis-derived astaxanthin synthases possessed the highest astaxanthin synthetic bioactivity in tobacco,the ones from H.pluvialis showed lower astaxanthin synthetic bioactivity,and those from X.dendrorhous displayed undetectable astaxanthin synthetic bioactivity.5.TLC and UPLC analyses of pigments in the leaves of transgenic tobacco plants showed that A.aestivalis-derived astaxanthin synthases could stably synthesize astaxanthin in tobacco.Yet,the astaxanthin synthase-expressing tobacco plants exhibited a slowed growth and development processes than that of the control tobacco plants,and their net photosynthetic rate was also significantly lower than that of the control tobacco plants.