Metabolic engineering of astaxanthin biosynthesis in tobacco by coexpression of two key enzyme genes (CrBKT and HpBHY) together with a transcription factor CsMADS6

Natural astaxanthin exhibiting superior antioxidant activity is a liposoluble ketone carotenoid only synthesized in a few marine bacteria and algae, which is widely used in food, feed, health and other industries. Here, we report a new strategy for metabolic engineering of astaxanthin biosynthesis in tobacco ( Nicotiana tabacum ) plants by coexpression of two key enzyme genes, CrBKT and HpBHY, together with a transcription factor CsMADS6. CrBKT and HpBHY encode the enzymes β-carotene ketolase and β-carotene hydroxylase in microalgae Chlamydomonas reinhardtii and Haematococcus pluvialis , respectively, while CsMADS 6 is a positive regulator of carotenoid synthesis pathway in Citrus sinensis . The engineered tobacco plants newly accumulated high levels of astaxanthin in leaves (717.12 μg/g DW), along with biosynthesis of lycopene and capsorubin. Most intrinsic carotenogenic genes were upregulated, followed by carbon flux efficiently directed into carotenoids, leading to a significant increase ( P < 0.01) of flavonoids, anthocyanins, and carotenoid esters in the transgenic tobacco leaves, with the biosynthesis of significant amounts of the ketone carotenoid astaxanthin when compared to the non-transformed tobacco lines. The transgenic tobacco plant showed normal growth and development as well as net photosynthetic rate (Pn) ( P > 0.05) though chlorophyll content was decreased ( P ＜ 0.05). Our findings offer robust theoretical support for the efficient engineering of higher plants to produce high-valued astaxanthin. • Coexpression of CrBKT , HpBHY , and CsMADS 6 to astaxanthin biosynthesis. • Tobacco leaves enriched with astaxanthin, lycopene, and capsanthin. • Reddish-brown tobacco leaves increase the content of flavonoid and anthocyanins. • Reddish-brown tobacco leaves that reduce the chlorophyll content. • Tobacco leaves enriched with astaxanthin increase carotenoid ester by 2.23 times.