1. ASTA biosynthesis
Astaxanthin (3, 3′-dihydroxy-β-carotene-4, 4′-dione) is a natural keto-carotenoid which is found in some microalgae, shrimps and salmons (Figure 1). This compound is insoluble in water while soluble in most of organic solvent. Astaxanthin is synthesized merely in some specific species such as algae (Haematococcus pluvialis) , and yeast (Phaffia) . It cannot be synthesized in animals, but many marine animals uptake astaxanthin in via their diets of algae and acquire the red pigmentation. Astaxanthin is a powerful antioxidant. Thus, astaxanthin has been claimed a good commercial prospect for its value in medical and health care.



Figure 1   Astaxanthin wildly exists in microalgae Haematococcus pluvialis(left) , shrimps (middle) and salmons (right), but only some microalgae can synthesize astaxanthin.


Currently, the industrial productions of astaxanthin are extracted from microalgae H.pluvialis, Phaffia yeast, shrimp processing waste and chemical product. However, these ways are not safe enough and the purification is difficult. While higher plants are supposed to be an efficient and safe bioreactor to produce astaxanthin, because it has advanced protein processing system to produce complex products, such as senior terpenoids. In nature, many precious products are terpenoid, such as carotenoid, microbial A, paclitaxel, etc. But many complex products could not be synthesized by animals.


Although higher plants such as Zea mays are capable to synthesize zeaxanthin, which is the metabolic precursor of astaxanthin. However, due to their lack of β-carotene ketolase, astaxanthin still cannot be synthesized in these higher plants.



Figure 2   The biosynthesis pathway of astaxanthin formation in transgenic rice endosperm. The dotted arrows indicate pathway limitations in rice endosperm. The solid arrows indicate the existence of carotenogenic reactions. The red arrows indicate the reactions catalaysed by four exogenous transgenes Psy, CrtI, BHY and BKT.


The biosynthesis of astaxanthin from pyruvate needs more than ten kinds of enzymes. However, based on the study of Golden Rice, we deduced that its biosynthesis in rice endosperm requires four key enzymes (Figure 2). The PSY (phytoene synthase) catalyzes geranylgeranyl-PP into phytoene. The CrtI gene from Erwinia uredovora encodes phytone desaturase that could complete the catalysis process from phytoene to lycopene. Moreover, the β-LCY gene, encoding β-lycopene cyclases, is expressed and active in rice endosperm. Therefore, when the two genes (PSY and CrtI) are drived by endosperm-specific promoters in rice, the β-carotene (pro-vitamin A) was synthesized to produce the famous Golden Rice. From β-carotene to astaxanthin, there are still two steps: BHY (β-carotenehy droxylase) catalyze β-carotene to zeaxanthin, and BKT (β-carotene ketolase) catalyzes zeaxanthin directly to synthesize the end product astaxanthin. Because the expression of the endogenous rice gene BHY is very low, the biosynthesis of astaxanthin in rice endosperm requires at least 4 genes (PSY+CrtI+BKT+BHY, BBPC). For the combination of three genes (PSY+CrtI+BKT,BPC) maybe produce little astaxanthin (even nothing!). Therefore, we utilized a multigene vector system to assemble and transformed these four genes into rice to study the metabolic synthesis of astaxanthin in endosperm.



2. Rice-based bioreactor
According to the advantages listed below, we took rice (Oryza sativa) endosperm as the bioreactor for astaxanthin production.


● Rice is a crop plant of low-cost, high-yield, high-safety.
● Rice is easy to plant on a large scale with a very high yield
● Rice endosperm is an excellent biomass container for Astaxanthin.
● Genetic modification technology is quite mature in rice.
● As a special nutrition storage organs, the products in rice seed are convenient to store, extract and purify
● Astaxanthin accumulation in seeds would not interrupt the normal growth of the whole plant


The astaxanthin biosynthetic key gene BKT, encoding a β-carotene ketolase, does not exist in rice and other plants. Meanwhile, the expression levels of a large amount of endogenous genes involved in carotenoid synthesis are very low or of no expression in rice endosperm. Thus, astaxanthin cannot be produced in wild-type rice. However, it is possible to biosynthesize astaxanthin in rice by using multigene metabolic engineering to stack multigenes involved in astaxanthin pathway. In this project, we assembled four astaxanthin biosynthetic genes to transform rice callus, which all genes are under the control of four different endosperm-specific promoters. In this way, rice endosperm serves as a special container for astaxanthin, which provides conveniences for later storage and extraction.






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