1. ASTA biosynthesis

Astaxanthin (3, 3′-dihydroxy-β-carotene-4, 4′-dione) is a kind of naturally occurring keto-carotenoids which is found in some microalgae, shrimps and salmons (Figure 1). This compound is insoluble in water while soluble in most of organic solvent like pyridine, ethanol and benzene. In fact, it is synthesized merely in some specific species of algae, bacteria and yeasts. For animals, astaxanthin could not be synthesized. Astaxanthin is responsible for the pigmentation of many marine animals which acquire the red pigment from their diets. In some organisms, astaxanthin takes on a colour of brown or blue as it forms into some types of pigment-protein complexes. For humans, astaxanthin is a powerful antioxidant with broad health implications. 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, shrimps and salmons (from left to right), but only some microalgae can synthesize astaxanthin.

Currently, the industrial ways to produce astaxanthin are extracted from microalgae Haematococcus pluvialis, Phaffia yeast, shrimp processing waste and chemical product.However these ways aren’t safety 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, there are many precious products were terpenoid, such as carotenoids, microbial A, paclitaxel, etc. But many complex products could not be synthesized by animal.

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

Figure 2The 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 enzymes. However, based on the study of Golden Rice, we deduced its biosynthesis in rice endosperm require 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 expression 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 transform these four genes into rice to study the metabolic synthesis of astaxanthin in endosperm.

2.Rice-based bioreactor

According to the advantages listing below, we take rice (Orazy sativa) endosperm as the bioreactor for astaxanthin production.

● Rice is a low-cost, high-productivity, high-safety and commonly used model plant

● Rice is easy to plant on a large scale and has very high yield

● Rice seed is an excellent biomass container

● Genetic modification technology is quite mature in rice

● As a special nutrition storage organs, rice seed is convenient to store, extract and purify

● Astaxanthin accumulation at seed would not interrupt the growth of the whole plant

The astaxanthin biosynthetic key gene BKT, encoding a β-carotene ketolase, does not exist in rice and plants. Meanwhile, the expression levels of a large amount of endogenous genes involved in carotenoid synthesis are very low or 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 stacking 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 storage and extraction.

references

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