Team:SCAU-China/Project

Astaxanthin is a kind of naturally-occurring keto-carotenoids which is found in some microalgaes, shrimps and crabs. This compound is insoluble in water while soluble in most of organic solvent like pyridine, ethanol and benzene. In fact, it is merely in some specific species of algaes, bacteria and yeasts that astaxanthin can be synthesized. It is impossible for animals to synthesize astaxanthins on their own so that the accumulation of astaxanthin in the body of animal is the consequence of diet. 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.

Currently, the industrial ways to produce astaxanthin are extract 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 product, such as senior terpenoids. In nature, there are many precious products were terpenoid , such as carotenoids, microbial A, paclitaxel, etc. And in the low-level and annimal unable to synthesize some complex product.

Although higher plants such as Zea mays are capable to synthesize zeaxanthin, which is the metabolic precursor of astaxanthin, due to their lack ofβ- carotene ketolase, astaxanthin still can not be synthesized in these higher plants unless with the help of metabolism engineering.
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



Genes responsible for carotenoid synthesis are inactive in rice endosperm. So it is impossibleo harvest astaxanthin from wild-type rice. Using multiple-gene metabolic engineering, we introduced the astaxanthin biosynthesis pathways which is specifically expressed in rice endosperm. Thus, possible negative effects of astaxanthin would not influence the growth of plants. In this way, rice endosperm serves as a special container for astaxanthin, which provides conveniences for storage and extraction.
PSY (phytoene synthase) catalyzes Geranylgeranyl-PP into Phytoene. The gene CrtI from Erwinia uredovora could finish the catalysis process from Phytoene to Lycopene. Therefore, when these two genes(PSY and CrtI) with specific promoters of endosperm ahead ,they will express CRTISO and β-LCY enzymes which synthesizeβ-Carotene to produce the famous Golden Rice. There are still two steps from β-Carotene to astaxanthin: BHY (β-carotenehy droxylase) catalyze β-Carotene to Zeaxanthin. And BKT catalyzes directly to synthesize the end product astaxnthin. The expression of the endogenous gene BHY in rice is still unknown, accordingly it needs at least 3 genes (PSY+Crt I+BKT，BPC) or 4 genes(PSY+Crt I+BKT+BHY, BBPC) to synthesize astaxanthin. For the combination of three genes, if the endogenous gene BHY of rice has little expression, maybe there will be just a little astaxanthin produced(even nothing!). But for the combination of four genes, it will create a complete metabolic pathway for astaxanthin production. Surely, it could produce astaxanthin. Thus we use the systems of Assembly of multiple genes and transformation, and the specific promoters of endosperm to construct three vectors(380-PC,BCP and BBCP) to study the metabolic of the synthesis of astaxanthin in the endosperm of rice.
The system of multiple genes vectors is composed of one acceptance vector which could transform artificial chromosome and two donor vectors(322-d1/322-d2) carrying genes. Using Cre/loxP recombination system, acceptance vector accept the gene from donor vectors alternatively to accomplish the construction of objective vectors(Lin et al., PNAS, 2003, 100: 5962-5967；Zhu et al., unpublished). Assembling the four genes(CrtI,PSY,BKT and BHY) and the specific promoters of endosperm by the principle of Gibson Assembly(Gibson, Methods Enzymol., 2011, 498: 349–361). We got those vectors,(Ⅰ)pYL322d1-CrtⅠ,(Ⅱ)pYL322d2-PSY,(Ⅲ) pYL322d1-BKT and (Ⅳ)pYL322d2-BHY via assembling expression cassettes to donor vectors alternatively. Another time, using the assembly of Cre/LoxP system, transfer objective genes to acceptance vector according to the line of Ⅰ\Ⅱ\Ⅲ\Ⅳ to finish every steps. After the steps ofⅠ\Ⅱ, we got the binary expression vector 380-PC. The second step of Ⅲ, we got 380-BPC. After the Ⅳ, we got the 380-BBPC. Then we transformed the three vectors into Agrobacterium tumefaciens EHA105. We successfully got the rice which can produce astaxanthin in endosperm by Agrobacterium-mediated transformation of rice callus.If you want to know more about it, please click here!
We get the callus by callus induction of rice seed, after that we will transfer the carrier into callus by agrobacterium-mediated transformation. If we get positive callus, we should cultivate it to complete plant by plant tissue culture. Finally we will obtain the transgenic rice plants and harvest the rices.
In this part, we mainly introduce the work we used cre/loxp recombination，a site-specific recombinase technology to delete the selective marker.You can read more about this part here!
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Astaxanthin supplementation enhances adult hippocampal neurogenesis and spatial memory inmice. Molecular nutrition & food research. 60 , 589-599(2016)