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      <div class="text">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.<br><br>
 
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.<br><br>
 
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.
 
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  According to the advantages listing below, we take rice (Orazy sativa) endosperm as the bioreactor for astaxanthin production.<br><br>
 
  <p>Rice is a low-cost, high-productivity, high-safety and commonly used model plant</p>
 
  <p>Rice is easy to plant on a large scale and has very high yield</p>
 
  <p>Rice seed is an excellent biomass container</p>
 
  <p>Genetic modification technology is quite mature in rice</p>
 
  <p>As a special nutrition storage organs,rice seed is convenient to store, extract and purify</p>
 
  <p>Astaxanthin accumulation at seed would not interrupt the growth of the  whole plant</p><br><br>
 
  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.
 
 
 
 
 
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      <div class="text">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.
 
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<div class="h1_font_size">1. ASTA biosynthesis</div>
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<div class="p_font_size"style="text-indent:0em">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 (<font style="font-style:italic">Haematococcus pluvialis</font>) , and yeast (<font style="font-style:italic">Phaffia</font>) . 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.
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      <div class="text">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.<a href="https://2016.igem.org/Team:SCAU-China/PART">If you want to know more about it, please click here! </a>
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      <div class="text">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.
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<div class="p_font_size" ><small> <font style="font-weight:bold">Figure 1</font> &nbsp;&nbsp;Astaxanthin wildly exists in microalgae <font style="font-style:italic">Haematococcus pluvialis</font>(left) , shrimps (middle) and salmons (right), but only some microalgae can synthesize astaxanthin.</small>
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      <li><a href="#vecter">Introduction of astaxanthin</a></li>
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      <li><a href="#tissue">Bioreactor in rice endosperm</a></li>
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Currently, the industrial productions of astaxanthin are extracted from microalgae <font style="font-style:italic">H.pluvialis, Phaffia </font>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.
      <li><a href="#pcr">Pathway</a></li>
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Although higher plants such as <font style="font-style:italic">Zea mays</font> 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.
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<p>&nbsp;</p>
      <div class="text">In this part, we mainly introduce the work we used cre/loxp recombination,a site-specific recombinase technology to delete the selective marker.<a href="https://2016.igem.org/Team:SCAU-China/Safety#pcr">You can read more about this part here! </a>
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<div class="p_font_size" ><font style="font-weight:bold"><small>Figure 2</font> &nbsp;&nbsp;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 <font style="font-style:italic">Psy, CrtI, BHY</font> and <font style="font-style:italic">BKT.</font></small>
   
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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 <font style="font-style:italic">PSY </font>(phytoene synthase) catalyzes geranylgeranyl-PP into phytoene. The <font style="font-style:italic">CrtI</font> gene from <font style="font-style:italic">Erwinia uredovora </font>encodes phytone desaturase that could complete the catalysis process from phytoene to lycopene. Moreover, the <font style="font-style:italic">β-LCY</font> gene, encoding β-lycopene cyclases, is expressed and active in rice endosperm. Therefore, when the two genes (<font style="font-style:italic">PSY</font> and <font style="font-style:italic">CrtI</font>) 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: <font style="font-style:italic">BHY</font> (β-carotenehy droxylase) catalyze β-carotene to zeaxanthin, and <font style="font-style:italic">BKT</font> (β-carotene ketolase) catalyzes zeaxanthin directly to synthesize the end product astaxanthin. Because the expression of the endogenous rice gene <font style="font-style:italic">BHY</font> is very low, the biosynthesis of astaxanthin in rice endosperm requires at least 4 genes <font style="font-style:italic">(PSY+CrtI+BKT+BHY</font>, BBPC). For the combination of three genes <font style="font-style:italic">(PSY+CrtI+BKT</font>,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.  
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2. Rice-based bioreactor
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According to the advantages listed below, we took rice <font style="font-style:italic">(Oryza sativa)</font> endosperm as the bioreactor for astaxanthin production.
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<p>&nbsp;</p>
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<div class="p_font_size" >● Rice is a crop plant of low-cost, high-yield, high-safety.</div>
 +
<div class="p_font_size" >● Rice is easy to plant on a large scale with a very high yield
 +
</div><div class="p_font_size" >● Rice endosperm is an excellent biomass container for Astaxanthin.
 +
</div><div class="p_font_size" >● Genetic modification technology is quite mature in rice.
 +
</div><div class="p_font_size" >● As a special nutrition storage organs, the products in rice seed are convenient to store, extract and purify
 +
</div><div class="p_font_size" >● Astaxanthin accumulation in seeds would not interrupt the normal growth of the whole plant
 +
<p>&nbsp;</p>
 +
<div class="p_font_size" >
  
    <div class="vecter">
+
  The astaxanthin biosynthetic key gene <font style="font-style:italic">BKT</font>, 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.
      <ul class="nav">
+
</div>
      <a id="references"></a>
+
<p>&nbsp;</p>
      <li><a href="#vecter">Introduction of astaxanthin</a></li>
+
<p>&nbsp;</p>
      <li><a href="#tissue">Bioreactor in rice endosperm</a></li>
+
<p>&nbsp;</p>
      <li><a href="#pcr">Pathway</a></li>
+
<div class="h2_font_size" > <font style="font-weight:bold">References</font></div>
  <li><a href="#Genes">Multiple Genes Vector</a></li>
+
<p>&nbsp;</p>
  <li><a href="#Methods">Methods</a></li>
+
<div class="p_font_size" >【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002)
  <li><a href="#marker">marker free</a></li>
+
</div><div class="p_font_size" >【2】Salim Al-Babili, Peter Beyer. Golden Rice–five years on the road–five years to go? Trends in Plant Science. 10, 12, 565-573 (2005)
  <li><a href="#references" class="on">references</a></li>
+
</div><div class="p_font_size" >【3】Jacqueline A Paine, Catherine A Shipton, Sunandha Chaggar, Rhian M Howells, Mike J Kennedy, Gareth Vernon, Susan Y Wright, Edward Hinchliffe, Jessica L Adams, Aron L Silverstone, Rachel Drake. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology. 23, 4, 482-487 (2005)
      </ul>
+
</div><div class="p_font_size" >【4】Cong-Ping Tan, Fang-Qing Zhao, Zhong-Liang Su,  Cheng-Wei Liang, Song Qin. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol . 19, 347–355 (2007)  
      <div class="text">【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002)<br><br>
+
</div><div class="p_font_size" >【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering.  Plant Biology.  19, 111–117 (2014)
  【2】Salim Al-Babili, Peter Beyer. Golden Rice–five years on the road–five years to go? Trends in Plant Science. 10, 12, 565-573 (2005)<br><br>
+
</div><div class="p_font_size" >【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki,  Cho Joon Yong, Soya Hideaki.  
  【3】Jacqueline A Paine, Catherine A Shipton, Sunandha Chaggar, Rhian M Howells, Mike J Kennedy, Gareth Vernon, Susan Y Wright, Edward Hinchliffe, Jessica L Adams, Aron L Silverstone, Rachel Drake. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology. 23, 4, 482-487 (2005)<br><br>
+
</div><div class="p_font_size" >【7】Astaxanthin supplementation enhances adult hippocampal neurogenesis and spatial memory inmice. Molecular nutrition & food research. 60 , 589-599(2016)
  【4】Cong-Ping Tan, Fang-Qing Zhao, Zhong-Liang Su,  Cheng-Wei Liang, Song Qin. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol . 19, 347–355 (2007) <br><br>
+
</div></div>
  【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering.  Plant Biology.  19, 111–117 (2014)<br><br>
+
  【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki,  Cho Joon Yong, Soya Hideaki. <br><br>
+
  Astaxanthin supplementation enhances adult hippocampal neurogenesis and spatial memory inmice. Molecular nutrition & food research. 60 , 589-599(2016)
+
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Latest revision as of 05:29, 18 October 2016

SCAU

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.

 

 

 

References

 

【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002)
【2】Salim Al-Babili, Peter Beyer. Golden Rice–five years on the road–five years to go? Trends in Plant Science. 10, 12, 565-573 (2005)
【3】Jacqueline A Paine, Catherine A Shipton, Sunandha Chaggar, Rhian M Howells, Mike J Kennedy, Gareth Vernon, Susan Y Wright, Edward Hinchliffe, Jessica L Adams, Aron L Silverstone, Rachel Drake. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology. 23, 4, 482-487 (2005)
【4】Cong-Ping Tan, Fang-Qing Zhao, Zhong-Liang Su, Cheng-Wei Liang, Song Qin. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol . 19, 347–355 (2007)
【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering. Plant Biology. 19, 111–117 (2014)
【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki, Cho Joon Yong, Soya Hideaki.
【7】Astaxanthin supplementation enhances adult hippocampal neurogenesis and spatial memory inmice. Molecular nutrition & food research. 60 , 589-599(2016)