Creating a Biosynthetic Pathway in Oryza Sativ for β-Carotene: A Review Article of Golden Rice
A review article of why golden rice was made and the process of creating golden rice.
Table of contents
Consequences and further development
Introduction
Vitamin A deficiency kills 670 000 children under the age of 5 each year, the equivalent of 4500 children dying a day. [1] Vitamin A deficiency causes blindness in a quarter of a million to half a million children each year in developing countries, primarily Asia and Africa. [2] Vitamin A deficiency weakens the immune system, and it is estimated that two million people die a year from diseases that their body couldn’t fight off because of a severe vitamin A deficiency. [3]
To tackle this issue, professors Ingo Potrykus, from ETH Zürich, and Peter Beyer, from the University of Freiburg, created golden rice, a humanitarian project to engineer rice, a staple food in many developing countries, to include a high amount of vitamin A to combat vitamin A disease. Three plasmids were engineered to include the psy and crtI genes which would create the poly-cis pathway for β-Carotene, a precursor to vitamin A. The plasmids were inserted into the endosperm of immature rice by agrobacterium inoculation. One year after golden rice was created, golden rice 2 was created to improve the vitamin A content in golden rice by using a single plasmid with fewer genes that would target the endosperm specifically, instead of the whole plant. Golden rice is currently undergoing the regulatory phase of genetically modified organism commercialization, and has been approved in four countries and commercially approved in the Philippines. [4]
Vitamin A Disease (VAD)
The research that lead to the development of golden rice showed that 1.02 billion people worldwide are severely effected by micronutrient deficiencies, with vitamin A being third largest micronutrient deficiency globally. [5]
A study conducted in 2012 by the World Health Organization showed that over 250 million pre-school kids are affected by VAD, and that providing these children with vitamin A could could prevent a third of all deaths under the age of five, which amounts to 2.7 million children that could be saved from death because of vitamin A. [6]
4500 children die every day because of a lack of vitamin A, making it the leading cause of child mortality in the world.
The WHO has classified VAD as a public health problem affecting one third of children age 6 to 59 months in 2018, with the highest levels in sub-Saharan Africa (48 per cent) and South Asia (44 per cent), shown in figure 1 below.
β-Carotene
β-Carotene (beta-carotene) is a type of carotenoid that is essential to the human body. Carotenoids (tetraterpenoids) are yellow, orange and red pigments that are produced by plants, algae, bacteria and many fungi. Carotenoids are produced by chemical synthesis of enzymes in a process called a biosynthetic pathway. Carotenoids gives squash, carrots, oranges and other yellow/orange/red vegetables or fruits their orange color.[7]
β-Carotene is a precursor to vitamin A (known pro-vitamin A) which becomes vitamin A (retinol) during metabolization. [8] There are more than 70 carotenoids, but only 50 of them are precursors to vitamin A, with β-carotene being the most common retinol precursor found in high concentrations in carrots. β-carotene is cleaved from the retinol chain at the –C15 = C15′–location on the retinol genome. [9] β-carotene is a member of the terpenoid group and has eight isoprene groups and 40 carbons. β-carotene is recognized as one of the only carotenoids that has beta-rings at the end of the molecule. β-carotene contains no oxygen atoms. [10]
During metabolism, β-carotene is converted to vitamin A in the liver. β-carotene breaks down, loosing two carbon atoms becoming retinol. But at this stage in the process the compound only shows 16% retinol activity. [11] As the metabolization process continues, retinol from food travels to the β-carotene site in the genome, and the incoming retinol breaks down, creating more β-carotene. With added β-carotene, the compound starts to chemically synthesize to form retinol. Any excess retinol is transferred through the body in the form of lymph.
β-Carotene is a naturally occurring antioxidant found in many yellow, green and orange fruits and vegetables, but not starches or carbs such as wheat, cereal and rice (see figure 3). [12] In developing countries where the price of vegetables and fruits are high and the access is limited, people rely mostly on starches and carbs for their daily calories, leaving them very high risk of permanent blindness, severe infectious diseases and mortality due to a lack of vitamins (especially vitamin A).
Rice (oryza sativa)
Rice is a staple food for hundreds of millions of people. Oryza sativa is a starchy cereal grain of the grass family, with carbohydrate making up over 80% of the plant. Oryza sativa is made up of long chains of glucose called amylose and amylopectin. The remaining 20% of rice is made up of fiber. [13]
Rice is mostly consumed in it’s milled form with the outer layers (pericarp, tegmen and aleurone layers) removed. This leaves the endosperm as the only edible part of rice. This carbohydrate-dense plant makes it a perfect food source for people who do not have enough to eat and need a way to get the maximal number of calories for the smallest amount of food. Rice seeds are cheap to buy, and cheap and easy to grow, making it a perfect source of food and income for people in developing countries. [14]
The issue
The issue is that the rice endosperm lacks many essential vitamins and nutrients for human health, like vitamin A. [15] Rice is primarily made of carbohydrate and fiber, and only contains 0.06μg iron, 0.023μg manganese and 0.015μg calcium. Rice contains no vitamin A.
Heavy reliance on rice as the primary calorie source for people in developing countries leads to vitamin A deficiency, among many other deficiencies and diseases. A 2002 study shows that the average consumption of food for individuals in Bangladesh is 1–2 cups of rice per day. One cup of rice per day only provides 24% of an individual’s daily nutritional requirements, and no vitamin A. [16] This low-calorie, high-carb, low-nutrient diet leads to a weakened immune system, malnutrition, nutrient deficiencies, disease, and in many cases, eventually death. Out of the many nutrient deficiencies, VAD is the most common and the most dangerous.
The solution
Ingo Potrykus, from ETH Zürich, and Peter Beyer, from the University of Freiburg, created golden rice with the hope of creating strand of rice that had high levels of vitamin A to reduce VAD. Unlike oryza sativa, golden rice was engineered to contain β-carotene, the precursor to vitamin A. [17]
To engineer a pathway for β-carotene formation, it was necessary to activate four plant enzymes: phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene β-cyclase. [18] Usually there are seven enzymes that are required to create the pathway for β-Carotene, but to simplify this, Potrykus and Beyer reduced the number of enzymes required by using a bacterial carotene desaturases capable of introducing all four double bonds of the four enzymes, creating the poly-cis pathway. Each four enzymes would be introduced into the rice endosperm by a plasmid vector.
Poly-cis pathway
The poly-cis pathway is the pathway to create β-carotene, which involves four key plant enzymes: phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene β-cyclase. [19] Figure 4 (below) depicts the poly-cis pathway with all the proteins and enzymes involved.
Phytoene Synthase
The phytoene synthase (psy) sequence was isolated from the daffodil plant. psy is the first enzyme in the poly-cis pathway, and is a transferase enzyme involved in the biosynthesis of carotenoids. [20] It catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP, a molecule that is a precursor to carotenoids)[21] to phytoene (first step in creating carotenoids). [22]
psy diverts retinol and carbon entering an organism from a competing pathways (ex: photosynthesis gluconeogenesis, etc.) and instead towards the poly-cis pathway to form β-carotene. psy is the enzyme that controls the first step in the poly-cis pathway. [23]
Phytoene Desaturase
The phytoene desaturase (crtI) sequence was isolated from the soil bacterium erwinia uredovora. crtI catalyzes the conversion of 15-cis-phytoene (intermediate in the biosynthesis of carotenoid) into lycopene (red carotenoid in red fruits and vegetables like watermelon and tomatoes).
crtI is the second enzyme in the poly-cis pathway, and is responsible for attaching to liposomes and converting phytoene into phytofluene, and phytofluene into ζ-carotene. crtI catalyzes the conversion of the first two steps in the poly-cis pathway. [24]
ζ-carotene desaturase
The ζ-carotene desaturase enzyme catalyzes the formation of 7,9,7,9-tetra-Z-lycopene (poly Z-lycopene) in the ZDS section of the poly-cis pathway (figure 3). ζ-carotene desaturase is responsible for breaking down lycopene into β-carotene, completing the poly-cis pathway. Dehydrogenation (a chemical reaction that removes hydrogen(s) from an element) is the process that ζ-carotene desaturase uses to break down lycopene. [25]
Lycopene β-cyclase
Lycopene β-cyclase is an enzyme that catalyzes the chemical reaction: carotenoid psi-end group → carotenoid beta-end group [26]. This is the basic chemical reaction of β-carotene, which builds the hydrogen bonds of all carotenoids.
Lycopene β-cyclase catalyzes the β-cyclization of both ends of lycopene to produce β-carotene. Lycopene β-cyclase completes the poly-cis pathway, producing β-carotene which can be converted into retinol by the body. [27]
Vectors
Three plasmid vectors were constructed to transform phytoene synthase, phytoene desaturase, ζ-carotene desaturase, lycopene β-cyclase and other genes important for the creation of β-carotene, into the rice endosperm: pB19hpc, pZPsC, and pZCycH. Each plasmid is schematically depicted below in figure 9.
pB19hpc vector
The pB19hpc vector contained the phytoene synthase (psy) sequence, and the bacterial phytoene desaturase (crtI) sequence. Both the psy sequence and crtI sequence contained a sequence of complimentary DNA (cDNA) that would serve as a template during transcription. The psy cDNA contained a 5'-sequence coding for a transit peptide, and the crtI gene was fused to the transit peptide sequence of the pea rubisco small subunit (an enzyme that converts carbon dioxide to glucose). [28] The transit peptide will carry the psy and ctrI genes to the cell ribosome, so proteins can be coded for the genes and β-carotene can be created. [29]
The plasmid was under the control of the rice specific promoters endosperm-specific glutelin promoter (Gt1) and the CaMV 35S promoter. The Gt1 promoter was for psy and the CaMV 35S promoter was for crtI. The aphIV expression cassette selectable marker was also included in the plasmid which would give the plasmid the ability to grow and survive in toxins and chemicals. The entire pB19hpc plasmid (both psy and crtI) were under the hygromycin-selectable marker, a popular marker known for alerting transfection in eukaryotic cells by the gathering of tRNAs. [30]
The plasmid contained a total of nine other genes that would play important roles in silencing certain genes inside the endosperm and highlighting gene expression of the psy and ctrI gene. A few of these genes include (see figure 9 for all genes in pB19hpc) the nptII gene, which inactivates kanamycin (a harmful antibiotic in cells), by catalyzing ATP (cell energy currency) to create phosphates to protect the plasmid. [31] Another important gene in the plasmid is the 35Sp promoter gene which is derived from the cauliflower mosaic virus, a promoter that is used in 80% of all transgenic crops. It is known for it’s high level of RNA transcription initiated at the plasmid ORI. [32]
This plasmid should create the formation of lycopene in the endosperm plastids, at the site of geranylgeranyl diphosphate synthases (GGPP) formation. GGPP is an enzyme that regulates metabolites in a plant. [33] The GGPP formation site is located in the stroma, which is the internal section of the plastid double membrane. The lycopene β-cyclase enzyme will then convert lycopene into β-carotene. GGPP enzymes will carry the β-carotene to the ribosome so proteins can be coded for β-carotene, and transcription can be initiated, leading to the mass production of β-carotene in a cell. [34]
pZPsC and pZCycH vectors
The pZPsC vector and pZCycH vector are co-transformation vectors. amplifying the genes in the pB19hpc vector. pZPsC contained psy and crtI like pB19hpc, but lacked the selectable marker aphIV. The pZCycH plasmid was under the GUS glutelin promoter, a rice specific promoter that drives gene expression in the endosperm. It contained the lcy gene along with the aph gene, both genes that help in the expression of certain genes. It also contained a sequence isolated from the daffodil that codes for lycopene β-cyclase. Just like the psy gene in the pB19hpc vector, the lycopene β-cyclase gene had a complimentary transit peptide which will allow for easy entry into the plastid and stroma. [35]
Both vectors contained the 35Sp promoter for the same reasons that the pB19hpc vector contained the 35Sp promoter (mentioned above). All three plasmids contained the Gt1 promoter (Gt1p) , since Gt1p is one of the best rice-specific promoters for amplified genetic cloning. [36] Gt1p works with the Gus reporter (also included in all three plasmids), and the Gus reporter works with the 35Sp promoter. This makes the combination of promoters, selectable markers and reporters very efficient at replicating large quantities of plasmids and expressing genes inside a cell.
Transformation
Immature rice embryos were inoculated with agrobacterium, a transformation material isolated from soil that is used to aid in the transfer of genes to plants. Cell walls do not easily take up foreign DNA, so agrobacterium (working with the Gt1p promoter) will naturally infect weak or wound sites on a cell, creating pores in the cell membrane at weakened areas. The plasmids can then slip through the pores and into the cell. The process of using agrobacterium to transfect DNA into a cell is called transfer DNA (tDNA). [37]
For the transformation process 800 immature rice embryos were inoculated with an agrobacterium mixture of pB19hpc vectors. Fifty out of the 800 embryos were analyzed by the southern hybridization analysis technique, which is a technique used to detect specific DNA sequences in a genome. The technique uses restriction enzymes to cut a sample of DNA into pieces and then use electrophoresis gels to transfer the DNA fragments onto a filter which can then be examined under an X-ray machine or under a microscope. [38] The analysis showed that all of the plants had single gene insertions of at least one out of the four plasmids, and 24% of all seeds tested contained two or more of the four plasmids.
For the first co-transformation, 250 immature embryos were inoculated with an agrobacterium mixture of pZPsC vectors, carrying the psy and crtI genes. The final co-transformation consisted of 250 immature embryos which were inoculated with an agrobacterium mixture of pZCycH, containing lcy together with aph as well as the selectable marker.
Results
50 of the 800 pB19hpc transformation embryos were analyzed; the tests showed that all lines carried the transgenes and most of the plants had single insertions. 60 randomly chosen co-transformed plants were analyzed, and each of the 60 were positive for lcy. 12 out of the 60 co-transformation plants contained pZPsC.
On average each embryo took up 1–3 genes out of the 4 genes inoculated onto each plate. A total of 10 plants contained all four genes, and these ten plants were transferred into the greenhouse to settle seeds. (All plants showed normal fertility and normal phenotype.) The 10 seeds were dried, dehusked, and polished to isolate the endosperm. All seeds showed a notable yellow color, indicating carotenoid formation and eventual formation of β-carotene and retinol.
The pB19hpc single transformations showed a clear 3:1 (colored:non-colored) ratio, and pZPsC/pZCycH co-transformations showed a broad range of difference in the phenotype with no clear pattern. The pB19hpc transformations were further analyzed and showed a complete pathway to form β-carotene, which explains the yellow color that was observed. None of the transformations showed any detectable amount of lycopene (lcy), but did show formations of lutein and zeaxanthin, creating a carotenoid pathway quite similar to the pathway in green leafy vegetables, a pathway almost identical to spinach. This carotenoid pathway suggests that lycopene, β-cyclases and hydroxylase (all parts of the carotenoid pathway) were present somewhere in the genome, but in an un-detectable amount.
The co-transformation lines showed a much higher degree of variability (as expected). Some samples seemed like they had taken up none of the plasmids, while others had very clearly defined β-carotene pathways and had taken up all four pathways. The co-transformation line z11b was chosen as the winner (quantitatively) for β-carotene pathway. A carotenoid content of 1.6 μg/g was determined in the dry rice endosperm, which is the equivalent of 1.6g of total carotenoids per gram of grain, which is a 1:12 ratio of vitamin A to carbohydrate. This is significant increase of vitamin A from regular white rice that contains no vitamin A, and a tremendous feat for gene editing.
The daily requirement of vitamin A for the average female is 700μg, the requirement for children aged 4–8 is 400μg, and the requirement for children aged 8–13 is 600μg. This means that for a child to fulfil their daily requirements of vitamin A they would need to eat 200g of cooked golden rice, which equals 1 cup. The average adult would need to consume 400g of cooked golden rice (2 cups) to consume their daily requirements.
Testing has been done to compare golden rice with other sources of β-carotene like spinach, carrots, and lettuce, with research and testing from Mallikarjuna Swamy et.al showing that β-carotene from golden rice is converted into vitamin A by the body 5 times more efficiently than β-carotene found in spinach. [39][40]
Consequences and further development
The largest worry with golden rice is that the two co-transformation plasmids were not necessary to create a carotenoid pathway, so off-target effects and unnecessary added genes could be reduced without the co-transformation plasmids. With this in mind Ingo Potrykus and Peter Beyer reconstructed a single plasmid (pB19hpc) with a few modifications: the hygromycin-selectable marker gene was exchanged for the PMI gene, a rice specific selectable marker. In another golden rice attempt made in the early 2000s, the nptII gene was removed from the pB19hp plasmid, which was a gene that was not necessary for the β-carotene pathway. [41]
Golden rice 2
Golden rice was created to help control VAD, and following the development of golden rice, golden rice 2 was created to improve the vitamin A content in golden rice. Golden rice 2 contains 23 times more vitamin A than golden rice 1, which equals out to 37g of total carotenoids (31g is β-carotene) per gram of dry grain. [42] Golden rice 2 is very similar to golden rice 1 with only a few key differences: it contains the the phytoene synthetase gene (psy) isolated from maize, not isolated from daffodil like in golden rice 1. In golden rice 2, the carotene desaturase gene (crtl), isolated from Erwinia uredovora (same as golden rice 1). Both ctrI and psy were inserted into golden rice 2, in the same plasmid as the psy gene. There were no co-transformation plasmids in golden rice 2, and only the pB19hpc plasmid that contained all the necessary genes, enzymes, etc. Testing showed that one of the limiting factors of golden rice 1 was that the phytoene synthase pathway was not pronounced enough, which is a limiting factor of carotenoid biosynthesis. Much research showed that the most efficacious source of phytoene synthase to create the largest amount of carotenoids (specifically β-carotene), was found in maize. By inserting the psy gene isolated from maize instead of daffodil into golden rice 2, the efficiency of the β-carotene pathway immediately increased by 32%.
Golden rice 1 expressed β-carotene throughout the whole plant (roots, leaves, stem, etc.), not just the endosperm. To increase efficiency for golden rice 2, the psy gene and ctrI genes were only expressed in the endosperm, maximizing the amount of vitamin A in the edible part of the plant. Golden rice 2 used gene bombardment instead of inoculation to insert the plasmids into the rice endosperm. The psy and ctrI genes were inserted into A.tumefaciens, a natural soil bacterium. This A.tumefaciens plasmid was then inserted into the rice endosperm using gene guns. [43]
Eventual Dissemination and Legal Situation
It took 10 years, from 1980–1990, to develop the technology to introduce genes into rice. It took another 4 years, from 1990–1994, to identify which genes that create a biosynthetic pathway for β-carotene into rice. And it took another 5 years, from 1994–1999,to introduce those genes into the rice endosperm, resulting in golden rice. In 1999 Ingo Potrykus and Peter Beyer released their creation of golden rice. During the following 3 years, Potrykus and Beyer worked with Syngenta to improve golden rice. [44] Twenty years later, in 2022, golden rice is still not on the market. Golden rice has moved on to the regulatory phase, after two decades of rigorous testing. In 2019 golden rice was approved by the US Food and Drug Administration (FDA), and has been declared safe by three other countries including Australia, New Zealand and Canada. [45] In July 2021 the Philippines government issued a biosafety permit, officially declaring golden rice as safe as ordinary rice, becoming the first country to approve golden rice for commercial farming. [46]
The golden rice project was made possible by two agencies that funded the project, The Rockefeller Foundation and a research program of the European Community. Funding from the Rockefeller Foundation was free of obligations and allowed Potrykus and Beyer to do science and research. The partnership with the European Community required an industrial partner that would hold rights to inventions developed during the research. The industrial partner was Zeneca (recently merged with Novartis to form Syngenta). This partnership slowed down the Golden Rice Project as there were complications since golden rice is meant as a humanitarian project, not a commercial GMO created for profit or efficiency. In 2013 Greenpeace became the number one shareholder in Syngenta, gaining full control over the commercialization of golden rice. Greenpeace opposes the release of gene edited crops and refuses to play any role in commercializing golden rice. [47]
Asian communities are not accepting golden rice, which is another reason for golden rice’s delayed commercialization. The political, ideological and emotional connection to GMOs that society holds has greatly held back golden rice. Vandalism, boycotting, protesting, and media hype against the approval of golden rice has been a major setback to the potential of golden rice. [48]
Conclusion
Golden Rice is a genetically engineered strain of rice that has the potential to save millions of people from suffering, disease and death due to vitamin A deficiency. Golden rice now contains more vitamin A than maize, pepper, tomato and daffodil, because of the biosynthetic β-carotene pathway that was engineered by three plasmids (shown in figure 8): pB19hpc, pZPsC, and pZCycH.
Golden rice 2 was created in 2005 after golden rice 1 to increase the amount of vitamin A in golden rice. Golden rice is currently undergoing the regulatory phase, and has taken over two decades to be approved in four countries and commercially approved in the Philippines. Hopefully very soon golden rice will be approved in more countries and grown throughout the world as governments and people see the incredible potential of golden rice to save millions of people a year.
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My name is Rachel, I am a grade 10 student who’s main goal in life is to solve poverty and world hunger. I love being outside, spending time with my family, and learning about science and technology. If you have any suggestions, questions or just want to talk, you can email me at: runnerrachel.lee@gmail.com, or message me on LinkedIn. Don’t forget to sign up for my monthly newsletter here. Thanks so much for reading!
