• WHY: We want to develop a rice germplasm with excellent nutritional value, food safety, and taste quality

As the staple food in Asia, especially in East Asia, ensuring food security cannot only guarantee food production, but also requires improving food nutrition. The limitation of China's food security lies not in yield, but in quality. In 2022, the grain production reached 1373.1 billion kilograms, with a per capita grain possession of 486.1 kilograms, which is higher than the internationally recognized food security line of 400 kilograms. The self-sufficiency rate of rations is over 100%, and the self-sufficiency rate of grains is over 95%. However, the rice market presents homogenization and lacks high value-added varieties that are nutritionally fortified. In the early 1990s, FAO and WHO proposed to "pay attention to nutrition security on the basis of raising public awareness of nutrition, and ensure that all people can sustainably achieve adequate nutrition." They directly linked food security and nutrition issues, making nutrition security an important component of food security. (Please read human practices integrated human practices - Hubei Rural Planning Research Association) With the increasing demand for healthy food among consumers, there is a growing demand for nutritionally fortified rice varieties in the market. But strangely, currently, both nutrient fortified and functional rice varieties circulating in the market in China are very rare, and they are unfamiliar to all three ends of production, sales, and consumption. Through multiple large-scale online and offline background research and face-to-face communication with stakeholders, we have found that there is a significant gap between the nutritional value, food safety, taste quality, and consumer demand of various rice varieties as food, as current rice varieties often only meet 1 to 2 aspects. (Please read human practices integrated human practices market research (Offline&Face to Face)) There is an interesting phenomenon in the Chinese rice market. During our market research, we found at least 10 different rice varieties with different tastes, such as "soft glutinous", "chewy", "soft smooth", "refreshing", etc., indicating that traditional rice varieties are inwardly curled in taste quality, but have not paid much attention to nutritional quality. However, merchants and developers of nutritionally fortified and functional rice focus on emphasizing health concepts and nutritional value, while avoiding discussing taste quality. (Please read human practices integrated human practices - The 2023 23rd Jingchu Grain and Oil Boutique Exhibition and Trade Fair) The food safety issues that stakeholders are most concerned about for an unfamiliar rice variety are often overlooked by both types of rice varieties. There is a hidden contradiction here: often high protein rice varieties have stronger grain texture, rough and dry texture, lower rice viscosity, and higher hardness, and high protein food intolerance populations (more common among children) are prone to intolerance or even allergies to high protein rice. So how to break this contradiction and develop a rice variety that can meet consumer needs in terms of nutritional quality, food safety, and taste quality? This contradiction stems from rice protein, and we hope it is a heritable rice germplasm. We have set the goal of using plant synthetic biology to improve endogenous proteins in rice.

• WHAT Characteristics of Rice Endosperm Storage Proteins and Amino Acids

Since we want to improve the endogenous protein in rice, we first need to fully understand its characteristics.
Rice endosperm is the part of rice seeds that people eat. The proteins in rice endosperm storage proteins can be divided into four types: glutelin, gliadin, globulin and albumin. Their properties and storage locations are different (see table below).

The proportion of essential amino acids for the human body in grains is often unbalanced, with lysine being the most lacking essential amino acid. Therefore, lysine is also known as the first essential amino acid in grains; and so on: the second essential amino acid is methionine, and the third essential amino acid is methionine
Glutelin contains rich and balanced essential amino acids for human body. The content of lysine, the first essential amino acid, is also the highest among storage proteins. However, it is neither fat soluble nor water soluble, which means that the rice varieties rich in it (most high protein rice varieties are rich in glutelin, because about 80% of the total protein of rice endosperm storage protein is glutelin) cannot be fully absorbed and emulsified during the cooking process, which also explains why the current high protein rice has poor taste.
The gliadin is fat soluble, which is helpful for the emulsification stage in the rice cooking process. It makes the rice taste more delicate, but the digestion of gliadin is poor, and its nutritional value is also inferior to glutelin. Glutelin and gliadin have their own advantages and disadvantages, so any kind of protein cannot be removed blindly. Taking into account both nutritional quality and taste quality, a balanced ratio of them would be more advantageous.
The accumulation site of albumin is close to the bran layer and is almost removed after rice processing and polishing, so we no longer consider it. The distribution of globulin is closer to the interior of the endosperm, and there is more residue after processing, which needs to be taken into account. Globulin is non fat soluble and poorly water-soluble, with only one lysine residue. It has a low content of essential amino acids for the human body and an unbalanced ratio. Compared with glutelin and gliadin, the globulin content in processed rice is not high, which can be reduced to improve food safety without affecting the nutritional quality and taste quality of rice storage protein. Considering the nutritional quality, food safety and taste quality comprehensively, we need to adjust the proportion of three components of rice storage protein that remain considerable after processing, increase the content of gliadin until it is close to the content of glutelin, remove globulin, and keep the content of glutelin unchanged.
In terms of amino acids, we hope to increase the content of lysine, because lysine is an amino acid that can not be synthesized by the human body and can only be ingested from food. As the staple food of Chinese people (especially in southern provinces), it is undoubtedly a great regret that rice lacks lysine. On the other hand, lysine is also a precursor to ketogenic amino acids and carnitine. Ketones are important reserve energy sources for maintaining normal physiological functions of the brain due to the lack of carbohydrate supply. Carnitine participates in the β - oxidation of fatty acids and plays an important role in liver function. However, although lysine plays an important role in human health and growth and development, long-term lack of lysine in children's diet can easily lead to protein energy malnutrition (PEM). However, consuming more lysine is not necessarily better. Excessive intake can disrupt the balance of amino acids, lead to metabolic disorders, and even cause health problems such as decreased appetite, obesity (high calorie content of lysine), and weakened immunity. WHO/FAO recommends a dietary amino acid requirement pattern based on the relative proportion of essential amino acids needed by the human body, with lysine being 5.5%. However, due to the different dietary habits of countries around the world, for countries with underdeveloped animal husbandry and scarce meat, egg, and dairy food resources, the requirement for lysine in grains should be higher than 5.5%; However, countries with developed animal husbandry have a rate of less than 5.5%. Based on this recommended indicator, combined with China's economic situation and dietary structure, rice lysine levels of 0.4-0.5% can meet the demand with a combination of meat and vegetables.

• HOW: Our technological approach should balance effectiveness, safety, and minimize the impact on rice growth and development

The easiest thing to think of when adjusting the ratio of protein components in rice storage is metabolic engineering, but in rice metabolic engineering, precise overexpression/knockout/knockdown of proteins or key enzymes involved in free lysine synthesis are used to regulate the content of a certain product. But this means that if you want to change the yield of glutelin, gliadin, globulin and free amino acid, you have to overexpress/knock out/knock down multiple genes in rice. Because each metabolite in rice has a complex regulatory network, often accompanied by intertwined positive and negative regulation. This will seriously affect the physiological functions of rice and even lead to death. Our target trait is centered around proteins (rice has not only free lysine but also protein lysine in its physiological state). The synthesis of proteins is not only influenced by key enzymes, but fundamentally determined by the expression of coding genes. Therefore, the best strategy is to reduce genetic manipulation and screen up to two or even one gene that can effectively indirectly regulate these traits based on the interaction pattern between genes for genetic manipulation. This approach minimizes the impact on the growth, development, and physiological functions of rice while achieving the goal. Coincidentally, there are dose effects and compensatory expression on the transcriptome of genes in rice. Compared with metabolomic regulation, using genome regulation+expression group regulation can avoid interference from complex metabolic networks and reduce interference on the entire metabolic network. Improving target traits does not affect agronomic traits.
The metabolic engineering mentioned above requires overexpression of some key enzyme genes for synthesis. Taking free lysine as an example, when using metabolic engineering to increase lysine synthesis and accumulation in rice, it is necessary to overexpress exogenous enzymes to break the feedback inhibition of the rice's own key enzyme for lysine synthesis, in order to achieve good results. However, this approach often comes with safety concerns regarding the introduction of exogenous genes. Currently, biotechnology that introduces exogenous genes is considered transgenic technology and is not approved for production and strict regulation in rice; People are very panicked and resistant to this technology in the public interest, so using metabolic engineering strategies will bring many unnecessary troubles. (Please read Human Practices Integrated Human Practices Analysis of Genetically Modified Public Opinion)
In terms of effectiveness, the improvement brought by metabolic engineering is too significant, and we don't need such a large amount, just need to make minor adjustments. Taking the increase in lysine content as an example, the lysine content in traditional rice is around 0.3%. We only need to increase its content to 0.4-0.5%, but the improvement in metabolic engineering is generally around 50%, which will lead to excessive eutrophication and product overnutrition. In summary, the combination of genome regulation and expression group regulation can achieve appropriate, safe, and minimal damage to rice agronomic traits in terms of strategy. So, how to choose the specific technical changes?
Polyploid organisms are a typical case of genome regulated dose-response, and polyploidization of the rice genome to overall increase its biomass is an effective protective measure to reduce the damage to its growth and development caused by subsequent genetic improvements, because its stress resistance and metabolic basis are better than diploid.
However, polyploidization technology has the drawback of lacking directional selectivity for improving target shapes. Due to the immature application of plant synthetic biology in rice and the complex regulatory network of rice as a higher organism, we still need to model and predict whether its improvement effect meets our expectations after selecting the best modification target or combination. And it is necessary to choose a technology that does not introduce exogenous genes to precisely modify the target. Considering both accuracy and safety, we have chosen CRISPR/Cas9 gene editing technology because its application in plants is relatively mature and the precision of targeted effects meets our requirements.
In summary, we induced doubling of the rice genome through polyploidization technology and optimized the chassis at the genomic level; Then, through the combination of rational design and semi rational design, we found the regulatory target consisting of one or two CRISPR/Cas9 site directed editing mutation target genes, and realized the expression omics regulation of rice storage protein components and glutenin family members' proportion programming, and the proteomics regulation of changing the structure of glutelin B1 and globulin.
After determining the strategy and technology, we need to filter and iterate on the targets through rational design (please read the wet lab-design page)and semi rational design (please read the engineering page).

Reference

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