• 1、 The necessity of improving rice germplasm through synthetic biology.
Rice is an important global food crop, providing about 15% of the world's per capita protein. Its protein and amino acid content are important indicators for measuring nutritional quality. High protein rice is an important high-quality plant protein source with good nutritional and commercial value. However, the amino acid composition of rice is unbalanced, which limits the nutritional quality of rice. As is well known, germplasm is an important resource in the seed industry, and traditional breeding is difficult to break through this limitation, urgently requiring disruptive technologies and strategies. Under the guidance of modeling, we used polyploidization combined with CRISPR/Cas9 mediated gene editing technology, and based on the principles of genomics+transcriptomics, designed a three round iterative technology to reprogram the ratio of rice endosperm glutelin, gliadin, and globulin on the premise of ensuring rice agronomic traits, increased the content of total protein and lysine, removed allergen globulin, and created a rice germplasm that improved nutritional quality, food safety, and eating quality. Providing a thoughtful solution to the contradiction between high nutrition, good taste, and non allergenicity for the "willful" Chinese consumers.
• 2、 CRISPR-Cas9
In our survey, we found that the majority of people would choose to avoid genetically modified technology. Our investigators and interviewers always feel fearful about genetically modified technology, and they worry about whether consuming genetically modified rice will harm their health? Will it have any side effects on the body? Is genetically modified rice really reliable?
The concerns of investigators and interviewers have made us ponder and given us new inspiration. Why don't we avoid the genetically modified technology that everyone is worried about? So we turned our attention to CRISPR/Cas9 technology, which can improve the protein content in rice through gene editing without introducing foreign genes, truly eliminating the concerns of the public.
Among the numerous proteins in rice, we found globulin that can cause allergic reactions in humans. Therefore, we also used CRISPR/Cas9 technology, which can selectively knock out globulin genes, for gene editing to reduce the content of globulin in rice and thus reduce its allergenicity.
• 3、 Our chassis material: diploid rice
The reason why we chose the plant chassis is:
In the sustainable development of food production, the continuous improvement of germplasm is irreplaceable no matter how many plant nutritional supplements are produced. Although microbial fermentation is more efficient in producing plant nutritional supplements than breeding improvement, the nutrients obtained by crops from the environment are likely to be non heritable.
From the perspective of consumers, considering their interests, compared to taking dietary supplements like taking medicine to treat illnesses every day, improving the nutrition of staple foods in daily diet is obviously more satisfying.
Our diploid rice chassis also has unique advantages, as it comes from the core technology of our research group - PMeS strain (polyploid meiotic stable strain). Compared with traditional diploid rice, the use of colchicine to induce doubling often leads to a decrease in seed setting rate. It has the advantages of increased polyploid biomass, stronger stress resistance, and unaffected seed setting rate, which plays an important role in ensuring grain yield. The mechanism by which it maintains fruiting rate is related to the high expression of OsMND1 and the meiotic behavior of PMeS lines resembling diploid rice. OsMND1 stabilizes the meiosis process by maintaining a balance of pairing, synapses, and recombination, thereby improving pollen fertility and fruiting rate.
• 4、 Optimization of chassis: polyploidization
Due to the dose-response of genes, polyploidization induction can lead to an overall increase in rice biomass, making rice grains larger and fuller, and improving the protein and amino acid content of rice. Doubling the rice genome leads to an increase in gene copy number, which alters the expression patterns of genes and changes the plant's stress resistance through methylation levels. Thanks to the advantages of our diploid chassis, the optimized tetraploid chassis will not be compromised in terms of durability. Therefore, the optimized tetraploid chassis is more conducive to increasing the content of the target product.
• 5、 Strategic innovation and advantages
The advantages of polyploidization+gene editing (genomics+proteomics) and the limitations of metabolic engineering
Polyploidization can achieve the incremental effect of overexpression of lysine rich genes in metabolic engineering, and gene editing can achieve the screening effect of metabolic reprogramming by screening proteins with high lysine content.
1. Direct targeting key genes: The synthesis and accumulation of rice storage proteins is a complex regulatory network. Expression group regulation can target and control the coding genes related to the synthesis and accumulation of a certain component protein, directly regulate the expression of this protein (such as glutelin B1 and globulin in our project), and use the interaction network between it and the expression of other component protein coding genes to indirectly regulate the expression of a variety of other component proteins. This method, through transcriptional control, can achieve the regulation of target protein content with minimal genetic manipulation, adjust the ratio of storage protein components, and reduce the impact of excessive genetic manipulation on plant growth, development, and agronomic traits. Metabolic engineering often requires the introduction and removal of multiple components to reprogram metabolic pathways, but it often means putting pressure on plant growth and development, ultimately affecting agronomic traits.
2. Reduce the impact of by-products: Regulating lysine synthesis and accumulation through metabolomics often interferes with a wide range of metabolic pathways, potentially producing unnecessary by-products and affecting plant agronomic traits. The regulation of lysine synthesis and decomposition pathways is related to plant stress responses. For example, under environmental stress, plants may respond to energy and nitrogen demands by regulating lysine and other amino acid metabolism. Overexpression of these key enzymes rashly may affect the nitrogen demand of plants in non stress environments, thereby affecting plant growth and development. And expression group regulation is more precise, which can reduce the impact of these by-products on plant growth, thereby improving nutritional quality without affecting agronomic traits.
3. Less impact on overall metabolic balance: Metabolome regulation often involves changes in the entire metabolic network, which may disrupt the original balance and affect plant growth and development. The key enzymes involved in lysine metabolism in rice are closely related to the metabolic balance between amino acids, especially the interconversion of amino acids and the supply of metabolic energy. For example, the substrate generated by the decomposition of lysine can be interconverted with other amino acids such as glutamine or alanine under specific conditions, affecting overall nitrogen metabolism. And expression group regulation can reduce the impact on the entire metabolic network and maintain metabolic balance by regulating specific gene expression, which is beneficial for maintaining normal plant growth.
4. Safety issues: Currently, the widely used metabolic engineering methods in rice often increase the content of free lysine by overexpressing key endogenous synthesis enzymes. However, overexpression of endogenous key enzymes can enhance lysine synthesis, but the effect is usually limited. Due to the complex metabolic regulation of rice itself, the activity of endogenous enzymes is regulated by multiple factors, and there are some feedback inhibition mechanisms, which may lead to a lower upper limit of lysine synthesis.
Therefore, most studies choose to overexpress key enzyme genes involved in exogenous lysine synthesis, mainly for the following reasons:
1. Low endogenous key enzyme activity: Lysine synthases such as dihydropyridine carboxylic acid synthase (DHDPS) in rice have naturally low activity, and overexpression of endogenous genes may not significantly increase lysine accumulation.
2. Feedback inhibition mechanism: There is strict feedback inhibition regulation in the endogenous lysine synthesis pathway of rice, and overexpression of endogenous enzymes is easily restricted by these mechanisms, thereby inhibiting excessive accumulation of lysine.
3. Advantages of exogenous enzymes: Exogenous enzymes, especially those from non plant species such as bacteria and algae, often have high activity and are not affected by endogenous feedback regulation in rice, which enables them to more effectively promote lysine synthesis.
Most current research tends to overexpress exogenous genes, but this inevitably leads to issues of low safety and public acceptance.
Our strategy can bypass the safety issue of introducing exogenous genes and achieve the concept of metabolic engineering screening and increment by doubling the genome as a whole to increase biomass and reprogramming the ratio of rice endosperm storage proteins.
• 6、 Contribution to Plant Synthetic Biology
Regarding the contribution of plant synthetic biology, which we are very fond of and proud of, we chose diploid rice as the chassis material and used genome doubling combined with gene editing to build a high content plant protein germplasm material. We explored a safer and more effective strategy of genomics+expression genomics instead of metabolomics, which improved the nutrition of staple foods and reduced the workload of plant protein allergy testing, greatly helping the iGEM team who want to build plant chassis in the future.
Our understanding of plant synthetic biology is that by modifying plants, they should not only be containers for high-yielding certain products, but their own growth and development should be improved rather than destroyed. This idea also provides a reference example for future plant chassis iGEM teams.