BACKGROUND
Malnutrition and the Dual Dilemma
Malnutrition is an abnormal physiological condition caused by abnormal intake of nutrients and micronutrients, leading to weight loss. It includes undernutrition (stunted growth and thinness in children), deficiencies in vitamins and minerals, and overweight and obesity. Especially, nutritional deficiencies during early childhood impair brain development, impact learning ability and school readiness, suppressing life-long achievement potential and exacerbating health disparities and social inequality.
Currently, more than one-third of the global population faces the dual dilemma of malnutrition and low income. Constrained by the existing scarcity of natural resources and industries, individual efforts often seem futile and macroeconomic regulation by society is often difficult to implement as well.
“Malnutrition, on the other hand, hinders national progress and deeply compromises the health, development and well-being of present and future generations.”[1]
PUFA
Polyunsaturated fatty acids (PUFA), especially DHA and EPA, can treat cardiovascular and cerebrovascular diseases, reduce cholesterol, promote nervous system development, and have anti-inflammatory effects. For children, the intake of PUFA helps prevent chronic diseases such as ADHD and maintain normal immune function. For pregnant women, PUFA helps to promote the development of brains and retinal function, playing a role in prenatal nutrition intervention. Additionally, PUFA consumption is linked to higher birth weights, reducing the risk of low-birth weight infants. PUFA biosynthesis is subject to the dual constraints of oxygen, while nodules in a microaerobic environment are natural and high-quality factories for PUFA production.
N. FACTORY
Overview
We will harness synthetic biology to engineer rhizobia, enabling them to produce PUFAs, thereby transforming nodules into miniature yet intelligent microaerobic biofactories. This approach aims to concurrently address the dual challenges of malnutrition and low income, offering a novel and sustainable production paradigm.
We choose Sinorhizobium fredii CCBAU45436 as chassis, building up a set of regulation module in response to the changes of nitrogen and oxygen. Meanwhile, we select genes encoding PUFA synthase and dipeptidyl aldehyde to preliminarily demonstrate the feasibility of the “microaerobic factory". In order to improve its efficiency, we reprogram the metabolism by adjusting the expression of relevant genes, and we designed the suicide circuit to avoid chassis leakage.
Module 1: PHB Deletion
PHB (Poly-β-hydroxybutyrate) is an auto-metabolite of Sinorhizobium fredii CCBAU45436, which is highly expressed in chassis. We choose to delete the phaC2 gene encoding the key enzyme for PHB synthesis in S. fredii using homologous recombination double-exchange technology in order to achieve the accumulation of substances such as acetyl-coenzyme A, which will provide precursors for the synthesis module.
Module 2: Regulation and Synthesis Module
Based on the characteristics of endogenous components of rhizobia and the formation of a microaerobic consumption environment in symbiosis with the plant root system, we design the regulation module in response to the change of nitrogen and oxygen content in the environment, and verify the feasibility and effectiveness of the regulation and synthesis module by taking dipeptide aldehydes and PUFA as examples.
We choose the glnK promoter, which responds to low-nitrogen induction, and the nifH promoter, which responds to low-oxygen induction. We also introduce the CRISPRi system to regulate the expression of the downstream synthetic gene clusters, thus alleviating the stress of the introduction of this module on the normal life activities of the chassis themselves.
Module 3: Suicide Circuit
In the suicide circuit, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of these genes is regulated by the nifH and glnK promoters.
To avoid escape of chassis, we introduce the CRISPRi system and the endogenous TA (toxin-antitoxin) system of chassis for biosafety. In the suicide circuit, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of these genes is regulated by the nifH and glnK promoters.
For more information, please refer to the design page. Design
Advantages
- Production: Compared to general microbial fermentation, it eliminates the need for additional anaerobic fermentation equipment and human resources.
- Economics: It establishes the nodule economy, enhancing individual income and promoting local agricultural development. The sustainable nature of nodule factory is appealing and helps to promote financing.
- Technology: It provides a new approach of production, establishing a new framework for microaerobic production using root nodules. And we prove its feasibility. Other teams can build upon this framework to diversify downstream products in the future, so it is an inclusive production platform that can be continuously updated and developed.
- Environment: It is environmentally friendly and it avoids chemical pollution. So it is a responsible and sustainable production method.
- Society: Due to the fact that large equipment is not required for the production, governments can improve the economy with lower public spending. By producing relatively expensive microaerobic nutrients at low costs, it promotes affordable and healthy diets, contributing to reduce stunted children and increase breast milk nutrition, helping eliminate malnutrition and reduce the low-income population. And it helps narrow health disparities and reduce social inequality.
EXPECTATION
Transportation
We ultimately wish to transport DHA and EPA produced in root nodules into soybeans. Therefore, we firstly need to find relevant lipid transporter proteins that can mediate the transfer of fatty acids outside the root nodules.
For PUFA, a fatty acid exporting system has been discovered in Gram-negative bacteria. S. fredii is also a kind of Gram-negative bacteria. The physiological response to PUFAs of the Gram-negative pathogenic bacteria Acinetobacter baumannii was studied and revealed the upregulation of the adeJ gene, a component of the multidrug efflux pump adeIJK[2]. It was observed that the deletion of this gene increased the susceptibility to PUFAs. The adeIJK pump fulfills a similar role as the emhABC pump from Pseudomonas fluorescens, which controls lipid homeostasis through the efflux of endogenous long-chain fatty acids, both saturated and monounsaturated[3].
The results of the above studies on Gram-negative bacteria indicate that Gram-negative bacteria, including S. fredii, have mechanisms to transport fatty acids from inside the cell to outside. So, it is possible to transport DHA/EPA from rhizobia to soybeans by modifying our S. fredii to overexpress these specific transporter proteins.
With this possibility, we can get more nutritious soy, made into a range of soy products. (see more details in implementation)
Products and Promotion
The iGEM philosophy has never advocated building castles in the air, CAU-China actively engages with the global community, dedicated to thinking about how to truly bring our project to helping the world.
Therefore, we are concerned about the downstream of our project and envisioned the product, the rhizobia inoculants. we provided product instructions and supporting application equipment for the rhizobia inoculants to suit different situations. When the rhizobia inoculants need to be promoted, we plan to train agricultural technicians among local farmers. This will not only ensure the sustainability of our technology itself but also have long-term sustainable educational significance for local farmers.
This dual sustainability guarantees the continuous application of the project. Our nutrient-rich nodules and soybeans will become more competitive raw materials and are expected to be processed into higher yield downstream products beyond soybean oil and soybean milk. These products are expected to stimulate the emergence of new processing plants, realize the expansion of the chain, and bring more diverse and nutritious food to the local people, achieving sustainable development.(see more details in implementation)
References
- [1] Food and Agriculture Organization of the United Nations (FAO) etc. (2024). The State of Food Security and Nutrition in the World 2024: Financing to end hunger, food insecurity and malnutrition in all its forms. Rome: FAO.
- [2] Jiang, J. H., Hassan, K. A., Begg, S. L., Rupasinghe, T. W. T., Naidu, V., Pederick, V. G., Khorvash, M., Whittall, J. J., Paton, J. C., Paulsen, I. T., McDevitt, C. A., Peleg, A. Y., & Eijkelkamp, B. A. (2019). Identification of Novel Acinetobacter baumannii Host Fatty Acid Stress Adaptation Strategies. mBio, 10(1), 1–6. https://doi.org/10.1128/mBio.02056-18
- [3] Adebusuyi, A. A., & Foght, J. M. (2011). An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a. BMC Microbiology, 11, 252. https://doi.org/10.1186/1471-2180-11-252