Food is the foundation of people's livelihood. However, at present, farmland in many areas is being attacked by drought, diseases, insect pests, etc., especially people in backward areas are still hungry. Biopesticides/plant growth regulators are a class of substances used to regulate plant growth and development, which are generally produced by artificial synthesis or microbial fermentation, and their physiological and biological effects are the same or similar to natural plant hormones (a class of compounds naturally present in plants). It has the advantages of strong selectivity, low toxicity, high efficiency, low impact on humans, animals and the ecological environment, and is not easy to produce drug resistance. Among them, 5-aminolevulinic acid (5-ALA), as a typical class of biopesticides, can improve the resistance of crops to abiotic and biotic stresses, and promote crop yield. In this project, we have developed a method for the high yield of 5-ALA from microorganisms through synthetic biology techniques, and in this page, we will focus on the current problem and our solution.
1、What is the global agriculture facing?
A worsening global climate, salinization of arable land, drought, diseases and pests are threatening the sustainability of agriculture in many parts of the planet and are bound to cause a series of undesirable global problems.
Arable land resources are the basis for ensuring food supply. However, by 2050, more than 50% of the world's arable land is expected to be salinized, which will cause more soils to lose productivity [1]. In addition, low temperature stress can lead to the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) in plants, thereby disrupting the homeostasis of plants, damaging plant tissues, and even leading to plant death . As a major agricultural country, China will cause direct economic losses of 4.9 billion yuan due to low temperature freezing and snow disasters in 2023, and 20.974 million people will be affected by drought, with 38.037 million hectares of crops affected and direct economic losses of 20.6 billion yuan [3].Some fungi and weeds affect the physiology of crops by infecting plants or competing for nutrients, resulting in crops that cannot grow and develop properly. Therefore, improving the resistance of crops to abiotic and biotic stresses [6] is of great significance for promoting good food yields and alleviating the global food crisis.
2、What is the problem with current pesticides?
Often, chemical pesticides are used to kill germs, remove insects, and promote crop growth. However, improper use of these pesticides can lead to chemical residues, and long-term consumption of contaminated vegetables can increase the risk of cancer, arteriosclerosis, and cardiovascular disease. In addition, the loss of pesticides into the environment can pollute air and water sources, cause soil compaction, and even increase the resistance of bacteria and pests to pesticides [4]. Although traditional fungicides are able to destroy pathogenic bacteria, they also indiscriminately kill or inhibit beneficial fungi in plants and do not effectively regulate plant growth and development. Therefore, the development of a highly efficient, non-toxic and non-polluting biopesticide is of great significance to alleviate the global food security problem.
5-Aminolevulinic acid (5-ALA) is a natural, biodegradable non-protein amino acid that can be synthesized in plant metabolic pathways and plays an important role in plant seed germination, vegetative growth and fruit coloration. 5-ALA directly or indirectly regulates the photosynthesis, nutrient uptake capacity, antioxidant defense system and osmotic pressure [5] of plants, promotes the proliferation of plant probiotics, and inhibits the growth of pathogenic bacteria, thereby significantly improving the resistance of crops to abiotic and biotic stresses, and improving crop yield and quality. For example, in some walnut varieties, in order to maintain water balance, the leaf stomata are closed in order to maintain water balance, resulting in reduced carbon dioxide uptake and photosynthetic efficiency. The presence of 5-ALA can enhance the photosynthesis, osmoregulation and antioxidant system of walnut, and improve its tolerance to osmotic stress. In addition, 5-ALA can also selectively kill dicotyledon weeds in meadows such as wheat and corn, while causing biochemical and metabolic imbalances in lepidopteran and dipteran insects such as trichomoniasis, fruit flies, and cockroaches, leading to spasms and even death [7]. It is important to note that 5-ALA has very low toxicity to humans and the environment, is easily degraded and has no residue, so it can be safely used in agroforestry production.
At present, 5-ALA is mainly synthesized by chemical synthesis and microbial fermentation. The study of chemical synthesis began in the 50s of the 20th century, and was most active in the 90s. The researchers synthesized 5-ALA [9] using raw materials such as hippuric acid, succinic acid, tetrahydrofurfuramine, and levulinic acid. However, the chemical synthesis method has the disadvantages of high price, difficult to obtain, high toxicity, low production yield and harsh reaction conditions, and the synthetic products contain impurities that are difficult to determine, which may cause potential harm to agricultural and forestry [8]. In this project, E. coli was used as the chassis to screen strains that can produce stable and high yields of 5-ALA through enzyme modification, CRISPR-associated transposon (CAST) system, and microfluidic high-throughput screening. Finally, the efficient and sustainable production of 5-ALA will be realized, thereby reducing the cost of biopesticides and contributing to the alleviation of the global food crisis.
At present, with the rapid development of green and sustainable agriculture in the world, many effective but toxic chemical pesticides have been gradually withdrawn from the market, and biopesticides have emerged in sustainable agriculture. According to IHS Markit, the global biopesticide market is expected to grow at a compound annual growth rate of about 10% from 2020 to 2025, and the market size will exceed $8 billion by 2025. At this growth rate, the market size is expected to reach approximately USD 8.8 billion by 2026.
(unit: 100 million dollars)
Policy is one of the most important factors affecting the pesticide industry. With the country's increasing attention to environmental issues and the urgent need for sustainable development in all walks of life, China has introduced a series of policies since 2020 to vigorously promote the development of biopesticides. For example, in September 2020, the "Notice on Promoting the Implementation of Green Channel Management Measures for Pesticide Registration and Approval": clearly accelerate the registration and approval process of biological pesticides, highly toxic pesticide substitutes, and characteristic small crops, and promote the high-quality development and green development of pesticides;
2021.12 "The 14th Five-Year Plan for the Development of the National Planting Industry": decided to phase out 10 kinds of highly toxic pesticides such as phosphine and aldicarb in phases and batches, and vigorously develop high-efficiency/low-toxicity and low-risk pesticides;
2022.1 "The 14th Five-Year Plan for the Development of the National Pesticide Industry": decided to give priority to the development of biopesticides, including biopesticides (such as Beauveria bassiana, Metarhizium aeruginosa), agricultural antibiotics (such as spinomycin, chunleimycin), biochemical pesticides (such as plant anti-attractants), RNA and small peptide biopesticides.
All of the above shows that China has strong support for the development of high-efficiency, low-toxicity and sustainable biopesticides, and the biopesticide market is expected to grow rapidly. This also provides us with encouragement and support for the continuous development of our projects.
1. The engineering bacteria we selected: E.coli
At present, the production of 5-ALA in industry often uses plant endophytes, such as some photosynthetic bacteria. They often require light, and their growth is retarded, fermentation conditions are complex, and input costs are high, which limits the efficient production of 5-ALA. Escherichia coli is an important model industrial microorganism, with a clear genetic background, complete carrier receptor system, simple culture, rapid growth, and easy separation of products, and has a wide range of applications in medicine, chemical industry, agriculture, etc.
At present, the main pathways for microbial synthesis of 5-ALA are C4 and C5. In the C4 pathway, the precursors glycine and succinyl-CoA are catalyzed by 5-aminolevulinic acid synthetase (ALAS) in the presence of the essential cofactor pyritholaldehyde 5-phosphate (PLP) in a 1:1 ratio to form 5-ALA; The C5 pathway uses glutamic acid as a precursor and catalyzes the synthesis of 5-ALA by three enzymes: glutamine-tRNA synthetase (GltX), NADPH-dependent glutamine-tRNA reductase (HemA), and glutamine-1-semialdehyde aminotransferase (HemL). Considering that the C5 pathway is complex, the C4 pathway only needs a one-step enzyme-catalyzed reaction, and the added exogenous substrate glycine is relatively inexpensive, we chose the C4 pathway as the main pathway for the production of 5-ALA.
Among them, ALAS is a key enzyme in the synthesis of 5-ALA in the C4 pathway, so we screen highly active ALAS for stable expression in E. coli to improve catalytic efficiency.
2. Our technical means:
(1) Enzyme modification and CRISPR-associated transposon system
In order to improve the activity of ALAS enzymes, we decided to enzymatically modify ALAS and introduce E. coli BL21 (DE3) for fermentation validation.
When the plasmid enters the strain through transformation, the plasmid in the strain is often lost to varying degrees with the progress of fermentation, which seriously affects the stable synthesis of the product. Therefore, there is a need to adopt new strategies to improve the genetic stability of ALAS expression plasmids.
The CRISPR-Cas system has attracted extensive attention due to its excellent gene editing ability, but the limited homologous recombination efficiency has limited the application of the CRISPR-Cas system. We were inspired by two articles on CRISPR transposons published in Science and Nature in 2019. CRISPR-associated transposon (CAST) system is a unique system of mobile genetic elements that bind transposable proteins to Cas proteins lacking nuclease activity to catalyze the targeted integration of exogenous DNA in the genome. In this system, the cascade complex formed by the Cas protein is guided by the crRNA to a specific site in the DNA, which subsequently recruits the transposase,Integrate a donor DNA fragment with a transposase end recognition sequence downstream of the target site.Without relying on host DNA repair mechanisms,The CAST system allows for precise targeting of inserted DNA in the host genome, so that the exogenous genes are stable in the strain [10, 11]. We used this technique to integrate the ALAS gene into the E. coli chromosome, allowing it to be stably expressed, thus avoiding the negative impact of plasmid loss on yield.
(2) Exploring new targets: enzyme-constrained models
According to the report, the current modified targets to increase the yield of 5-ALA are mainly related enzymes directly involved in the synthesis, and many key participating factors have not yet been discovered, and these factors are likely to limit the further improvement of 5-ALA biosynthesis. Genome-scale metabolic network models have achieved remarkable success in guiding metabolic engineering, but the traditional flux equilibrium analysis method only considers the constraints of stoichiometry and reaction direction, and the results obtained are often theoretically optimal, rather than the optimal reaction under actual conditions [12]. By introducing more realistic constraints into the metabolic network model, such as the limitation of enzyme content and thermodynamic feasibility, we can more accurately model the behavior of cells in different environments [13]. We use enzyme-constrained models to predict new target genes that can effectively increase the yield of 5-ALA, and it is possible to further increase the yield of 5-ALA by high-profile or knocking them out.
(3)Establish a high-throughput high-yield strain screening method
A. ARTP mutagenesis
ARTP (Atmospheric and Room Temperature Plasma) is rich in active energy particles that cause damage to the genetic material of organisms and induce biological cells to initiate SOS repair mechanisms. The SOS repair process is a kind of high-fault tolerance repair, so a variety of mismatch sites will be generated during the repair process, and finally the inheritance will be stable and mutant strains will be formed.
Therefore, we use ARTP mutagenesis technology to mutagenesize the engineered-strains, so as to obtain more mutant strains, which provides a basis for subsequent screening.
B. Droplet microfluidics
After the modification of the engineered strains is completed, it is important to establish a method that can efficiently screen and obtain the engineered strains with the target traits.
Microfluidic technology uses a chip with a special microchannel design to consume only a small amount of fluid in a short period of time, and can realize the generation, mixing, storage, culture, transportation, fusion, splitting, detection, sorting and other manipulation of droplets, showing extremely high efficiency and integration. Compared with traditional screening methods such as agar plate screening method and microplate screening method, droplet microfluidic technology miniaturizes the experimental operation to the micro scale, which not only simplifies the operation process, but also greatly reduces the amount of reagents, shortens the experimental cycle, and improves the level of automation, which has the remarkable characteristics of fast speed, high throughput and low [14].
Through this technology, we will encapsulate a single molecularly engineered E. coli in an independent droplet microreactor , and screen out strains with high 5-ALA production by detecting biomass. This technology will greatly reduce the time to screen high-yielding strains and shorten the experimental cycle.
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2. Xue, X., et al. 5-ALA Improves the Low Temperature Tolerance of Common Bean Seedlings through a Combination of Hormone Transduction Pathways and Chlorophyll Metabolism. Int. J. Mol. Sci., 2023. 24: p.13189.
3. Statistical Communiqué of the People's Republic of China on the 2023 National Economic and Social Development, released by the National Bureau of Statistics of China.
4. Ahmad, M.F., et al., Pesticides impacts on human health and the environment with their mechanisms of action and possible countermeasures. Heliyon, 2024. 10(7): p. e29128.
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10. Yang, J., et al., Synthetic biology for evolutionary engineering: from perturbation of genotype to acquisition of desired phenotype. Biotechnol. Biofuels, 2019. 12: p. 113.
11. Strecker, J., et al., RNA-guided DNA insertion with CRISPR-associated transposases. Science, 2019.
12. Schuetz, R., L. Kuepfer, and U. Sauer, Systematic evaluation of objective functions for predicting intracellular fluxes in Escherichia coli. Mol. Syst. Biol., 2007.
13. Massaiu, I., et al., Integration of enzymatic data in Bacillus subtilis genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains. Microb. Cell Fact., 2019.
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