. Description .

1. Inspiration

In recent years, EI Nino phenomenon has caused drought and soil frost in several regions around the world. Soil frost and drought, derived from environmental and climatic disasters, can bring great hazards to agriculture and forestry. This year, we have decided to dedicate our research efforts to finding solutions for the severe challenges.

2. Background

Agriculture serves basic human needs by producing food, feed, and fibers. More resilient, sustainable, and inclusive food systems are critical to achieving the world's development goals.

The prevalence of food deficiency globally rose from 8.0% to 9.8% from 2019 to 2021, and it was estimated that about 828 million people worldwide were experiencing starvation in 2021 (1), which highlights the urgency and significance of addressing food issues. Agriculture is the primary source of food production and also accounts for 4% of global gross domestic product (GDP), and in some least developing countries, it can account for more than 25% of GDP (2). In China, the total output value of agriculture in 2022 reached 8.83 trillion yuan, accounting for 7.3% of that year's GDP (3). Agriculture development is one of the most powerful tools to end extreme poverty, boost shared prosperity, and feed projected 9.7 billion people by 2050 since the growth in the agriculture sector is two to four times more effective in raising incomes among the poorest compared to other sectors (4).

3. The problem

Seasonally frozen soil, freezing in winter and thawing in summer, alternating once a year is widely distributed in China. The thickness of seasonally frozen soil ranges from ~0.5 m to ~3 m in Northeast, North, and Northwest China. In 2022, a total of 870.7 thousand hectares of crops in China were damaged due to low temperatures, with a direct economic loss of 12.45 billion yuan. In addition, China encountered a nationwide cold wave in April 2023, which caused a large drop in temperature. Overall, about 1.899 million hectares of arable area were affected by low-temperature, with direct economic losses of 1.36 billion yuan (5).

Globally, frost and freezing temperatures caused severe damage to crops, including vineyards and fruit trees, oilseed rapes, potatoes, and sugar beets across Europe from early April 2021. In France, about 80% of the country's wine and fruit tree regions were affected, with the extent of the damage also substantial in Italy, the Czech Republic, and the UK (6).

Similarly, flash drought comes on fast—drying out soil in a matter of days to weeks. If a flash drought occurs at a critical point in the growing season, it could devastate an entire crop and cause huge economic losses (7). Fujian Province is particularly susceptible to droughts in autumn due to the rainless, hot weather caused by subtropical highs in the South China Sea. According to statistics, 6,090.2 thousand hectares of crops were affected by drought in China in 2022 (8), primarily concentrated in the south of the middle and lower reaches of the Yangtze River, with a direct economic loss of 51.28 billion yuan (9). Furthermore, we found that flash drought risk over cropland is expected to increase globally, with the largest increases projected across North America (change in annual risk from 32% in 2015 to 49% in 2100) and Europe (32% to 53%) (10).

In short brief, both soil drought and soil frost have significant adverse effects on agriculture globally, which can lead to decreased crop productivity, reduced food production, and economic losses for farmers. Additionally, these conditions can also result in soil degradation, erosion, and loss of soil fertility. Countermeasures that have the potential to decrease impacts are increasingly adopted, but there is still a lot of room for improvement.

4. The current solution

At present, people primarily resorts to traditional methods to address these problems in China. Methods for soil drought including constructing water conservancy projects, mobilizing water sources, growing drought-resistant crops, and using chemical water-retention material. However, these solutions either involve substantial engineering work or require expensive and complicated synthesis, separation, and purification techniques. As for frost damage, preventive measures like greenhouses, mulching films, and straw burning are currently used, which are neither convenient, efficient, nor eco-friendly. Therefore, we need to take action to develop new strategies to mitigate the effects of drought and freeze stress without delay.

5. Project

To address these challenges mentioned above, we design two production paths. Firstly, an antifreeze protein production path with a cold-responsive system is put forth. Meanwhile, an automated drought response system for water-retention material's production in terms of soil drought is designed. These two production paths are to tackle the soil frost and drought problems, respectively.

5.1 Anti-icing

Antifreeze proteins (AFPs) are extensively identified in different cold-tolerant species, which can facilitate the persistence of cold-adapted organisms by decreasing the freezing point of their body fluids (11). We choose two antifreeze proteins: TmAFP and SfIBP, noted for their high thermal hysteresis and ice recrystallization inhibitory activities, respectively (12), which can inhibit the occurrence of soil frost, effectively and environmental-friendly.

We expect to construct our engineered bacteria with an auto-low-temperature sensing function, so here we introduce the CspA low-temperature response system. The secondary structure of RNA's 5'-UTR and 3'-UTR is changed at low temperatures (13), which promotes the expression of downstream genes. Based on the cold shock response mechanism of RNA, we introduce the 5'-UTR and 3'-UTR sequences of the CspA system as a temperature control switch to realize the expression of antifreeze proteins in a low-temperature environment.

To optimize our engineered bacteria, we also take the stress resistance design and the surface display of cellulose-binding proteins design into consideration.

5.2 Anti-Drought

1. BC/HA complexity as water-retention material

Bacterial celluloses (BCs) have good water retention and repair properties, and in-situ modification of BC with hyaluronic acid (HA) improves the water-holding capacity of cellulose composite (14). E. coli Nissle 1917 and E. coli BL21(DE3) are co-cultured in the fermenters producing BCs and HA, respectively, eventually forming as BC/HA compounds.

2. Bio-factory for production

To reduce cumbersome chemical treatment procedures, we design a set of co-culture systems for two engineering bacteria to obtain BC/HA cross-linked polymers automatically (15).

We further equip the biological factory with monitoring Hardware, which can realize the functions of automatically synthesizing and deploying water-retention material.

6. Biosafety

Given that soil is an open environment, for the sake of biosafety, we are obliged to prevent engineering bacteria leakage. Additionally, due to the soil microbial diversity, it is also important to prevent horizontal gene transfer (HGT).

In that case, we use the ccdB/ccdA toxin/antitoxin system (16). The ccdA gene, which encodes the antitoxin CcdA conjugate to CcdB, is introduced into the genome of the engineered bacteria. The strong constitutive promoter J23106 is used to regulate its expression. The ccdB gene, which encodes a toxin with broad-spectrum toxicity, is regulated by a weak constitutive promoter J23109 and inserted into the gene expression vector. Once HGT occurs, the ccdB gene will encode toxins in genetically unrelated organisms, leading to their death.

7. Reference

1. https://www.fao.org/3/cc0639zh/online/cc0639zh.html
2. https://www.worldbank.org/en/topic/agriculture/overview
3. https://www.statista.com/statistics/270325/distribution-of-gross-domestic-product-gdp-across-economic-sectors-in-china/
4. https://www.worldbank.org/en/topic/agriculture/overvie
5. https://www.mem.gov.cn/xw/yjglbgzdt/202105/t20210508_384756.shtml
6. J. R. Lamichhane, Rising risks of late-spring frosts in a changing climate. Nat. Clim. Change. 11, 554-555 (2021).
7. Y. Qing, S. Wang, B. C. Ancell, Z.-L. Yang, Accelerating flash droughts induced by the joint influence of soil moisture depletion and atmospheric aridity. Nat. Commun. 13, 1139 (2022).
8. https://www.mem.gov.cn/xw/yjglbgzdt/202105/t20210508_384756.shtml
9. J. Xia, J. Chen, D. She, Impacts and countermeasures of extreme drought in the yangtze river basin in 2022. J. Hydraul. Eng. 53, 1143-1153 (2022).
10. J. I. Christian et al., Global projections of flash drought show increased risk in a warming climate. Commun. Earth Environ. 4, 165 (2023).
11. A. Baskaran et al., Anti freeze proteins (Afp): Properties, sources and applications - A review. Int. J. Biol. Macromol. 189, 292-305 (2021).
12. U. S. Midya, S. Bandyopadhyay, Elucidating the Sluggish Water Dynamics at the Ice-Binding Surface of the Hyperactive Tenebrio molitor Antifreeze Protein. The Journal of Physical Chemistry B. 127, 121-132 (2023).
13. A. M. Giuliodori et al., E. coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression. Front. Microbiol. 14, 72 (2023).
14. S. Tang et al., A covalently cross-linked hyaluronic acid/bacterial cellulose composite hydrogel for potential biological applications. Carbohydr. Polym. 252, 117123 (2021).
15. K. Liu, J. M. Catchmark, Bacterial cellulose/hyaluronic acid nanocomposites production through co-culturing Gluconacetobacter hansenii and Lactococcus lactis in a two-vessel circulating system. Bioresour. Technol. 290, 121715 (2019).
16. O. Wright, M. Delmans, G. B. Stan, T. Ellis, GeneGuard: A modular plasmid system designed for biosafety. ACS Synth Biol. 4, 307-316 (2015).