. Design .

1. Overview

Extreme catastrophic climates, such as drought and frost, not only become a major barrier to agricultural development but also bring enormous threats to global food security. Many economic agricultural products, such as tea and navel orange, suffer the negative effects of drought and frost. Unfortunately, the existing approach cannot protect the tea and navel orange trees from drought and frost absolutely, which would not only lower the production and quality but also cause the death of the trees. After brainstorming, we decided to design the gene circuit and regulate the expression of target products, combining with software and hardware equipment to protect the crop from the damage of extreme weather.

2. Solutions of soil frost

2.1 Antifreeze Proteins (AFPs)

The formation of ice crystals at subzero temperatures will cause frost damage, so we choose AFPs to mitigate damage from freezing. Their functions can be characterized by thermal hysteresis (TH) and ice recrystallization inhibition (IRI). TH is the ability to lower the freezing point (T_f) of water, causing a gap between the freezing point and melting point (T_m) (1). IRI is the ability to inhibit the growth of small ice crystals into larger ones, which can prevent further damage to cells from large ice crystals (2).

Two different AFPs, TmAFP from Tenebrio molitor and SfIBP from Shewanella frigidimarina were selected and applied in our project. TmAFP has a higher TH and can lower freezing point by 3-6 °C (3). SfIBP has a higher IRI, showing the lowest concentration that has IRI activity is 5 nM, which is much lower than most AFPs (4). We introduce both AFP genes into E. coli, allowing them to secrete both types of AFPs and completely playing an important role in their TH and IRI in low temperatures.

Since AFPs need to be bound to the surface of the ice to exert their functions, the engineered E. coli need to secrete AFPs into the soil. The signal peptide LMT, found from Vibrio natriegens by 2021 XMU-China has shown high secretion efficiency in E. coli. The other signal peptide Kp-SP, found in Kocuria sp. 3-3, has shown remarkable function to secret target proteins to the extracellular circumstance in previous research (5). Therefore, these two signal peptides are selected to accomplish the secretion of AFPs.

2.2 Low-temperature response

2.2.1 Cold-responsive elements (CREs)

In the low-temperature environment, the 5'-UTR of some mRNAs can form stem-loop structures (6), which can inhibit the translation of downstream genes and thus inhibit bacterial growth. However, relying on cold shock proteins, some bacteria can recover their physiological activities after a period of growth arrest. For example, E. coli can achieve this using the CspA protein, whose expression is controlled by the CspA CRE. CspA CRE can promote the expression of downstream genes in engineered bacteria at temperatures below 15 °C, which is lower compared to other existing microbial CREs. This excellent mechanism provides us with inspiration to prevent and deal with frost damage automatically.

Fig. 4a painted the CspA CRE at high temperatures, with the SD region hidden in the steam-loop region. Fig. 4b painted the situation at low temperatures, the SD region was exposed out due to the interaction between mRNA 5'-UTR and 3'-UTR, promoting the expression of downstream genes. Based on this mechanism (7), we design a low-temperature responsive expression system, referred by the literature-reported pCold plasmid (8). At the same time, to improve the expression level of target proteins, we introduced the TEE sequence to promote the binding of ribosomes to the mRNA (9).

The CspA CRE is effective in promoting gene expression around 15 °C, but it has a broad range of the response temperature, which may not only cause additional growth burden to our engineered bacteria but also affect the expression of AFPs due to leakage of a downstream gene above 15 °C. To solve this problem, we designed a logic AND gate, working with the CspA CRE.

2.2.2 AND gate of hypersensitive response and pathogenicity (hrp) system

The hrp system in Pseudomonas syringae includes the HrpR protein, HrpS protein, and the pHrpL. The HrpR or HrpS protein alone will not activate the pHrpL. But when both HrpR and HrpS proteins are expressed, they can form a complex that activates the pHrpL and induces downstream gene expression. Based on it, we design an AND gate to respond to low temperatures, namely, the hrp AND gate, in which the hrpR and hrpS genes are regulated by the CspA CRE. Under low-temperature conditions, the downstream genes can only be induced when both proteins are expressed, reducing the leaky expression (10).

2.2.3 AND gate of VSW-3 RNA Polymerase system

The VSW-3 RNA polymerase (VSW-3 RNAP) is found in a cold-adapted bacteriophage called VSW-3 (11). It generates fewer dsRNA byproducts compared to T7 RNA Polymerase (T7 RNAP), which also possesses stronger transcriptional and expressive activity at low temperatures.

Based on VSW-3 RNAP, we design another low-temperature response AND gate expression system. We cut the VSW-3 RNAP into two peptide fragments on suitable sites, which can be regulated by the CspA CRE. Then the full-length RNAP is reassembled using inteins NpuN from Nostoc putiforme and SspC from cyanobacterium PCC6803 (12). These inteins can improve the efficiency of protein trans-splicing. Thus, AFPs can be only expressed in the low-temperature environment when both peptide fragments are expressed and reassembled.

2.3 Optimization of Engineered Bacteria

2.3.1 Stress resistance improvement of E. coli

E. coli is a mesophilic bacterium that grows well in the temperature range of 21 °C to 49 °C. And its lowest observed growth temperature is 7.5 °C. To achieve the response to low-temperature signals (below 15 °C) and the secretion of AFPs, it is necessary to increase E. coli stress resistance in low-temperature environment.

Endogenous chaperone proteins GroEL and GroES can help E. coli correctly fold proteins or peptides, while activity is poor at low temperatures and cannot meet the normal growth requirements of E. coli. The homologous chaperone proteins Cpn60 and Cpn10 from Oleispira antarctica RB-8 T have been reported to have higher activity at low temperatures than GroEL and GroES. Furthermore, they can lower the lowest growth temperature of E. coli to -13.7 °C and extend its lower limit of suitable growth temperatures range to 4 °C (13).

Low-temperature environments can cause oxidative stress in E. coli, accumulating reactive oxygen species (ROS) in the cell. High levels of ROS can damage biomolecules and disrupt their functions. Previous studies have shown that expressing superoxide dismutase MpmMn-SOD from Micordera punctipennis in E. coli can efficiently erase ROS, thereby increasing E. coli's survival ability in low temperatures (14). Noticing that the product of MpmMn-SOD is H₂O₂, which is harmful to bacteria too. So, we further supplement katG in E. coli whose function is converting H₂O₂ to innoxious H₂O (15).

Therefore, we improve E. coli's growth ability in low-temperature environments from the aspects of increasing protein activity and maintaining the stability of the intracellular biomolecules. We plan to insert these genes into E. coli's genome to create a chassis that can adapt to cold conditions.

2.3.2 Crop root adsorption

To avoid irrigation water or rainwater washing away our engineered bacteria, we plan to make them bind to the surface of crop roots.

After investigation, we found that the surface of most crop roots contains cellulose (16). Cellulose-binding module (CBM) is a class of proteins with cellulose-binding function, most of which are structural domains in cellulases. Cellulose-xylanase from Cellulomonas fimi contains CBM, which has a strong ability to bind to cellulose irreversibly. At the same time, it performs excellently at low temperatures and in a wide pH range (17, 18), meeting the requirement of environmental conditions for our engineered bacteria applied.

To realize the adsorption of engineered bacteria on crop roots, CBM needs to be displayed on the surface of bacteria. E. coli's endogenous protein MipA is an outer membrane anchoring element used for protein display on bacterial cell surfaces. Its C-terminal truncated mutant MV¹⁴⁰ has a much higher surface display efficiency than other display systems, which can significantly stabilize displayed proteins (19). Therefore, we decided to use MV¹⁴⁰ to display the CBM on the surface of E. coli.

3. Solutions of soil drought

The main reason for drought in the soil is the deficiency of the water-retention ability of the soil (20).

3.1 Bacterial Cellulose (BC)

BC possesses excellent water-retention ability due to its high hydrophilic surface and porous three-dimensional structure. However, exorbitant prices limited its application as a water-retention material. Thus, E. coli Nissle 1917, a natural BC production strain, was selected as our chassis to generate BC. BcsA and BcsB are two catalytic subunits in BC synthase, in which BcsB also guides BC synthesized towards the outer membrane. These two proteins from Gluconacetobacter xylinus are more stable than their homologous proteins from Nissle 1917. Thus, we select bcsA and bcsB genes to improve BC production (21).

3.2 Hyaluronic Acid (HA)

Hyaluronic acid is a linear polysaccharide synthesized from D-glucose. It is a water-retention material with excellent performance due to its significant hygroscopicity. Previously, HA was extracted from organs of animals or produced through fermentation by Streptococcus spp. However, the high cost of separation and extraction and low productivity limit their applications. It's reported that E. coli owns the metabolic pathway for HA synthesis, except for the hyaluronan synthase HasA. Hence, we introduced the hasA gene from Streptococcus equi sub sp. Zooepidemicus (22) to E. coli BL21(DE3), which is a mature and non-toxic chassis, realizes the harmless production of HA.

3.3 Composite Water Retention Material

As BC has a unique porous three-dimensional network and abundant hydroxyl groups on its surface, when it is modified by HA, its water holding capacity will be enhanced, generating an excellent water-retention material (23). To save cost, and avoid complex manipulation of chemical crosslinking, we plan to co-culture these two engineered bacteria to achieve an assembly of HA on BC microfibrils.

Notably, we developed an unprecedented bio-factory for E. coli Nissle 1917 and BL21(DE3) co-culture fermentation, which can be combined with an irrigation system then is applicable to many kinds of farms.

As the ratio of HA and BC in the co-culture system is crucial for the property of the composite water retention material, it is necessary to regulate the production of HA and BC. To achieve this, we insert two fluorescent proteins RFP located on kanamycin resistance plasmid and GFP located on chloramphenicol resistance plasmid into E. coli Nissle 1917 (EcNP type) and BL21(DE3) respectively. By measuring the real-time red fluorescence intensity and green fluorescence intensity, we can obtain the density of the two strains. Hence, we can regulate the bacterial population density by adding different kinds of antibiotic.

4. Biosafety

There are two potential biosafety hazards associated with our engineered bacteria: one is the leakage of engineered bacteria into the environment, and the other is the potential horizontal gene transfer (HGT). These may pose potential risks to the environment and biodiversity.

In our implementation, we plan to mix engineered bacteria or products into the soil. To avoid the leakage of engineered bacteria, MazF is selected as the toxin by cleaving the mRNA of bacteria inside.

For the anti-icing part, we introduce the inverter to make engineered bacteria survive when arabinose is present and die when it runs out. It is worth mentioning that this idea is inspired by 2020 XMU-China.

While for anti-drought, as glucose is the production ingredient, we choose to induce expression of MazF directly by arabinose. When glucose exhausts, arabinose will play its role to lead bacteria to death.

Given that soil contains countless microorganisms with diverse forms and a wide range of species, it is even more important to prevent HGT (24). We use ccdB/ccdA toxin/antitoxin genes to prevent HGT. Gene ccdB encodes a broad-spectrum toxin CcdB, which is inserted into a gene expression vector and its expression is regulated by a weak constitutive promoter J23109. Gene ccdA encodes the corresponding antitoxin CcdA, which is introduced into the genome of the engineered bacteria and its expression is regulated by a strong constitutive promoter J23106. In the engineered bacteria, the expression level of ccdA exceeds that of ccdB, and the toxicity of CcdB is inhibited. However, once the gene expression vector undergoes HGT, the ccdB carried on it will express in other microorganisms and lead them to death, preventing HGT effectively.

5. Designs that fail to be used

We also considered another CRE, RNase E-cleavable RNA Thermosensor (RNAT) (25), but we were unable to implement it due to experimental limitations. It is a low-temperature responsive post-transcriptional regulatory element, whose 5'-UTR contains a RNase E binding site and its complementary sequence. At different temperatures, the RNase E binding site and its complementary sequence form different structures, which determine whether the RNA is degraded or not.

Besides, we chose the ParE/ParD toxin/antitoxin system to avoid HGT. ParE is a broad-spectrum toxic protein that can inhibit DNA gyrase, while ParD is its corresponding antitoxin. However, due to the high toxicity of ParE, the construction of the plasmid failed. Hence, we have to give up this system.

6. Reference

1. A. K. Gruneberg et al., Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis. Cyobiology 99, 28-39 (2021).
2. M. Chow-Shi-Yée et al., Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells. Protein Sci. 25, 974-986 (2016)
3. U. S. Midya, S. Bandyopadhyay, Elucidating the sluggish water dynamics at the ice-binding surface of the hyperactive Tenebrio molitor antifreeze protein. J. Phys. Chem. B 127, 121-132 (2023).
4. T. D. R. Vance, L. A. Graham, P. L. Davies, An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin. FEBS J. 285, 1511-1527 (2018).
5. Y. Cui et al., Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide. Protein Expr. Purif. 129, 69-74 (2017).
6. C. W. K. David, Y. J. Wui, D. V. Linh, P. C. Loo, Thermogenetics: Applications come of age. Biotechnol. Adv. 55, 107907 (2022).
7. Q. Guoliang et al., Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877-882 (2004)
8. A. M. Giuliodori et al., Escherichia coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression. Front Microbiol. 14, 1118329 (2023).
9. J. P. Etchegaray, M. Inouye, Translational enhancement by an element downstream of the initiation codon in Escherichia coli. J. Biol. Chem. 274, 10079-10085 (1999).
10. W. Baojun, K. R. I, J. Nicolas, B. Martin, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Commun. 2, 508 (2011).
11. X. Heng et al., Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. RNA Biol. 19, 1130-1142 (2022).
12. S. Yolanda, G. Magüi, I. Mark, A split intein T7 RNA polymerase for transcriptional AND-logic. Nucleic Acids Res. 42, 12322-12328 (2014).
13. Ferrer, M. Chernikova, T. N. Yakimov, M. M. Golyshin, P. N. Timmis, Kenneth N., Chaperonins govern growth of Escherichia coli at low temperatures. Nat. Biotechnol. 21, 1267 (2003).
14. X. Zilajiguli, M. Ji, L. Xiaoning, Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells. PeerJ 8, e8507 (2020).
15. M. Asma et al., Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis. Structure 29, (2021).
16. R. Maiti, P. Satya, D. Rajkumar, A. Ramaswamy. Crop plant anatomy. Wallingford, UK: CABI. 2012.
17. E. Ong, N. R. Gilkes, R. C. Miller Jr, R. A. J. Warren, D. G. Kilburn, The cellulose-binding domain (CBD(Cex)) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide. Biotechnol. Bioeng. 42, 401-409 (1993).
18. T. Reinikainen, K. Takkinen, T. T. Teeri, Comparison of the adsorption properties of a single-chain antibody fragment fused to a fungal or bacterial cellulose-binding domain. Enzyme Microb. Technol. 20, 143-149 (1997).
19. H. M. Jung, Novel bacterial surface display system based on the Escherichia coli protein MipA. J. Microbiol. Biotechnol. 30, 1097-1103 (2020).
20. M.-T. Luo et al., Bacterial cellulose based superabsorbent production: a promising example for high value-added utilization of clay and biology resources. Carbohydr. Polym. 208, 421-430 (2018).
21. S. Elaheh et al., Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacterxylinus in E. coli Nissle 1917. Biotechnol. Bioprocess Eng. 42, (2019).
22. Lai, Z. W., Teo, C. H., Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture. Malaysian J. Sci.. 15, 575-582 (2019).
23. K. Liu, J. M. Catchmark, Bacterial cellulose/hyaluronic acid nanocomposites production through co-culturing Gluconacetobacter hansenii and Lactococcus lactis under different initial pH values of fermentation media. Cellulose 27, 2529-2540 (2020).
24. W. Oliver, D. Mihails, S. Guy-Bart, E. Tom, GeneGuard: A modular plasmid system designed for biosafety. ACS Synth. Biol. 4, 307-316 (2015).
25. H.-O. C. Allison, H. Kristina, K. Lukas, M. T. Seok, De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic Acids Res.. 43, 6166-6179 (2015).