Design
Fresh produce keeps respiring after harvest and is very sensitive to gases like ethylene. Climacteric fruits release ethylene, which accelerates their ripening and deterioration nearby produce. Since we often judge freshness only by appearance, edible food is sometimes thrown away. And if deteriorated food is left alone, safety risks like foodborne illness increase. To solve this problem, we developed PreserVEG, a Cre/loxP based Biosensor for climacteric fruits and vegetables. We designed a biosensor that detects ethylene metabolites emitted by fruits and vegetables, and a GFP expression circuit that activates upon sensing them. This enables immediate visual assessment of ripening-induced deterioration at retail stores and in homes. Additionally, we incorporated nisin expressions to suppress microbial growth on fruit and vegetable surfaces, enhancing antimicrobial properties.
Ethylene-inducible EtnR1 and EtnR2 were used to detect ethylene-induced deterioration. EtnR1 and EtnR2 utilize genes involved in ethylene metabolism from Mycobacterium chubuense NBB4 (1). EtnR2 detects epoxyethane (ethylene monoxide), sending a signal to EtnR1. Upon receiving the signal, EtnR1 becomes phosphorylated, binds to EtnP, and activates it(1)(2).
EtnR2 is a transcription factor that does not respond to ethylene itself but detects epoxyethane. Therefore, ethylene released from fruit and vegetable must be oxidized to epoxyethane. In a previous study using Mycobacterium(2), this conversion was performed by etnABCD (alkenes monooxygenase). However, since this enzyme was found to have low expression and function in E. coli, we designed our system based on the enzyme Toluene o-xylene monooxygenase (TOM). TOM is derived from Burkholderia cepacia G4 and is an iron-centered monooxygenase(3). It activates O₂ using NADH as an electron donor and epoxidizes hydrocarbons by inserting one oxygen atom into the C=C bond. However, TOM's active site is large, while ethylene is a small molecule. This poses a problem that ethylene struggles to bind to the reaction center with the proper orientation. Therefore, we designed codon-optimized variants A113 and V106F. A113F is a mutation where alanine at position 113 is changed to phenylalanine, and V106F is a mutation where valine at position 106 is changed to phenylalanine(4). This has been reported to enhance enzymatic activity. (5)
The Cre-loxP system is a genetic recombination experimental system that utilizes the site-specific recombination reaction occurring when Cre, a DNA recombination enzyme, acts on DNA sequences called loxP sequences. When two loxP sequences are oriented in the same direction, Cre excises the gene between the loxP sequences, resulting in its circularization. The components used in the plasmid are listed in the table below.
| Parts | Explanation | References |
|---|---|---|
| T7_Promoter-lac_operator | Promoter activated by IPTG induction | BBa_K4437002(6) |
| NusA-NisinQ | Gene encoding the antimicrobial protein NisinQ, co-expressed with NusA to enhance solubility, and containing a His-tag and TEV protease cut site inserted between NusA and NisinQ. | BBa_K4437002(6) |
| loxP | DNA sequence that serves as the recombination target for Cre recombinase | Nathan Tague et al., 2023(7) |
| T7_terminator | Terminator sequence that terminates transcription by T7 RNA polymerase | Nathan Tague et al., 2023(7) |
| SuperfolderGFP | Green fluorescent protein derived from Aequorea victoria | Nathan Tague et al., 2023(7) |
When ethylene levels are low (below the threshold), EtnP which regulates Cre expression remains inactive. Consequently, Cre is not expressed, and nisin expression between the loxP sites continues, maintaining antimicrobial activity.
When ethylene levels reach the threshold, EtnP is activated by EtnR2's detection of epoxyethane, triggering Cre expression. The expressed Cre binds to both ends of the loxP sequence and excises the loxP region. This halts nisin expression and simultaneously removes the terminator upstream of GFP, allowing GFP expression to occur.
We focused on the antimicrobial peptide Nisin Q to not only detect deterioration with ethylene but also simultaneously enhance safety. Nisin Q exhibits broad antimicrobial activity and spectrum against Gram-positive bacteria, is effective for quality preservation, and is actually used as a food additive. The system was designed to incorporate Nisin Q into the loxP site, maintaining the antibacterial effect of Nisin Q until degradation is detected, and switching functions at the point of degradation detection. Nisin inhibits bacterial growth by forming pores in the cell membrane of target bacteria, causing the leakage of intracellular substances, thereby exhibiting antibacterial activity against Gram-positive bacteria. The detailed mechanism of action is as follows: (8)
1. The N-terminus of Nisin binds to lipid II, a cell wall precursor, through interaction with its pyrophosphate group.
2. Using the N-terminal binding as a foothold, the highly hydrophobic C-terminal side reorients itself inward toward the membrane and inserts. Intermolecular hydrogen bonds form and strengthen between Nisin molecules and between Nisin and Lipid II. This results in the formation of a complex comprising 8 Nisin molecules and 4 Lipid II molecules. 3. The Nisin-Lipid II complex forms a pore approximately 2.5 nm in diameter. The leakage of small molecules and ions such as H⁺, K⁺, and ATP through this pore causes the collapse of the membrane potential, thereby inhibiting bacterial proliferation.
3. The Nisin-Lipid II complex forms pores approximately 2.5 nm in diameter. The leakage of small molecules and ions such as H⁺, K⁺, and ATP through these pores causes the collapse of the membrane potential, thereby inhibiting proliferation.
This system enables the oxidation of ethylene emitted from fruits and vegetables using a monooxygenase strain, followed by detection in a sense strain expressing EtnR1/R2. In this way, the ageing of fresh produce can be visualized, and the chain reaction of deterioration caused by ethylene can be prevented. Furthermore, prior to visualization, the antimicrobial action of NisinQ can lessen concerns regarding microbial contamination on the surface of fresh produce. The application of this system can contribute to the reduction of FLW.
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