Description

Methanol poisoning is a globally underreported health threat, responsible for thousands of cases each year, many resulting in permanent blindness or death [1]. Most incidents arise from improperly produced alcoholic beverages, where methanol is inadvertently or deliberately introduced due to poor quality control, economic pressures, or illicit production practices. Even minimal exposure, less than 0.1 mL per kg of body weight, can cause severe toxicity [2].

Global attention to this crisis typically only surfaces when tragedy strikes tourists, such as the Australian travellers whose deaths briefly made headlines [3]. Yet, methanol poisoning remains a persistent problem, with outbreaks occurring frequently worldwide and disproportionately affecting vulnerable populations, especially in Asia where the highest prevalence is recorded, including Indonesia, India, Cambodia, Vietnam, and the Philippines [1]. Untreated fatality rates range from 20% to 40% [4].

Despite the severity of this issue, there are currently no widely available or affordable detection methods. Existing technologies are either extremely expensive or require specialised expertise and equipment, putting them out of reach for many communities most at risk. In the absence of rapid, affordable, and robust screening tools, methanol poisoning will remain a significant threat to global public health.

The Detection Dilemma

Methanol (CH₃OH) is a transparent, colourless, volatile liquid that is indistinguishable from ethanol to human senses. When consumed, methanol is metabolized by alcohol dehydrogenase to formaldehyde, which is then converted to formic acid—the toxic compound responsible for metabolic acidosis, blindness, and death [5].

Current Methods

Laboratory Methods (HPLC, GC):

• Highly accurate and regarded as the gold standard for methanol quantification.
• Require expensive equipment, specialised training, and typically 30–60 minutes per analysis.
• Impractical for rapid or routine field screening [6].

Portable Devices (e.g., Spark M-20):

• Allow on-site analysis without laboratory infrastructure.
• Cost around $7000AUD, which is prohibitive for most at-risk communities.

Sigma CS0007 Methanol Quantification Assay Kit:

• Provides sensitive detection in trained hands.
• Reagents are acutely toxic, flammable, require cold storage, and pose regulatory and handling challenges.
• Best suited to professional laboratories with specialist personnel—not routine, field, or consumer use [7].

The whole cell biosensor design is built around a bacterial two-component system (TCS), inspired by successful biosensors in the literature but incorporating key innovations. These systems consist of a sensing histidine kinase and a response regulator that together allow bacteria to detect external stimuli and trigger specific genetic responses [8] [9] [10].

In its natural context, the E. coli CusS histidine kinase detects elevated copper ions and, upon activation, transfers a phosphate group to the response regulator CusR. Phosphorylated CusR then activates transcription of an efflux pump operon, enabling the cell to quickly export toxic metals [10].

For our biosensor, we have repurposed this system by replacing the native copper-sensing domain of CusS with the methanol-sensing domain from MxaY of Paracoccus denitrificans, creating a chimeric kinase that responds to methanol [9]. Upon methanol detection, the chimeric kinase phosphorylates CusR, which in turn drives expression of an amilCP reporter gene under the control of the CusR-dependent promoter. The result is a bright blue colour change that serves as a simple and visible signal for methanol detection (Figure 1).

The main components of the design are as follows and the interaction between them at molecular level is shown in Figure 2.

Chimeric Histidine Kinase:

• MxaY: Methanol sensing domain from Paracoccus denitrificans
• CusS: Native E. coli copper sensing histidine kinase, phosphorylates response regulator

Response Regulator:

• CusR: Native E. coli response regulator, induces expression of the CusR promoter

Reporter Gene:

• amilCP: An iGEM standardised part originating from Acropora millepora (Part: BBa_K592009).

The detection system is built around a methanol-specific methanol dehydrogenase (MDH) enzyme, which uses pyrroloquinoline quinone (PQQ) and Ca²⁺as essential cofactors [11]. This enzyme catalyses the conversion of methanol into formaldehyde, which can then be measured via a colorimetric assay, enabling rapid and selective methanol detection (Figure 3). By leveraging the catalytic power of MDH, this approach provides a sensitive, user-friendly method suitable for both field applications and consumer use.

The essential components for MDH expression and functionality are as follows and the methanol oxidation mechanism at molecular is illustrated in Figure 4.

MxaF: The large alpha subunit of the MDH, the primary catalytic component.
MxaI: The small beta subunit that joins MxaF to form the full MDH.
MxaA: Crucial for incorporating PQQ and Ca²⁺ into the MDH [12].
MxaK: Crucial for incorporating PQQ and Ca²⁺ into the MDH [12].

PQQ: The redox cofactor needed for methanol oxidation to formaldehyde.
Ca²⁺: Stabilises the PQQ and increases its activity, forming part of the active site.
Cytochrome C: Electron acceptor for the PQQ allowing continued redox ability.

Methanol poisoning is an underreported global health issue affecting thousands every year. Current testing methods are inaccessible to those who want to ensure their safety. MethaNO is engineering an accessible and affordable biosensor using synthetic biology. We aim to address this gap with practical solutions suitable for consumer use.
Our whole-cell biosensor uses a two-component system to detect and respond to methanol poisoning with a colorimetric output. This engineered E. coli system is still in proof-of-concept and has considerable GMO regulatory and safety concerns that will limit its use.
In contrast, the enzyme-based biosensor is the practical solution, utilising a purified MDH to convert methanol into detectable formaldehyde. This design is in the proof-of-concept stage but is the most promising option moving forward.
For a more detailed view of our process, see our Engineering, Experiments, and Results pages. MethaNO has been actively engaging with potential customers while developing a business plan, see our Entrepreneurship page. Our team has also enjoyed communicating synthetic biology in an accessible and engaging way to students, see our Education page.

1. Médecins Sans Frontières, “Outbreaks worldwide – Methanol poisoning,” MSF, 2024. [Online]. Available: https://methanolpoisoning.msf.org/en/outbreaks-worldwide/ (accessed Oct. 8, 2025).

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3. F. Drury, “Holly Bowles sixth to die of suspected methanol poisoning in Laos,” BBC News, Nov. 22, 2024. [Online]. Available: https://www.bbc.com/news/articles/ced94znq424o

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7. Merck/Sigma-Aldrich, "Safety Data Sheet: Methanol Quantification Assay Kit, Product Number CS0007, Version 6.6," Dec. 1, 2023. [Online]. Available: https://www.sigmaaldrich.com

8. V. Selvamani, I. Ganesh, S. H. Hong, and S. Park, “Construction of methanol sensing Escherichia coli by the introduction of novel chimeric two-component system,” Korean Journal of Chemical Engineering, vol. 34, no. 3, pp. 830–835, 2017.

9. I. Ganesh, S. H. Hong, V. F. Wendisch, and S. Park, “Construction of methanol-sensing Escherichia coli by the introduction of a Paracoccus denitrificans MxaY-based chimeric two-component system,” Journal of Microbiology and Biotechnology, vol. 27, no. 6, pp. 1106–1111, 2017.

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12. C. J. Morris, Y. M. Kim, K. E. Perkins, and M. E. Lidstrom, “Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1,” Journal of Bacteriology, vol. 177, no. 23, pp. 6825–6831, 1995, doi: 10.1128/jb.177.23.6825-6831.1995.