Problem Statement and Definition
Formaldehyde, a colorless chemical with a pungent odor, is highly reactive and possesses toxic properties. It is the simplest form of aldehyde (H-CHO) and is produced through the catalytic oxidation of methanol. This chemical is soluble in water, and a solution containing 37% formaldehyde is commonly used as a preservative, pesticide, and disinfectant.
Commercially manufactured, formaldehyde is a key ingredient in a wide range of products, including resins, plastics, textiles, wood products, adhesives, medications, and cosmetics. The primary exposure to formaldehyde occurs through inhalation, which can happen in both environmental and occupational settings. Indoor exposure to formaldehyde is more common than outdoor exposure due to the widespread use of formaldehyde-containing products. The World Health Organization recommends an indoor formaldehyde limit of 0.1 mg/m3 (0.08 ppm) [1].
Occupational exposure to formaldehyde varies and is prevalent in numerous industries, including manufacturing. Small quantities of formaldehyde are naturally produced in living organisms through normal metabolic processes such as DNA/RNA/histone demethylation and oxidative deamination. The concentration of endogenous formaldehyde in human blood is approximately 2-3 mg/L (0.1 mM) [2]. Given its ubiquitous nature, many people are continually exposed to varying amounts of formaldehyde in their daily lives [3].
Ideation
The NCDriven-deHCHO project has a primary goal of addressing formaldehyde pollution through genetic and protein engineering methods. E.coli, a laboratory strain included in the iGEM whitelist of organisms, is both safe and easy to work with. This specific E.coli strain has been genetically modified to produce enzymes capable of catalyzing the synthesis of a non-natural coenzyme, Nicotinamide cytidine dinucleotide (NCD).
Furthermore, these engineered bacteria are also equipped to produce formaldehyde dehydrogenase, formate dehydrogenase, and malic enzyme, all of which can utilize NCD. These enzymes empower the engineered bacteria to metabolize formaldehyde while maintaining the cellular redox balance. To ensure environmental safety, a suicide switch has been incorporated to prevent any potential release of the engineered bacteria into the environment.
Design and Prototype
A bioreactor has been designed to create a suitable living environment for the engineered bacteria. The expression of relevant proteins is regulated using a lactose operon, enabling protein expression as needed. Additionally, a suicide switch activated by arabinose has been implemented to ensure that the engineered bacteria perish upon exiting our designed bioreactor, thus preventing environmental contamination.
For the verification of dehydrogenase activity, we have selected NBT as the colorimetric substrate. This choice is based on the fact that formaldehyde dehydrogenase and formate dehydrogenase can convert the oxidized non-natural cofactor (NCD+) into the reduced non-natural cofactor (NCDH) during the catalysis of formaldehyde to formate and formate to carbonate, respectively. NCDH subsequently reduces Nitroblue tetrazolium (NBT) to form a purple formazan derivative through the hydrogen carrier phenazine methosulfate (PMS). The resulting formazan derivative exhibits a significant absorption peak at a wavelength of 570nm. By measuring the OD values and conducting calculations, we can determine the dehydrogenase activity.
Testing and Analysis
To assess the growth conditions of the engineered bacteria, we introduced the plasmid related to the protein into E.coli and cultured it on a medium containing kanamycin resistance for 12 hours. Subsequently, we conducted an examination of the results.
Next, we cultured in 1 L of LB medium for 12 hours, and induced protein expression at 18℃ with 0.2 mM IPTG. Translate this into the language of the literature.
After collecting the bacterial cells, we subjected them to pressurized lysis and collected the protein-containing supernatant following centrifugation at 12,000 rpm. Subsequently, we carried out protein purification utilizing a nickel column, and observed that the Ncds-2 protein eluted from the nickel column at 40 mM imidazole.
After ultrafiltration and centrifugation, the Ncds-2 protein was purified by molecular sieve.
To determine the concentration of the purified Ncds-2 protein, we initially performed a tenfold dilution of the purified protein. We subsequently assessed the concentration of the diluted protein using the BCA method, resulting in a measurement of 0.58 mg/ml. This outcome establishes favorable conditions for subsequent modeling endeavors and in vitro experiments.
Proof of Concept with Lab Work Results
To demonstrate that formaldehyde dehydrogenase and formate dehydrogenase can utilize the non-natural coenzyme NCD for the metabolism of formaldehyde and formic acid, we used NBT as a colorimetric substrate. The change in absorbance values at 570 nm was measured to determine enzyme activity.
Over time, there is a gradual increase in the malic acid content, as depicted in Figure 7. Given the absence of NADH in our system, malic enzyme exclusively relies on NCDH as a cofactor. Consequently, the rise in malic acid content signifies the capacity of malic enzyme to catalyze the synthesis of malic acid from pyruvate, carbon dioxide, and NCDH.
References
[1] Abdul-Wahab, S. A., Chin Fah En, S., Elkamel, A., Ahmadi, L., & Yetilmezsoy, K. (2015). A review of standards and guidelines set by international bodies for the parameters of indoor air quality. Atmospheric Pollution Research, 6(5), 751-767. https://doi.org/10.5094/apr.2015.084
[2] HECK, H. d'A., CASANOVA-SCHM1TZ, M., DODD, P. B., SCHACHTER, E. N., WITEK, T. J., & TOSUN, T. (1985). Formaldehyde (CH2O) Concentrations in the Blood of Humans and Fischer-344 Rats Exposed to CH2O Under Controlled Conditions. American Industrial Hygiene Association Journal, 46(1), 1-3. https://doi.org/10.1080/15298668591394275
[3] Kang, D. S., Kim, H. S., Jung, J.-H., Lee, C. M., Ahn, Y.-S., & Seo, Y. R. (2021). Formaldehyde exposure and leukemia risk: a comprehensive review and network-based toxicogenomic approach. Genes and Environment, 43(1). https://doi.org/10.1186/s41021-021-00183-5