Project overview

Relevant data shows that air pollutants indoor are five to ten times higher than that outdoor, and the number of indoor air pollutants is up to 500. The indoor air pollutant formaldehyde (HCHO), a major indoor air pollutant, attracts worldwide attention because the exposure to formaldehyde is known to cause irritation, allergic asthma and neurasthenia, as well as to induce carcinogenicity and carcinogenesis (Fig.1) (Tang et al., 2009). The National Cancer Institute (NCI) published a survey report of 25,000 people who are exposed to formaldehyde while working in chemical plants in 2009 showed that the people who are often exposed to formaldehyde are 37% more likely to die from leukemia or lymph cancer. In China, the national standard concentration of indoor formaldehyde was published in 2002 and the concentration should below 0.1mg/m3. However, a survey in China during the period of 2002–2004 revealed that indoor formaldehyde levels in more than 69.4% of all new or newly remodeled houses exceeded the national standard of China (Xu et al., 2011).

Based on above information, it is important to remove formaldehyde to a safe level. Purification technologies commonly used for indoor air pollution control include adsorption, chemisorption, photo catalytic oxidization, plasma and thermal catalytic oxidization. However, these methodologies still remain a challenge due to either low degradation efficiency or environmental safety (Lu et al., 2012). Therefore, a more efficient and more environmental friendly technology is desired.

As we all know that various plants can remove formaldehyde from indoor air by means of the uptake and metabolism (Xu et al., 2010). It has been proved that formaldehyde as a central intermediate of photosynthetic carbon dioxide fixation in green plants can be removed by forming HM-GSH and finally turned to water and carbon dioxide (Fig.2A). FALDH and FDH are the key enzymes for formaldehyde fixation. For the wildtype plants, they have limited capacity to metabolize formaldehyde and it’s hard for them to survive in an environment with high concentration of formaldehyde. At the same time, formaldehyde is also a key intermediate in the metabolism of several one-carbon (C1) compounds in methylotrophic microorganisms. Those microorganisms have a special metabolic pathway named ribulose monophosphate (RuMP) pathway (Fig.2B). 3-hexulose-6-phosphate (HPS) and 6-phospho-3-hexuloisomerase (PHI) are the key enzymes that affect the metabolism of formaldehyde. In the pathway, HPS fixes HCHO and D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose 6-phosphate (Hu6P), and 6-phospho-3- hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P). Interestingly, Ru5P and F6P are also included in the Calvin-Benson cycle, so bacterial RuMP pathway and plant Calvin-Benson cycle can be connected if HPS and PHI can be expressed in plants. By this way, the capacity of formaldehyde uptake and metabolism will be greatly enhanced. In addition, the use of green plants to remove toxins from the air is more likely to be accepted by the public.

In our iGEM project, our objective is to further increase plant's ability on the uptake and metabolism of formaldehyde by using synthetic biology methods. We used tobacco as a model system to construct our super plant. The key enzyme genes related to formaldehyde metabolism in methylotrophic microorganisms as well as in plants are integrated. Consequently, the two formaldehyde metabolic pathways can work together, making our super plant even more efficient in removing formaldehyde. Our project includes not only the genes mentioned above but also some other genes. The gene AHA2 from Arabidopsis thaliana which can enlarge the stomatal opening is introduced into our project, endowing our super plant a super power on the metabolisation of formaldehyde. For safety reasons, we also included the gene AdCP into our project because of its capability of leading to pollen abortion. In that way, we can guarantee the biosafety of our super plant (Fig.3).

In total, we constructed 11 different vectors, including two backbones, six mono-gene expression vectors and three multi-gene expression vectors. The production of HPS, PHI, and FDH are located in chloroplast, while FALDH is located in cytoplasm. Chloroplast transit peptides were used for the purpose of chloroplast orientation. For comparison, those vectors carrying the genes of HPS, PHI, and FDH without the presence of transit peptide were also constructed. Those genes were inserted into tobacco via Agrobacterium-mediated leaf disk transformation method. By performing DNA and RT-PCR analysis, we got about 30 positive plants for each vector. The formaldehyde absorbance ability of our super plants was explored by both qualitative and quantitative detection. The results showed that our super plants have remarkable increased abilities of formaldehyde tolerance and can dramatically reduce the concentration of air formaldehyde (Fig. 4). Due to the time limitation, the following investigations are under the way: 1) the effect of individual four key enzymes on the metabolic efficiency of formaldehyde; 2) whether the presence of transit peptides can affect the metabolic efficiency of formaldehyde; 3) the expression of the gene AHA2 in tobacco.

References

Lu, N., J. Pei, Y. Zhao, R. Qi and J. Liu (2012). "Performance of a biological degradation method for indoor formaldehyde removal." Building and Environment 57: 253-258.
Tang, X., Y. Bai, A. Duong, M. T. Smith, L. Li and L. Zhang (2009). "Formaldehyde in China: production, consumption, exposure levels, and health effects." Environ Int 35(8): 1210-1224.
Xu, Z., N. Qin, J. Wang and H. Tong (2010). "Formaldehyde biofiltration as affected by spider plant." Bioresour Technol 101(18): 6930-6934.
Xu, Z., L. Wang and H. Hou (2011). "Formaldehyde removal by potted plant-soil systems." J Hazard Mater 192(1): 314-318.