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 surveys 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 wild-type 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 formaldehyde uptake and metabolism ability using synthetic biology methods. We use tobacco as our receptor plants for experiment. The key enzyme genes related to formaldehyde metabolism in methylotrophic microorganisms as well as in plants are integrated, as a result, the different formaldehyde metabolic pathways can work together and form a new one. Our project includes not only the genes mentioned above but also some other genes. The gene AHA2 from arabidopsis which can enlarge the stomatal opening is added to our project. If the gene expresses correctly, much more formaldehyde are absorbed, which provides sufficient substrate for the new formaldehyde metabolic pathway and boosts the metabolic rate .For security reasons, we include the gene AdCPinto our project because of its capability of leading to pollen abortion. In that way, we can guarantee biosafety of our super plant (Fig.3).

We construct 11 different vectors including two backbones, six mono-gene expression vectors and three multi-gene expression vectors. The product of HPS, PHI, and FDH are located in chloroplast, while the product of FALDH are located in cytoplasm. We used chloroplast transit peptides to locate these productions of genes. So we constructed different vectors with and without transit peptide. We hope to compare the ability of metabolizing formaldehyde of transgenic tobacco between different transgenic lines. Those genes are inserted into tobacco via Agrobacterium-mediated leaf disk transformation method. At last, after DNA and RT-PCR detection, we got about 30 positive plants for each vector. Then qualitative detection and quantitative detection are used to explore whether our super plants have better ability of absorbing formaldehyde. From the experimental results, we can draw the conclusion that formaldehyde assimilation pathway and oxidative pathway super plant was strongly enhanced, and the super plant has remarkable formaldehyde tolerance and can dramatically reduce the concentration of formaldehyde in the air (Fig. 4). Due to the time limited, the activity of the four key enzymes in the formaldehyde metabolic pathway and whether the transit peptides can make a difference or not are currently being researched. How to let the gene AHA2 expresse in tobacco is what our future effort focus on.

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.