Overview

When the human body is exposed to elevated levels of formaldehyde for extended periods, it can potentially lead to significant adverse effects, such as respiratory damage and the development of leukemia. In pursuit of our goal to design an effective and health-conscious biological approach for formaldehyde removal, we've modified the cells to integrate a relevant enzyme system for formaldehyde metabolism. This incorporation empowers the engineered cells with the capability to clear formaldehyde [1].

Figure 1. The whole pathways designed

Formaldehyde Metabolism

Using modified formaldehyde dehydrogenase and formate dehydrogenase (originating from Pseudomonas malodorans) convert formaldehyde to CO2 and H2O.

  • Formaldehyde dehydrogenase can oxidize formaldehyde in the environment to formate, and formate dehydrogenase can oxidize formate to carbon dioxide and water.
  • Employing these two enzymes can effectively achieve the oxidation of formaldehyde and the conversion of toxic compounds into non-toxic ones. This streamlined reaction pathway, involving only two enzymes, minimizes cellular stress.
  • Modified formaldehyde dehydrogenase and formate dehydrogenase both prefer to NCD instead of NAD [2][3].
Figure 2. The pathway of formaldehyde metabolism

NAD Equilibrium

Introducing a non-natural coenzyme NCD system to maintain the NAD balance and achieve the orthogonality of biochemical reactions [4].

  • The oxidation of formaldehyde triggers a redox reaction, which results in a competition for NAD among formaldehyde dehydrogenase, formate dehydrogenase, and some endogenous enzymes, finally interfering with other physiological reactions. The imbalance of intercellular NAD has a significantly negative impact on the survival of the engineered cell.
  • Utilizing the synthetic coenzyme NCD, formed by substituting the original adenine in NAD with cytosine and the NCD-preferred formaldehyde dehydrogenase and formate dehydrogenase, offers a promising solution to this challenge [3].
    • Figure 3. The difference bwtween NAD and NCD
  • To incorporate NCD effectively and maintain the optimal functioning of these two dehydrogenase enzymes, we introduced CtCTPS, FtNadE, and Ncds-2 to facilitate NCD synthesis.
    • Figure 4. The genetic circuit of NCD synthesis
  • Meanwhile, we also imported the modified malic enzyme to recycle NCD, thereby converting the produced NCDH back into NCD. These strategic measures ensure a continuous and ample supply of NCD for the desired processes [2].
    • Figure 5. The genetic circuit of formaldehyde metabolism and NCD balance

Safety

Designing the suicide genetic circuit to prevent potential impacts on external environments and protect biological safety.

  • The proposed suicide genetic circuit achieves by combining elements from the arabinose operon with the MazF toxin and MazE antitoxin genes. In the system, the T7 strong promoter governs the expression of the lethal MazF toxin, while the expression of the protective MazE antitoxin is regulated by the arabinose operon.
  • Under the condition of high L-arabinose concentration, the engineered bacteria can express the antitoxin MazE. This protein associates with the continuously generated MazF toxin, resulting in the formation of a non-toxic complex.
  • Under the condition of low L-arabinose concentration, the absence of MazE allows MazF to trigger cellular damage, leading to the demise of the bacterial population [5].
    • Figure 6. The genetic circuit of kill switch
  • The engineered bacteria survives in the medium containing high concentrations of L-arabinose and suicides when the bacteria escapes to environment, which contains low concentrations of L-arabinose.

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.

[2] Guo, X., Liu, Y., Wang, Q., Wang, X., Li, Q., Liu, W., & Zhao, Z. K. (2020). Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate. Angewandte Chemie International Edition, 59(8), 3143-3146.

[3] Wang, J., Guo, X., Wan, L., Liu, Y., Xue, H., & Zhao, Z. K. (2022). Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide. Chembiochem: A European Journal of Chemical Biology, 23(7), e202100697.

[4] Wang, X., Feng, Y., Guo, X., Wang, Q., Ning, S., Li, Q., Wang, J., Wang, L., & Zhao, Z. K. (2021). Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide. Nature Communications, 12(1), 2116.

[5] Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nature Reviews Microbiology, 9(11), 779–790.