Part:BBa_K4257011
RPD
RPD is the thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506. It catalyzes the nearly irreversible oxidation of phosphite to bioassimilable phosphate. Therefore, it can be used to support the growth of E.coli in the medium with phosphite as the phosphorus source. It has been reported that RPD had high intracellular activity in phosphite oxidation (Hirota et al. 2012). This year, our team wants to develop an engineered metabolic pathway that can produce phosphate using phosphite as the substrate. Therefore, phosphite dehydrogenase with high intracellular activity is especially desired.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Data:CPU-Nanjing 2022 TEAM
The RPD gene was cloned into plasmid pBBR1MCS2 using E. coli K12 as the host cell and the derivative strain was designated MR.
1. Growth test use phosphite (P, +3 valence) as the solo phosphorus source
Growth test was performed in synthetic municipal wastewater (SMW, a nutrient-poor synthetic medium) with phosphite as the solo phosphorus source (Wang et al. 2018). Wilde type E. coli K12 served as the control.
As shown in Figure 1, the expression of RPD can indeed support the vigorous growth of MR in SMW (P, +3 valence). Due to the inability of E. coli K12 to assimilate phosphite, it showed no sign of growth in such medium.
2. Supernatant phosphite
As expected, concurrent with the growth of MR was the decrease of supernatant phosphite (Figure 2). Given that the form of phosphorus available to life is phosphate, we concluded that the growth of MR was attributed to its capability to oxidize phosphite into bioassimilable phosphate.
3. Supernatant phosphate
Interestingly, we found that no phosphate was detected in the supernatant of the MR culture (Figure 3). This means that although MR possesses the capacity of phosphite oxidation, it does so just to meet its own growth needs.
References
Hirota, R., Yamane, S.-t., Fujibuchi, T., Motomura, K., Ishida, T., Ikeda, T. and Kuroda, A. (2012) Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506. Journal of Bioscience Bioengineering 113(4), 445-450.
Wang, X., Wang, X., Hui, K., Wei, W., Zhang, W., Miao, A., Xiao, L. and Yang, L. (2018) Highly effective polyphosphate synthesis, phosphate removal, and concentration using engineered environmental bacteria based on a simple solo medium-copy plasmid strategy. Environmental Science Technology 52(1), 214-222.
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