Part:BBa_K2587009
ptxD_opt
ptxD allows microorganisms to start metabolizing phosphite, also known as phosphonic acid, an alternative phosphorus source not commonly metabolizable by most organisms. In industry applications, the use of antibiotic resistnace markers is not widely accepted. Therefore, a system which avoids contamination by other microorganisms in a different manner, while still functioning as a reliable selection marker, is required. We present the use of the ptxD gene from Pseudomonas stutzeri together with phosphite media (which reduces growth of contaminants). This could abolish the use of antibiotics. In this case, ptxD is codon optimized for S.cerevisiae1.
Usage and Biology
- ptxD encodes phosphonate dehydrogenase
- Oxidation of phosphite (phosphonate) using NAD+ and H2O to phosphate and NADH
- Selection marker for budding and fission yeast
- Phosphite-oxidizing ability
- Environmentally safe culture
- Antibiotic free system
- pH optimum: 7.25 - 7.75
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 6
Illegal BamHI site found at 1044
Illegal XhoI site found at 216 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 714
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 22
Illegal BsaI.rc site found at 1051
Results Phosphite Measurement
Experimental Design
In our three-way co-culture, we want to use phosphite as a non-metabolizable phosphorus source. Only our engineered S.cerevisiae strain is able to convert it to phosphate for itself, as well as providing it to the other organisms.
To test if our construct with the codon optimized ptxD gene (ptxD_opt)1 works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. S. cerevisiae and E. coli, both used as negative control, were cultivated in standard M2 medium2 with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid. The same supplements were used to make the media differ only in the phosphorus source. Medium lacking uracil was used in the samples containing our construct, to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. As sample size 200 µl were chosen. Every time, OD600 and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.
Table 2: Loading scheme of the 96 well plate for the OD600 measurement of different cultures, different colors represent different media compositions:
Data
First it had to be tested whether other organisms in the co-culture were able to use phosphite as a phosphorus source. To test this, we compared growth of E. coli and S. cerevisiae in normal M2 medium with M2 medium where phosphite was the only phosphorus source.
Figure 1: Growth of E. coli over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but only with phosphite instead of phosphate (yellow). The cell density was measured using OD600.
As shown in Figure 1, E. coli shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours in standard M2 medium. After 20 hours, E.coli reaches a plateau with an OD600 of nearly 0.4. In M2 medium with phosphite, the bacteria stay in the lag phase and only reach an OD600 of less than 0.1. At the end of the measurement a slow decrease of the population is visible.
Figure 2: Growth of S. cerevisiae over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but containing phosphite instead of phosphate (yellow), measured with OD600.
S. cerevisiae shows a growth curve similar to E. coli, with a lag phase up until 10 hours and a stationary phase after 20 hours (Figure 2). For cells incubated in M2 medium, supplemented with phosphite as the sole phosphorus source, no growth is detectable.
To figure out the strongest one, different promoters from the YTK toolbox3 were tested: TDH3 (BBa_K124002, Link: https://parts.igem.org/Part:BBa_K124002), CCW12, PGK1 (BBa_K122000, Link:https://parts.igem.org/Part:BBa_K122000), HHF2, TEF1 controlling the ptxD_opt gene in S. cerevisiae. All previously described experiments were also performed using M2 medium with phosphite as phosphorus source.
Figure 3: Growth of five S. cerevisiae strains containing ptxD_opt under the control of different promoters over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite. Strains with promoter TDH3 (blue), CCW12 (red), PGK1 (green), HHF2 (gray), TEF1 (yellow), measured at OD600.
All constructs with the five promoters show a constant, but slightly different growth in M2 medium containing phosphite (Figure 3). With the strongest promoter TDH3, S. cerevisiae achieves an OD600 of nearly 0.025, which is more than 100% more growth than the weakest promoter of the five: CCW12. The strains with the promoters PGK1 or TEF1 reach an OD600 of a little bit over 0.015, representing medium strength.
The final question is if our modified S. cerevisiae strains show a better growth on phosphite than the progenitor S. cerevisiae. Therefore, the growth of the strain with the ptxD_opt gene and the strongest promoter TDH3 was compared with the progenitor strain.
Figure 4: Growth of the S. cerevisiae control strain (yellow) and S. cerevisiae with the ptxD_opt and TDH3 (blue) on M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite over 52 h, measured at 600 nm. For the S. cerevisiae BY4742 background strain, uracil (18 mg/l) was added.
In Figure 4, the graph shows an initial growth of the progenitor strain S. cerevisiae, but the growth stops at an OD600 of less than 0.03 after 10 hours. After 10 hours, the cell density of S. cerevisiae starts to decrease, slowly in the beginning, but faster towards the end (around 50 h). At the end of the experiment the progenitor strain has an OD600 of less than 0.02. The modified S. cerevisiae strain with ptxD_opt under the control of the TDH3 promoter starts at nearly the same OD600 as the progenitor strain but after a short lag phase of around 5 hours, the strain grows, slowly in the beginning and faster towards the end, where it reaches an OD600 of over 0.02. Both strains start at the same OD600 level. The non-modified strain grows faster than the modified strain at the beginning, but then decreases more and more, while the modified strain needs some time but shows a slow increase of growth afterwards.
Conclusion
Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro nutrient essential for microorganisms. Creating a dependency based on the ability of S. cerevisiae to utilize an otherwise unusable phosphorus source like phosphite by expressing ptxD and making it metabolically available for E. coli is therefore a very promising approach.
In order to build a stable dependency, the other organisms in this system have to lack the enzymes for the catalysis from phosphite to phosphorus. The experiment with E. coli (Figure 1) and S. cerevisiae (Figure 2) demonstrates that neither E. coli nor S. cerevisiae show any growth in M2 medium with phosphite. Figure 3 shows the growth of engineered S. cerevisiae with different promoter_ptxD_opt constructs. All show growth regardless of what promoter was used but the promoter TDH3 showed the strongest growth rates. This indicates that our approach can work.
When comparing the growth of both S. cerevisiae strains (Figure 4), some differences can be detected. The non-modified S. cerevisiae grows much faster in the beginning but soon thereafter begins to die. The modified strain however shows slow but constant growth. As a consequence, we assume that the modified strain would outcompete contaminating microorganisms and possibly the other members of the co-culture. To add to that, we have designed our co-culture with more than one dependency, for example with nitrogen or carbon.
In general the growth was minimal, so for further experiments, higher concentrations of nutrients and phosphite might lead to better results. The slow growth rate of the modified strain is as we expected, because it is comparable to what the literature shows. There, a ptxD construct with the TEF1 promoter in S. cerevisiae was used and growth was monitored over 40 hours4. A constant temperature of 30°C also might increase growth. Moreover, a longer measurement time would also show the behavior of the culture over a longer time. This would be interesting because we would like to create a stable culture which can be maintained for as long as possible. In addition, co-culture experiments would likely lead to other results than monocultures. In this case it would be interesting to perform them as well with the same experimental design. For a future application in the co-culture, we suggest to use the phosphate exporter XPR1 from Homo sapiens5,6. It may help to provide for the other organisms, due to the export of the produced phosphate.
References
1. Kanda, Keisuke, et al. "Application of a phosphite dehydrogenase gene as a novel dominant selection marker for yeasts." Journal of biotechnology 182 (2014): 68-73.:
https://www.sciencedirect.com/science/article/pii/S016816561400193X
2. Weiss, Taylor L., Eric J. Young, and Daniel C. Ducat. "A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production." Metabolic engineering 44 (2017): 236-245.
https://www.sciencedirect.com/science/article/pii/S1096717617301763
3. Lee, Michael E., et al. "A highly characterized yeast toolkit for modular, multipart assembly." ACS synthetic biology 4.9 (2015): 975-986. https://pubs.acs.org/doi/abs/10.1021/sb500366v
4. Shaw, A. Joe, et al. "Metabolic engineering of microbial competitive advantage for industrial fermentation processes." Science 353.6299 (2016): 583-586. http://science.sciencemag.org/content/353/6299/583
5. Giovannini, Donatella, et al. "Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans." Cell reports 3.6 (2013): 1866-1873.
https://www.sciencedirect.com/science/article/pii/S2211124713002684
6. Legati, Andrea, et al. "Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export." Nature genetics 47.6 (2015): 579.
https://www.nature.com/articles/ng.3289
| None |