Part:BBa_K2587009

ptxD_opt

Most of the microorganisms, especially common used model organsims like Escherichia coli or Saccharomyces cerevisiae grow only very slow on phosphite as phosphorus source. Moreover in industry is not widely accepted to use antibiotic resistances as markers. Therefore a system is required, which allows avoidance of contamination by other microorganisms and at the same time represents a reliable selection marker. We present the use of the ptxD gene from Pseudomonas stutzeri together with a phosphite media (reduces growth of contaminants), which could abolish the use of antibiotics in the future and the more efficient use of a phosphorus source in the medium. In this case ptxD is codon optimized for S.cerevisiae¹.

Usage and Biology

• ptxD codes for 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 93
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 6
  Illegal BamHI site found at 1044
  Illegal XhoI site found at 216
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 714
  
• 1000
  INCOMPATIBLE 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 only differ 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 are 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 is 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 in standard M2 medium E. coli shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours. After 20 hours E. coli reaches the stationary phase 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 the common growth curve similar to E. coli, with a lag phase until 10 hours and a stationary phase after 20 hours (Figure 2). Cells incubated in M2 medium, supplemented with phosphite as 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 controle 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. Strain with promoter TDH3 (blue), CCW12 (red), PGK1 (green), HHF2 (gray), TEF1 (yellow), measured with OD₆₀₀.

All constructs with the five promoters show a constant, but different growth in M2 medium with 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 and have middling strengths.

The final question is if our modified S. cerevisiae strains shows 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 were compared with the progenitor strain.

Figure 4: Growth of S. cerevisiae (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 with OD₆₀₀. For the S. cerevisiae BY4742 background strain, uracil (18 mg/l) were added.

In Figure 4 the graph demonstrates 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 S. cerevisiae starts to decrease, in the beginning slowly, but in the end after over 50 hours a faster decrease is recognisable. 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 and TDH3 starts nearly at 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 a bit faster in the end, where it reaches an OD₆₀₀ of over 0.02. Both strains start at the same OD₆₀₀ level. The not modified strain grows initially faster than the modified strain, but then decreases more and more, while the modified strain needs some time but then shows a slowly rise of growth.

Conclusion

Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro element essential for microorganisms. Creating a dependency based on the ability of S. cerevisiae to utilise 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 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. But for that reason there are more than one dependency in the co-culture, for example with nitrogen. 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 could 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 as long as possible. In addition, co-culture experiments could 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 sapiens^5,6. It may help to feed the other organisms, due to the secretion 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