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Pyrene degradation operon

Encodes an operon with in total nine protein coding sequences. These encode enzymes, forming a complete metabolic pathway which degrades pyrene to 3,4-dihydroxy-phenanthrene, a phenanthrene degradation intermediate. [1, 2] In this way this part shall enable bacteria, already able to degrade phenanthrene (like Pseudomonas vancouverensis DSM8368 [3, 4]), to degrade pyrene, too.

Sequence and Features

Usage and Biology

Genes were chosen based on Mycobacterium vanbaalenii Pyr-1’s pyrene degradation pathway and mainly derived from this bacterium. As shown in Figure 1 pyrene gets subsequently degraded to 3,4-dihydroxy-phenanthrene by the different enzymes/enzyme complexes in the operon. [1] The resulting 3,4-dihydroxy-phenanthrene shall be channeled into phenanthrene degradation pathway. [2]

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Pyrene tolerance

Description

The OD600 values with different pyrene concentration were plotted over time for P. vancouverensis and P. putida including the unmodified and the engineered strain bearing this composite part. The primary objective was to compare the growth rates in the presence of pyrene, determining if the part enhances the strain's tolerance to pyrene. Furthermore, the minimal bactericidal concentration (MBC) was determined for pyrene and phenanthrene to have a qualitative value.

P. vancouverensis DSM8368

Results

Figure 2: Growth rate comparison of P. vancouverensis unmodified and possessing pyrene degradation plasmid. Incubation at 180rpm, 28°C in LB media with different pyrene concentrations.

Figure 3: Growth curve of P. vancouverensis unmodified at 3 and 4g/L pyrene in LB Medium. Incubation at 180rpm, 28°C.

Figure 4: Growth curve of P. vancouverensis with pyrene degradation operon at 0, 3 and 4g/L pyrene in LB Medium. Incubation at 180rpm, 28°C.

Growth curves of P. vancouverensis in LB medium containing pyrene are displayed in Figure 3 and Figure 4. In Figure 2 growth rates of P. vancouverensis unmodified and transformant are compared. Time error of 3min was assumed. For OD600 error of 0.05 was assumed, corresponding to standard deviation observed in sterile control.

Discussion

Due to the high variability observed between the growth rates, no definitive conclusion can be drawn regarding the pyrene degradation capability of the transformed strain. This variability suggests that the observed increase in growth between 0 g/L and 3 g/L of pyrene for the P. vancouverensis transformant may not be reliable or conclusive.
However, some interesting assumptions for P. vancouverensis can be made: The unmodified strain exhibited a significantly higher growth rate at 3 g/L compared to 0 g/L, which could suggest that its native metabolic pathways might be involved in the degradation of pyrene. Previous studies have shown that enzymes responsible for the breakdown of certain PAHs may also be capable of degrading structurally similar compounds, [5, 6] Additionally this would also indicate potential co-metabolization of pyrene, where pyrene is used additional to the nutrients provided by the LB medium [7], which may resulted in a higher biomass and thus growth. Nonetheless, these theories remain speculative, and given the lack of direct evidence of pyrene degradation, the higher growth rate is more likely due to pipetting errors or inconsistencies in the experimental setup
At a pyrene concentration of 4 g/L, the growth rate of both strains declined compared to 3g/L, indicating a potential inhibitory effect of pyrene at higher concentrations. To further investigate this decline, the minimal bactericidal concentration (MBC) of pyrene was determined to show the concentration threshold where pyrene inhibits the growth.
Table 1: Comparison of the MBC value from p. vancouverensis unmodified and transformed for phenanthrene and pyrene after 16h Compound MBC P. vancouverensis WT [g/L] MBC P. vancouverensis transformed [g/L] phenanthrene 5 5 Pyrene 3 3
Different pyrene concentrations, derived from a pyrene-isopropanol stock solution, were incubated for approximately 16 hours, followed by plating on LB Agar to assess viable cell counts. At a concentration of 3 g/L pyrene, no viable cells were observed. This supports the observed decline in growth rate at 4 g/L, as pyrene concentrations at or above the minimal bactericidal concentration of 3 g/L leads to toxic effects that impair the metabolic activity of both strains, resulting in complete cell death under our experimental conditions (after 16 hours). Prior to cell death, a reduced growth rate is due to increased cell death rate and/or reduced reproductive capacity. Despite this, the starting decline at 4g/L could be due to the isopropanol which could have resulted in lower MBC values, whereas in the LB Medium the pyrene was added as a solid. Additionally, no defined CFU was inoculated for the determination of the MBC values. A higher CFU density could have contributed to increased pyrene tolerance. All in all, no statement about the degradational capability of the P. vancouverensis transformant could be made thus a comprehensive analysis using high-performance liquid chromatography (HPLC) will be conducted. This analysis will allow for the precise quantification of pyrene and its degradation over time.

P. putida KT2440

Results

Figure 5 growth curve of P. putida unmodified and with the pyrene-degradation plasmid 1mg/L Pyrene in LB Medium. The growth conditions were 180rpm at 28°C.

Table 2 Comparison of the MBC value from p. putida modified and transformed for phenanthrene and pyrene after 16h Compound MBC P. putida unmodified [g/L] MBC P. putida transformed [g/L]

phenanthrene 2 5 Pyrene <0.0001 2

In Figure 5 the growth behavior of unmodified and transformed P. putida strain in 1mg/L pyrene is plotted. Errors were assumed as above. The growth rate for the unmodified P. putida is at 0.25±0.02 1/h and for the transformant at 0.36 ±0.05 1/h.

Discussion

The growth rate of the engineered P. putida strain is 28% higher compared to the unmodified P. putida in 1mg/L pyrene, suggesting a higher tolerance of pyrene. This could also be seen at the MBC Value for pyrene were initially even at pyrene concentration of 0.1mg/L no cell viability after an incubation of 16h could be seen. Whereas the engineered strain tolerates concentrations up to 1g/L of pyrene. The growth observed in the unmodified strain at 1 mg/L pyrene, despite its lower tolerance indicated by the MBC test, have the same reasoning as above (variable CFU and the isopropanol solution). Interestingly, the engineered strain also demonstrated an increased MBC value for phenanthrene from 2g/L (unmodified) to 5g/L. This finding highlights the engineered strain's potential for broader applications in the bioremediation of PAH-contaminated environments.
Overall, the higher growth rate and MBC values indicate a greater tolerance to pyrene in the engineered strain. This enhanced tolerance could be attributed to the transformant ability to degrade pyrene, due to the introduction of pyrene degradation pathway indicating a successful pathway design.

Pyrene Degradation HPLC Assay

Description

To test pyrene degradation abilities of transformed P. vancouverensis and P. putida, HPLC-analysis of bacterial cultures for pyrene concentration was carried out.

Method

Liquid cultures of P. vancouverensis and P. putida in LB and M9 media with different pyrene concentrations were incubated at 28°C, 180rpm. Probes were taken regularly and centrifuged. As pyrene is poorly water-soluble [8], most of it should be found in pellets after centrifuging. Cell pellets were resuspended in dimethyl sulfoxide (DMSO) and cells were lysed via ultra sonic device. Lysed cells were centrifuged again, and supernatant was applied to Zorbax SB-C18 HPLC column.

Results

Pyrene showed 7.8 to 8.1 minutes retention time on HPLC column. Pyrene peak was best visible at 270nm absorbance. In Figure 6 and Figure 7results of HPLC-analysis are shown. Pyrene A270 peak area correlating to pyrene concentration in probe is plotted against incubation time. A time error of 1h is assumed. For pyrene peak area an error of 10% as result of pipetting errors is assumed.

Figure 7: Pyrene peak area over time is shown for M9-medium cultures with 100mg/l pyrene.

HPLC-analysis for P. putida in LB-medium indicates constant pyrene concentration in media. P. vancouverensis analysis shows also constant pyrene concentration for transformed bacteria but great fluctuations for unmodified control. Both exhibit unusual high peak areas (>3500mAU*s) after 21h of incubation (not shown in diagram).
All probes in M9-medium show initial pyrene decrease which is followed by constant pyrene amounts. Data for unmodified P. putida strain shows great fluctuations.

Discussion

Data cannot provide proof of pyrene degradation for any of the examined bacterial cultures. Pyrene decrease in all M9-probes including sterile-control could indicate spontaneous decomposition of pyrene. Though this seems unlikely as pyrene is thermodynamically stable [1] and decline could not be observed in LB-cultures.
It stands out, that especially graphs for P. vancouverensis in LB medium and unmodified P. putida in M9 medium show great fluctuations, beyond assumed error tolerances. This most likely indicates that our chosen extraction method does not work quantitatively reliably. This could also explain why pyrene peak area does not correlate with pyrene concentration in medium well.
In summary, data suggests that no bacterial strain, neither unmodified nor bearing pyrene degradation plasmid, is capable of degrading pyrene. Therefor graphs in all cultures, except P. vancouverensis in LB-medium, show approximately constant pyrene concentrations. That the pyrene degradation plasmid does not show success here could be due to several reasons.
At first, due to time constraints we were not able to prove protein expression by transformed strains. As amilGFP was successfully produced using the same expression system, most likely at least some of pyrene pathway’s proteins get produced. Yet there is no proof, the whole operon gets expressed.
In addition, as mentioned earlier, pyrene is poorly water soluble (135μg/l at 25°C) [8]. This means only few substrate will be available for degradation resulting in overall slow pyrene degradation, so that no pyrene decrease could be observed during our chosen incubation times. In this case, production of biosurfactants enhancing PAH-solubility like shown by 2015 iGEM-team from Uppsala, could be helpful. [9]
Furthermore, cellular uptake of pyrene could also be a problem. As all expressed degradation enzymes are cytosolic, pyrene is required to pass bacterial cell membrane. As pyrene shows high octanol-water partition coefficient [8], it can be suspected to diffuse passively through membranes. However for naphthalene active uptake by Pseudomonas fluorescence has been reported [10], indicating an active PAH import system could be necessary for efficient degradation.

Conclusion

In conclusion, most data shows that our transformed bacteria are not capable of degrading pyrene. Only growth curves and MBCs of transformed P. putida indicate a higher pyrene tolerance. This could be due to pyrene degradation or at least detoxification. In summary further analysis, improvements and iterations of the DBTL-Cycle are necessary to implement successful pyrene degradation.

References

[1] W. C. EVANS, H. N. FERNLEY, and E. GRIFFITHS, "OXIDATIVE METABOLISM OF PHENANTHRENE AND ANTHRACENE BY SOIL PSEUDOMONADS. THE RING-FISSION MECHANISM," The Biochemical journal, vol. 95, no. 3, pp. 819–831, 1965, doi: 10.1042/bj0950819.
[2] S.-J. Kim, O. Kweon, R. C. Jones, J. P. Freeman, R. D. Edmondson, and C. E. Cerniglia, "Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology," Journal of bacteriology, vol. 189, no. 2, pp. 464–472, 2007, doi: 10.1128/JB.01310-06.
[3] W. W. Mohn, A. E. Wilson, P. Bicho, and E. R. Moore, "Physiological and phylogenetic diversity of bacteria growing on resin acids," Systematic and applied microbiology, vol. 22, no. 1, pp. 68–78, 1999, doi: 10.1016/S0723-2020(99)80029-0.
[4] Y. Yang, R. F. Chen, and M. P. Shiaris, "Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816," Journal of bacteriology, vol. 176, no. 8, pp. 2158–2164, 1994, doi: 10.1128/jb.176.8.2158-2164.1994.
[5] M. Vazquez-Vilar et al., "GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data," Nucleic acids research, vol. 45, no. 4, pp. 2196–2209, 2017, doi: 10.1093/nar/gkw1326.