Overview

    NCKU_Tainan aims to make E. coli synthesize selenomelanin in vivo to improve its tolerance of radiation. There are two goals to achieve when proving this concept. One is to synthesize Sec and selenomelanin. The other is to prove that the survival rate of the engineered E. coli (Se coli) really improves under UV tests and GABA verification. With these two goals achieved, it is certain that the design improves the potential of synthetic biology space application.

Biomanufacturing Selenocysteine

Sec Synthesis

    To synthesize Sec, one of the precursors of selenomelanin, NCKU_Tainan designed in vitro and in vivo pathways, and succeeded in the in vitro pathway.

I. In vitro Sec synthesis

    In the in vitro pathway, Sec was synthesized through tRNA synthesis, aminoacylation of Ser and tRNA, conversion of seryl-tRNA to selenocysteinyl-tRNA, and deacylation. To measure the result, Urea PAGE (Fig. 1 and 2) and HPLC (Fig. 3) were used as the main measuring methods.

Fig. 1. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNA^Ser; Lane2: seryl-tRNA^Ser; Lane3: selenocysteinyl-tRNA^Ser;Lane4: tRNA^Sec; Lane5: seryl-tRNA^Sec; Lane6: selenocysteinyl-tRNA^Sec

Fig. 2. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNA^UTuX; Lane2: seryl-tRNA^UTuX; Lane3: selenocysteinyl-tRNA^UTuX

    Urea PAGE can measure the production of tRNA, seryl-tRNA, and selenocysteinyl-tRNA. The bands of seryl-tRNA and selenocysteinyl-tRNA have upshifted compared to the band of the tRNA, which indicates that both experiments are successful.

                (A)

                (B)

Fig. 3. Confirmation of Sec by HPLC (A) Sec synthesized by tRNA^Ser; (B) Sec synthesized by tRNA^UTuX

    In these results, the retention time of the Sec standard is 10.5 minutes (the peaks at 3.5 minutes and 4.5 minutes are solvent peaks), which is the same as the retention time of Sec synthesized via the in vitro pathway. It proves that the pathway is successful.

II. Future plan

    There are still a lot of obstacles as tRNA in vivo pathway and Sec in vivo pathway did not succeed eventually. Therefore, NCKU_Tainan manages to optimize the methods, hoping to improve them in the future.

    It is difficult to induce certain modifications to tRNA structure through in vitro synthesis, which may reduce its affinity towards amino acids.

    In order to synthesize tRNA more precisely and scalably, NCKU_Tainan also designed a tRNA in vivo synthesis pathway, in which tRNA was put in the middle of lpp promoter and rrnC terminator, a way to overexpress tRNA sequence that had been proved in many papers. After the construction, the protocol based on the accompanying paper[1] was followed to extract the final tRNA modified by E. coli.

    However, owing to technical difficulties, construction of the promoter and terminator did not succeed (see Engineering page). In the future, more troubleshooting and adjustment of the experimental process will be conducted.

    In order to synthesize Sec in vivo and yield it more easily, another pathway which mimics the native Cys synthesis pathway, an analog of Sec, was designed.

    The concept was based on the similarity between Sec and Cys. cysK, cysE*, and ydeD were overexpressed. By measuring the growth curves and performing CFU assay, the function of these three proteins were verified. However, the results did not fit to the expectations, as the production of Cys and Sec have failed. As reasons of failures are analyzed, NCKU_Tainan strives to improve the Sec and selenomelanin synthesis for future application. The conclusion and hypothesis are summarized as below.

    First, the efficiency for selenide to intrude sulfide metabolism pathway is not as good as expected. The idea of synthesizing Sec through Cys synthase has been confirmed in plants[2], but not in E. coli. NCKU_Tainan will try to engineer other hosts or challenge other Cys synthesis pathways in E. coli.

    Second, during the experiments, sodium selenite was added in order to form Se^2-, but chances were that it had been deoxidized before being transformed into Sec since the medium turned red significantly. Since it is the intrinsic property of E. coli, the substrate should be changed in order to avoid this circumstance.

Melanin Production Optimization

Model

    Since NCKU_Tainan aims to culture E. coli under space radiation, the production of melanin should be sufficient to protect bacteria. By using the optimized parameters, NCKU_Tainan has increased melanin production significantly (Table 1) (see Model page).

Table 1. Comparison of melanin yield produced from dual-plasmid bacteria (melA and gadB).

GABA Production in Se coli

    The plasmid for TyrP and MelA (BBa_K4171024) expression was constructed to get higher melanin production. By adding Sec to melanin, selenomelanin was polymerized in Se coli.

    Since the goal is to biosynthesize crucial substances in space, it is important to ensure the ability of Se coli factory. GABA was chosen as a verification of our concept. With GAD overexpressed from Se coli as well as precursor MSG and cofactor PLP, Se coli was able to produce GABA (Fig. 4). The quantification of GABA was completed with an OD meter at the wavelength 630 nm after dyeing the cells (see Experiments page).

    GABA-synthesizing Se coli was placed under UV irradiation with the comparison of non-melanized as well as melanized bacteria to verify if selenomelanin protects microorganisms from radiation effectively. As the result indicates (Fig. 4), even though the amounts of GABA decrease because of melanin and selenomelanin production, the efficacy of GABA synthesis is still satisfying. This demonstrated the excellent radiation tolerance of GABA-synthesizing Se coli.

Fig. 4. GABA production in Se coli

(A)

(B)

Fig. 5. Bacteria under exposure of UV-C (A) Survival rate (B) CFU comparison.

    As the results shown above, Se coli had the best radiation tolerance towards UV irradiation exposure for an hour (12 W, UV-C) among the three groups. 17.9% of Se coli survived, while only 5% of melanized bacteria and 3.2% of non-melanized bacteria survived respectively. In general, selenomelanized bacteria (Se coli) is 3.6 times more tolerant to radiation than melanized-bacteria, and 6 times than non-melanized ones. This indicates the success of selenomelanin biosynthesis in Se coli.

Space Environment Simulation

    NCKU_Tainan also designed MerStage, a simpler simulator that can test survivability of Se coli in space. Radiation and microgravity should be taken into consideration[3] since they are two of the most critical factors that can affect the bacteria functions in space.

    The growth curve of E. coli without 3D cell culture and aggregation was compared with the growth curve of E. coli in chip in Fig. 6. The growth of bacteria with 3D aggregation was faster in the beginning but slowed down after 10 hours of cell culture, for the bacteria in the middle of the clusters had less contact area with nutrients[4].

Fig. 6. Growth curve (Aggregation/ No Aggregation)

References

[1] Avcilar-Kucukgoze, I., Gamper, H., Hou, Y. M., Kashina, A. (2020). Purification and use of tRNA for enzymatic post-translational addition of amino acids to proteins. STAR Protocols. 2020;1(3):100207. doi:10.1016/j.xpro.2020.100207
[2] Ng, B. and Anderson, J., 1978. Synthesis of selenocysteine by cysteine synthases from selenium accumulator and non-accumulator plants. Phytochemistry, 17(12), pp.2069-2074.
[3] Ohnishi K, Ohnishi T. The biological effects of space radiation during long stays in space. Biol Sci Space. 2004;18(4):201-205. doi:10.2187/bss.18.2011
[4] Shao X, Mugler A, Kim J, Jeong HJ, Levin BR, Nemenman I. Growth of bacteria in 3-d colonies. PLoS Comput Biol. 2017;13(7):e1005679. Published 2017 Jul 27. doi:10.1371/journal.pcbi.1005679