Team:Tsinghua-E/Result

Strains

BL21(DE3)(mut-bitter30(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3)(mut-bitter32(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-32-tetA

Negative control：

BL21(DE3) pSC101wt-ori-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3) pSC101wt-ori-sfGFP/pTrc99A-trpss-32-tetA

Each evolution was conducted in three parallel with only one parallel of negative control.

Results

By using the parts constructed by us, following the protocol described, we conducted the continuous evolution derived by mutD based mutation for two E. Coli BL21 (DE3) strains carrying bitter defender part 30 and 32. The results were shown below:

It was obvious that tryptophan productivity evolution indeed occurred with the increase productivity trend along the time course compared with the control group. From the wild type E. Coli BL21 (DE3) with nearly zero productivity, this sensor-based evolution approach successfully increase the productivity to about 3mg/L after only three rounds with totally irrationally knowledge-free fashion. Meanwhile, the control group without bitter defender part showed no growth during the three rounds of evolution, which gave strong proof that it was our constructed system which contributed to the evolution process.

However, the strains carrying mutation part showed no difference in final tryptophan titer than that of strains with naturally occurring mutation. According to the report of Leconte A. M.et. al[1], in certain conditions, higher mutation rate contributed significantly to the final desired phenotype obtainment along the designed evolution process of biomolecule. However, for our whole-cell level evolution process, considering the inhibition caused by mutation to cell growth, it was possible that in our designed conditions the fitness of higher mutation was not such significant compared with inhibition in fitness. Thus, it was worthy considering resetting our evolution conditions to find the best ones for fast evolution derived by super mutator in the future work.

We further plated the evolved strains on agar plate, and picked 24 single colonies to culture in 24-well plate for 48h and detected tryptophan production by HPLC.

Figure 1 Tryptophan production of rbs30 evolved stains

Figure 2 Tryptophan production of rbs32 evolved stains

As shown in Fig.1 and Fig.2, each single colony has different productivity of tryptophan, as the evolved strains shown in Table 1 and Table 2 are mixed culture. Unfortunately, as the amount of the selected strains was only 24, in these two figures, we did not get the productivity as high as that of mixed culture. We finally found that evolved strain of rbs30-16 and rbs32-3 have the highest tryptophan productivity. We then selected these two strain, and confirmed there productivity comparing with the controls. The result were shown in figure 3.

Figure 3. Tryptophan production of control and mutation stains

The control of E. coli Bl21 (ancestor of evolution) cannot produce tryptophan, but the other two controls with bitter defender part rbs30 and rbs32 showed very low tryptophan production of about 0.4mg/L. Obviously, the two evolved strains had much higher productivity of tryptophan. The productivity of the evolved strain of rbs30-16 was 2.4 times higher than that of control, while that of the evolved strain rbs32-3 was 2.7 times higher than the control. These results clearly proved our evolution strategy successfully evolved a strain with higher tryptophan from their ancestors which cannot produce tryptophan at all.

[1] Leconte, A. M. et al. A Population-Based Experimental Model for Protein Evolution: Effects of Mutation Rate and Selection Stringency on Evolutionary Outcomes. Biochemistry52, 1490-1499, doi:10.1021/bi3016185 (2013).