Complete conversion of sugars synthesized by photosynthesis into
commodity productsShigeki EHIRA
Graduate School of Science, Tokyo Metropolitan University
Research objective
MazF, a protein toxin of the bacterial toxin-antitoxin system, is a sequence-specificendoribonuclease that recognizes “ACA”. Given that ACA sequences are present in almost all
mRNAs, most mRNAs are degraded in MazF-expressing cells. In contrast, mRNAs lacking
the ACA sequence do not undergo cleavage by MazF and are translated in large quantities byoccupying the ribosome. Accordingly, by removing ACA sequences from all genes encoding
enzymes in a particular metabolic pathway, we could create cells in which a particular
metabolic pathway functions preferentially. In the present study, we developed a system toinhibit cellular functions using MazF to create cells in which specific metabolic pathways
preferentially function.
Methods
First, we examined whether the expression of MazF improves the translation
efficiency of mRNAs lacking the ACA sequence in Escherichia coli using GFP as a model.
There are 26 ACA sequences in the gfp gene. All ACA sequences in the gfp gene were replacedwith other sequences without changing the amino acid sequence of GFP to create the gfpPgene. Plasmids pKMgP and pKMg, which express gfpP and gfp, respectively, from thearabinose-inducible promoter PBAD, were created based on pBR322. In addition, we createdthe plasmid pKMmR, which expresses MazF from the tetracycline-inducible promoter PR0040,
based on pACYC177. A control plasmid, pKM, was created by removing PR0040 and mazF
from the pKMmR vector. E. coli strains KMmR+g and KMmR+gP were generated bytransforming pKMg or pKMgP, respectively, into E. coli HST08 carrying pKMmR. Strains
KM+g and KM+gP, harboring pKM instead of pKMmR, were used as controls. In 96-wellplates, each strain was cultured in 200 µL of LB medium, supplemented with 100 µg/ml
ampicillin and 30 µg/ml kanamycin, for 2 h at 37°C with shaking. Subsequently, arabinose(final concentration 0.1%) and anhydrotetracycline (aTc) were added to induce the expressionof GFP and MazF. GFP fluorescence intensity was normalized to an optical density at 600 nm.

Results
To determine whether the translation efficiency of the gfpP gene lacking the ACA
sequence was similar to that of the original gfp gene, GFP expression was induced by adding
arabinose (range, 0.001–1%) in KMmR+g and KMmR+gP, followed by the measurement of
GFP fluorescence intensity. In both strains, GFP fluorescence intensity increased withincreasing arabinose concentrations, and there was no significant difference in fluorescenceintensity at each concentration. Accordingly, there was no difference in translation efficiency
between gfpP and gfp.
To examine the effect of MazF expression on GFP expression, aTc and arabinose
were added simultaneously to induce MazF expression (Figure 1). GFP fluorescence increased
immediately after induction in KM+g but did not increase in KMmR+g until 60 min after
induction. These results suggested that MazF could suppress protein synthesis from gfp.
However, GFP fluorescence increased after 60 min, and there was no significant difference inthe fluorescence intensity between KM+g and KMmR+g at 120 min. Therefore, the MazF
function was considered to be suppressed after 60 min. Moreover, the MazF-mediatedsuppression of GFP fluorescence occurred independent of the aTc concentration, suggestingthat differences in MazF expression levels had no effect on the suppression of GFP expression
and that markedly low levels of MazF expression could suppress GFP synthesis even in theabsence of aTc.
Next, we compared the effects of MazF on gfp and gfpP expression. GFP fluorescenceincreased at 60 min in KMmR+gP, indicating that deleting the ACA sequence reduced theMazF-mediated suppression of GFP synthesis. Moreover, in KMmR+gP, GFP fluorescenceintensity was significantly increased at an aTc concentration of 1 µg/ml than at lower aTcconcentrations. Thus, higher MazF expression was associated with enhanced gfpP translation,
thereby suggesting that degradation of endogenous mRNA by MazF increased the number ofribosomes binding to gfpP mRNA.
Conclusion
The present study revealed that MazF expression could suppress protein synthesis. However,
the inhibitory effect was abolished after 60 min, suggesting that mazF mRNA was alsodegraded because there were eight ACA sequences in the mazF mRNA. Sustained suppressionof protein synthesis would be achieved by removing ACA sequences from mazF. In addition,MazF could suppress protein synthesis even in the absence of aTc, thereby implying that MazF
functions well, even at markedly low expression levels. At high MazF expression levels,
protein synthesis from gfpP was preferred over that from other proteins. These results indicatethat protein synthesis from genes lacking the ACA sequence occurs preferentially in MazFexpressing cells. However, given that the effect of MazF is significant even at low expression
levels, it is necessary to develop a strict gene expression control system in cyanobacteria toapply the results of this experiment to cyanobacteria.
References1) Zhang, Y., Zhang, J., Hoeflich, K.P., Ikura, M., Qing, G., and Inouye, M. (2003) MazF
cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli.
Mol. Cell 12: 913-923.