Development of a novel fermentation technology using
mechanosensitive channels

Ken-ichi HASHIMOTO

Department of Green and Sustainable Chemistry,Tokyo Denki University

Research aims
Inosinate and guanylate are commonly used worldwide as seasonings
because they evoke umami, one of the five basic tastes. Inosinate is anumami component of the taste of katsuobushi, and guanylate is an umami
component of the taste of shiitake mushrooms; these are two of the mosttraditional seasonings in Japan. These compounds are produced bymicroorganisms; however, the mechanisms by which these compounds areexported out of cells have not yet been found.
When some compounds are produced as a result of direct fermentation, it
is essential to know how the compound is exported; however, the analysis
and development of procedures to study membrane proteins are challenging.
The NCgl1221 channel is a glutamate exporter, first described in

Corynebacterium

glutamicum—aglutamate fermentation.

microorganism

usedforindustrial

We have previously discovered that aspartate, phenylpropionate, andlysine can also pass through the NCgl1221 channel. We hypothesized thatthe yield of a substance can be improved by using this channel as a novel
exporter in the substance-producing cell. In this study, we focused on
inosinate and guanylate production, because it is still unknown how thesesubstances are exported by cells. NCgl1221 channels were expressed in astrain of Escherichia coli with enhanced biosynthesis of inosinate andguanylate(1); we analyzed the effects of NCgl1221 on the production of
inosinate and guanylate, and on the gating properties of NCgl1221, for thedevelopment of a versatile exporter using this mechanosensitive channel.
Methods
1. Effects of NCgl1221 gene transfer on inosinate and guanylate production

C. glutamicum does not produce glutamate in normal growth

conditions; however, glutamate overproduction can be induced in thisstrain by biotin limitation, Tween 40 supplementation, or penicillin
supplementation. Studies have shown that these culture conditions
induce NCgl1221 channel opening; this is essential for NCgl1221 to be
used as an exporter of inosinate and guanylate. Two mutations ofNCgl1221 (A111V and W15CSLW) were identified, which induce
glutamate overproduction in normal growth conditions(2); these mutantproteins were expected to form the channel opening. These mutants ofthe NCgl1221 gene were expressed in a strain of E. coli with enhanced

biosynthesis of inosinate and guanylate (E. coli FADR add edd yicP pgi
xapA ushA aphA/ pMWKQ); culture experiments were performed withthis strain.
2. Analysis of the gating properties of NCgl1221

A detailed analysis of the gating properties of NCgl1221 was needed
for the development of the novel, versatile exporter. The influence ofmembrane conditions on the gating properties of NCgl1221 wasconsidered, because the channel is opened by alterations of membranetension. In this study, we analyzed the gating properties of NCgl1221
present in the membrane of C. glutamicum or E. coli.

Results
1. Effect of NCgl1221 gene transfer on inosinate and guanylate production
The A111V or W15CSLW mutant gene was introduced into inosinateand guanylate-producing E. coli, using a plasmid vector. As shown in Fig.
1, the data suggest that this channel forms a path for the excretion ofinosinate and guanylate across the cytoplasmic membrane in E. coli. Thedata also suggest that the excretion of inosinate and guanylate is arate-limiting step in inosinate and guanylate production in this strain,
and that intracellular accumulation of inosinate and guanylate inhibits

growth.

Fig. 1. Effects of introducing mutant NCgl1221 genes into inosinate- andguanylate-producing E. coli

2. Analysis of the gating properties of NCgl1221
We analyzed the gating properties of NCgl1221 using the patch clamp
technique for prokaryotic cells. This method requires the preparation ofgiant spheroplasts, because normal prokaryotic cells are too small to beaccessed by the patch pipette. We have previously been able to prepare
giant spheroplasts of E. coli and Bacillus subtilis, but not of C.
glutamicum. We developed a method to prepare giant spheroplasts of C.
glutamicum using the SI method(3), with supplementation of ethambutol.

Fig. 2 Giant spheroplast of C. glutamicum

We analyzed the gating properties of NCgl1221 by comparing the giant
spheroplasts of C. glutamicum to those of NCgl1221-expressing E. coli.

When a pipette voltage of +80 mV was used, NCgl1221 in the native
membrane of C. glutamicum was maintained in the open state for 0.5 s
without membrane tension, after the channel was opened once.

Fig. 3. Gating property of the NCgl1221 channel in the membraneof C. glutamicum or E. coli

Conclusion
In this study, we developed improvements in inosinate and guanylate
production by the introduction of genes encoding mutant forms of NCgl1221.
This study suggests that NCgl1221 can function as a novel exporter ofnucleotides.
However, the amount of inosinate and guanylate obtained was lower than
the amount of glutamate obtained by the cultivation of C. glutamicum. Our

patch clamp data showed that NCgl1221 was maintained in the open statefor 0.5 s without membrane tension, after the channel was opened once in
the C. glutamicum membrane; this suggests that NCgl1221 is difficult toclose in this membrane. Because membrane conditions vary among
microorganisms, we believe that 1) the improvement of NCgl1221 for eachhost strain, and 2) the optimization of culture conditions for altering
membrane conditions of host strains are important goals for improving
productivity.

References
1) Kakehi, M., Usuda, Y., Tabira, Y., and Sugimoto, S. (2007) Complete
deficiency of 5’-nucleotidase activity in Escherichia coli leads to loss ofgrowth on purine nucleotides but not of their excretion. J. Mol. Microbiol.
Biotechnol. 13:96–104.
2) Nakamura, J., Hirano, S., Ito H., and Wachi, M. (2007) Mutations of theCorynebacterium glutamicum NCgl1221 gene, encoding amechanosensitive channel homolog, induce L-glutamic acid production.

Appl. Environ. Microbiol. 73:4491–4498.
3) Kuroda, T., et al. (1998) J. Biol. Chem. 273:16897–16904.