Abstract
Although synthetic biology has made remarkable progress in recent years toward the design of microbial cell factories (MCFs) that produce a variety of valuable channels, many unexplored areas in the field of exporter engineering still remain due to the difficulty of research. We focused on mechanosensitive channels (Msc), which are responsible for L-Glu export in industrial L-Glu-producing MCF. In this study, we characterized the properties of Msc and explored its potential as a versatile exporter. The introduction of Msc genes carrying a gain-of-function (GOF) mutation into MCFs successfully increased the production rate of valuable chemicals such as L-Lys, and inosine 5′-monophosphate (5′-IMP). In contrast to the conventional view that high specificity is required for an exporter, our results demonstrated that even a channel with low selectivity can function effectively as an exporter in MCF.
Furthermore, our findings on the dynamic gating behavior of mechanosensitive channel of large conductance (MscL) obtained by all-atom molecular dynamics simulations provide clues to understanding the mechanism by which G46D substitution of MscL leads to GOF. Specifically, the G46D substitution causes a kink at A38 of the first transmembrane helix (TM1), which may affect the interaction between TM1 and TM2 and destabilize the gate in closure state, resulting in a GOF. To verify this, we introduced a double substitution gene with A38V and G46D, which interferes with A38 kink, into 5′-IMP-producing MCF, and the effect of G46D on 5′-IMP production rate per cell was abolished. These demonstrate that molecular dynamics simulation is effective in understanding the complex mechanism of the Msc gating and has great potential as a tool for designing functionally improved Msc.
