Engineering a Bifunctional ComQXPA-P<i>_srfA</i> Quorum-Sensing Circuit for Dynamic Control of Gene Expression in <i>Corynebacterium glutamicum</i>

Corynebacterium glutamicum is an important industrial workhorse for the production of amino acids and other chemicals. However, the engineering of C. glutamicum is inflexible due to the lack of dynamic regulation tools. In this study, a quorum sensing (QS) circuit and its modulated hfq-sRNA cassette were constructed, and the dynamic control of gene expression by these bifunctional circuits was researched. First, the ComQXPA-PsrfA QS system of Bacillus subtilis was harnessed and modified to create an upregulating QS circuit, in which the transcription of genes controlled by the PsrfA promoter may be promoted at high cell density. This QS circuit successfully activated the expression of green fluorescent protein (GFP) to 6.35-fold in a cell density-dependent manner in C. glutamicum. Next, the hfq-sRNA-mediated downregulating circuit under the control of the ComQXPA-PsrfA QS system was established, and the expression of GFP was autonomously repressed by 96.1%. Next, to fine-tune these two QS circuits, a library of synthetic PsrfA based promoters was constructed, and a series of mutant PsrfAM promoters with 0.4-1.5-fold strength of native PsrfA were selected. Subsequently, the ComQXPA-PsrfAM QS circuit was utilized to upregulate the expression of red fluorescent protein, and the same QS-based hfq-sRNA system was utilized to downregulate the expression of GFP simultaneously. Last, this bifunctional ComQXPA-PsrfAM QS circuit was verified again by fine-tuning the expression of α-amylase. Therefore, the engineered ComQXPA-PsrfAM QS cassette can be applied as a novel bifunctional QS circuit to flexibly control gene expression in C. glutamicum.