No abstract

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-P srfA QS system of Bacillus subtilis was harnessed and modified to create an upregulating QS circuit, in which the transcription of genes controlled by the P srfA 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-P srfA 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 P srfA based promoters was constructed, and a series of mutant P srfA M promoters with 0.4–1.5-fold strength of native P srfA were selected. Subsequently, the ComQXPA-P srfA M 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-P srfA M QS circuit was verified again by fine-tuning the expression of α-amylase. Therefore, the engineered ComQXPA-P srfA M QS cassette can be applied as a novel bifunctional QS circuit to flexibly control gene expression in C. glutamicum.

ObjectivesTo explore an L-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. ResultsFour Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying P tacM -driven IleRS1 or IleRS3 (14.09±1.07, 15.20±0.93 g 4-HIL L -1 ) were similar with control strain S-D5 I (15.73±2.66 g 4-HIL L -1 ). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated L-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5 I and KIRSA-3-9 I, and Ile concentration was maintained less than 35 mmol L -1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9 I produced 22.46±0.96 g 4-HIL L -1 . ConclusionThe screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.