Quorum sensing-mediated dynamic regulation of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum

With the development of synthetic biology, some quorum sensing (QS) systems have been studied and applied to coordinate growth and production. Recently, a novel ComQXPA-P_srfA system with different response strengths was constructed in Corynebacterium glutamicum. However, the plasmid-harbored ComQXPA-P_srfA system lacks genetic stability, which restricts the application of this QS system. In this study, the comQXPA expression cassette was integrated into the chromosome of C. glutamicum SN01, resulting in QSc chassis strain. The green fluorescence protein (GFP) was expressed by the natural and mutant P_srfA promoters (P_srfAM) with various strengths in QSc. All the expressions of gfp were activated to the related level in a cell density-dependent manner. Therefore, ComQXPA-P_srfAM circuit was applied for modulating the dynamic biosynthesis of 4-hydroxyisoleucine (4-HIL). First, the expression of ido encoding α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase was dynamically regulated by P_srfAM promoters, resulting in QSc/_NI. The 4-HIL titer (125.18 ± 11.26 mM) increased by 45.1% compared to static ido expression strain. Then, to coordinate the α-KG supply between TCA cycle and 4-HIL synthesis, the activity of α-KG dehydrogenase complex (ODHC) was dynamically inhibited by regulating the expression of ODHC inhibitor gene odhI under QS-responsive P_srfAM promoters. The highest 4-HIL titer of QSc-₁₁O/₂₀I (145.20 ± 7.80 mM) increased by 23.2% compared to QSc/₂₀I. This study modulated two critical genes expression in both cell growth and 4-HIL de novo synthesis pathways by the stable ComQXPA-P_srfAM system, and 4-HIL was produced responsively with the cell density. This strategy enhanced the 4-HIL biosynthesis efficiently without additional genetic regulation.