Development Of Quorum Sensing System Applied In Corynebacterium Glutamicum And Its Application

Quorum sensing(QS)system is an autoinducible system that can sense the increase of cell density and thereby regulates the expression of its downstream gene(s)in a cell density-dependent manner.The Com QXPA system of Bacillus subtilis can dynamically up-regulate the expression of its downstream genes such as srfA in a cell density-dependent manner.In this study,the Com QXPA-P_srfA QS auto-induction system was designed,constructed and introduced into Corynebacterium glutamicum to dynamically upregulate gene expression.This system was then used to control hfq-sRNA module of target gene to dynamically downregulate the expression of this gene.Next,a P_srfA promoter mutation library was constructed,and natural and mutant P_srfA promoters were used to dynamically upregulate and downregulate different target genes simultaneously.Finally,this biofunctional auto-induction system was used to regulate the?-amylase genes amy E and isoleucine dioxygenase gene ido to further verify its regulatory effect.The main research contents and results are as follows.(1)First,the com QXPA operon of B.subtilis and its target promoter P_srfA were modified and introduced into C.glutamicum SN01 to construct the Com QXPA-P_srfA QS system.This QS system was used to regulate the expression of green fluorescent protein encoding gene gfp.The gfp expression was up-regulated in a population density-dependent manner and was activated by 6.35 folds.(2)The above-mentioned QS auto-induction system regulated positively by Com QXPA-P_srfAwas used to mediate the expression of hfq-sRNA module targeting gfp,and a QS negative regulation system was constructed.One or both of the hfq gene and the sRNA(gfp)gene were controlled by the population density-dependent promoter P_srfA,and three QS negative regulation strains were obtained.When P_srfA was used to regulate the expression of hfq gene in the hfq-sRNA module,the fluorescence level of the strain significantly reduced,and the inhibition efficiency was 96.1%.(3)To fine-tine the dynamic regulation of gene expression in a population density-dependent manner,a P_srfA promoter mutation library was designed and constructed.Through random selection and fluorescence level analysis,18 mutant promoters,i.e.Mn,of P_srfA with different strengths were screened out.The strength of these promoters were 0.4-1.5 folds that of the natural P_srfApromoter.(4)On the basis of the negative regulation of gfp expression by the hfq-sRNA mediated by Com QXPA-P_srfA QS system,the expression of m Cherry gene was also positively regulated simultaneously by the same Com QXPA-P_srfA QS system to test the biofunctional regulation performance of this QS system toward different target genes.For the positive regulation of m Cherry and negative regulation of gfp,four promoters with different strengths,i.e.mutant M1,M11,M22 and natural P_srfA promoters,were chosen;thereby,16 bidirectional QS strains regulated auto-inducibly by different combinations of these promoters were constructed.When the expression of gfp was negatively regulated by the M11 promoter,the cell density-dependent down-regulation effect of green fluorescence was the best.When the expression of m Cherry was positively regulated by each of the four promoters,the red fluorescence was all enhanced in a cell-density-dependent manner.(5)To further verify the regulatory effect of this biofunctional auto-inducible Com QXPA-P_srfA QS system,the expression of the?-amylase gene amy E and the isoleucine dioxygenase gene ido were then regulated by this QS system.The M14,M20,M28,and P_srfA promoters were used to positively regulate the expression of amy E in an auto-inducible manner.The M14regulated bacterium showed the highest enzyme activity of 3.02 U·m L^-1.Then M1 and M11were used to regulate the hfq-sRNA module targeting amy E.The enzyme activity of these strains reduced by 8.29-100%.Among them,M11 showed the best inhibitory effect.Finally,M1,M11,M20,M22,M28,and P_srfA were used to positively regulate the expression of ido in a population density-dependent manner,and the titer of 4-hydroxyisoleucine,the final product of ido expression,was detected.The ido-expressing strain regulated by the natural P_srfA showed the highest titer of 24.93 g·L^-1.