| Bacillus licheniformis is an important industrial strain,but currently wild-type and its traditional mutagenesis-derived strains are mainly used in research or production.Less understanding of regulation and the lack of standardized biological elements severely limit the application of B.licheniformis.As an expression system,the quorum sensing system don’t need to monitor the growth of the bacteria and add exogenous inducers,which is suitable for the production in fermentation engineering.This study excavated and identified the endogenous Lan QS system of B.licheniformis,and developed artificial regulatory elements based on the Lan QS system.The specific results are as follows:1.Mine the endogenous quorum sensing system of B.licheniformis CICIM B1391 and study its characteristics.By blasting,the lan gene cluster has a certain homology with the reported Agr QS system,and lanC,lanA,and lanB are expressed after reaching a certain cell density at16 h.Its downstream element,the Ptr1 promoter,shows extremely weak expression intensity in the early stage(5 h)and a pulse-like growth in the late stage(48 h),and the fluorescence intensity can reach up to 30 times that of the constitutive strong promoter Pshuttle-09;at the same time,the expression of Ptr1 relying on extracellular signal molecules,after removing the original medium,the fluorescence intensity of Ptr1 decreased to 30% of the control at the later stage(48 h).2.Identify the regulatory function of the lan gene cluster on Ptr1 promoter in vivo and in vitro binding experiments.The function of each element in the lan gene cluster was identified by the density-independent constitutive promoter,and it was found that the expression of the complete gene cluster of lanCBDA,lanC,lanBD and lanA all advanced the time of high expression of Ptr1,among which lanC and lanA have the most significant effect,and the unit fluorescence intensity at 16 h was 24 times and 11 times that of the control respectively,indicating that the lan gene cluster regulates the expression of Ptr1.Gel retardation and footprinting was used to study the regulation of lan gene cluster on Ptr1 vitro.The results showed that the regulator LanA directly binds to the promoter Ptr1,and its prediction regulatory binding site is the GGGCATTTTG-CAAAATGCCC converted repeating sequence.Therefore,the promoter Ptr1 and the lan gene cluster belong to the endogenous Lan QS system of B.licheniformis.3.Research on artificially regulated expression elements based on Lan QS system.Mutant LanA regulates the binding site and screened to obtain a mutant library whose was different from that of the original Ptr1 of expression time.The time when the specific productivity of fluorescent protein in the library reached its peak could be delayed from 40 h to 52 h at most,and advanced to 34 h,but the expression intensity appeared.The results showed that the mutation and modification of the LanA regulatory binding site could affect the expression time and intensity of Ptr1.Using Pshuttle-09 as the backbone,a LanA regulatory binding site was inserted between the-35 and-10 regions and upstream of the-35 region to construct artificial elements ST-2 and ST-3,whose expression intensity pulse grew in late period that show the function of LanA regulatory binding site.In summary,this study excavated the endogenous Lan QS system of B.licheniformis,which includes the lan gene cluster and the Ptr1 promoter.Ptr1 does not express when the cell density is low,and turns on expression after reaching a certain cell density at 16 h.And the lan gene cluster regulates the expression of Ptr1 by direct binding between the regulator LanA and the promoter Ptr1.At the same time,based on the mutation of the regulation binding site and its insertion into the constitutive promoter,the function of the artificial regulatory element was verified.This study lays a foundation for the development of new regulatory and expression elements for B.licheniformis. |
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