| Bacillus licheniformis,which is not only a model organism in Gram-positive strain but also a food safety strain,has been widely applied to various industrial fermentation.Rapid advances in synthetic biology,which can remodel metabolism network to achieve established goal.However,the transcriptional reprogramming for metabolism substrate caused by carbon catabolite repression(CCR)may fluctuate the present metabolism network.The CCR was an effect commonly found in various microorganisms for distributing carbon flow.Although the CCR methods were different,the internal logical was consistent.In Gram-positive bacteria,this effect was driven by CcpA-cre interaction.Only we clearly known the molecular mechanism of CcpA-cre interaction,the carbon flow redistribution could achieve by rational design.This study selected the B.licheniformis that has a broad substrate spectrum as object and systematic studied the molecular mechanism of CcpA-cre interaction.The main findings are as follows:(1)Modeling and characterizing a cre motif in B.licheniformis.Firstly,the cre motif was identified as ‘TGWMARCGHTWWHW’ by constructing a library with 20 cre sites that were reported explicitly in B.licheniformis closely related strains.According to this motif,126 cre sites were identified in B.licheniformis genome,revealing the cre sites have global distribution.Secondly,the CcpA-cre interaction was identified as key factor for CCR effect in the presence of glucose.The physiological meaning of this effect was accelerated the substrate metabolism of inhibited carbon sources(such as maltose and mannitol)after glucose depletion.Finally,cre element engineering improved the activity of the indicator product by 101.41% by eliminating the sensitivity of the CcpA protein.(2)Identification of a new class of cre site in B.licheniformis for achieving programmable multi module collaborative regulation for different effectors.Firstly,a target consisted a conservative palindrome regions(AGCTTT/AAAGCT)and non-conservative spacer regions was identified.Secondly,the CcpA-cre interaction mediated carbon catabolite activation(CCA)in the presence of glucose,while the inversion of the palindrome regions of the cre motif would reverse CCA to CCR.Finally,a hybrid genetic loop with cre sites and mannitol elements could switch the ON/OFF states through effectors.After optimization,the OFF value reduced by 90.69% comparing to the base-line,which eliminated the background interference.(3)Mining and identifying a class of cre degenerated sites for revealing the alternative strategies for CcpA-cre.Firstly,we compared the substrates utilization difference between wild strain and CcpA-deletion strain and conducted molecular analysis in difference substrates.A common sequence ‘ATAA-N7-TTAT’ was found in these difference substrates operons and identified as CcpC binding sites.Secondly,81 targets were found in B.licheniformis genome,in which 34.62% differed from the cre motif in bases within 3.The CcpC protein was identified as bidirectional transcription regulator.(4)Relieving the CCR effect of glucose for xylose by CRISPRi-TF systems based on cre element.Firstly,the xylose inducible promoters were constructed by Xyl R boxes with different sources.Secondly,we constructed CRISPRi-TF that induced by xylose,which can relieve the CCR effect for xylose by targeting sgRNA guide sequence to three cre sites in xylose metabolism genes.51.46% xylose could co-uptake with glucose after optimizing the addition time of glucose and xylose.B.licheniformis exhibited significant carbon substrates selectively,which mainly driven by CcpA protein.In the presence of glucose,CcpA mediated CCR and CCA effect,which was caused by different cre types.Targeted competition of cre site based on CRISPRi-cre was a novel way for revealing the CCR effect. |
