Research Of Bacitracin And Pulcherriminic Acid Biosynthesis Regulation In Bacillus Licheniformis

Nonribosomal peptides synthetase(NRPS)and cyclodipeptides synthetase(CDPS)were the two main synthetases of nonribosomal peptides in Bacillus licheniformis.The NRPS-dependent bacitracin and CDPS-dependent pulcherriminic acid were the representative antimicrobial peptides in B.licheniformis.However,there was poorly understanding about the regulation of their biosynthesis pathways.This research was based on the bacitracin industrial strain B.licheniformis DW2.Gene in-frame deletion,RT-qPCR and electrophoretic mobility shift assay(EMSA)were applied to examine the functions of several transcriptional regulators in the regulation of bacitracin and pulcherriminic acid biosynthesis.For the first time,this study provided insights into the detail regulation network of CDPS synthesis pathway and explained the decision mechanism of pulcherriminic acid biosynthesis.The major findings of this research are listed as follows:1.Regulation of SpoOA-AbrB-SigH system on bacitracin biosynthesis.bacT is a putative type Ⅱ thioesterase gene which located on the upstream of bacitracin synthetase operon bacABC.The deletion of bacT did not affect the transcription of bacABCbut led to an 83.01%decrease of bacitracin synthesis efficiency,it indicated that the putative type Ⅱ thiesterase BacT is a component of the bacitracin synthetase.In the SpoOA-AbrB-SigH system,AbrB repressed the expression of bacT and bacABC by binding directly to their promoters and SpoOA influences bacitracin biosynthesis by negative regulating abrB.In the DW2△abrB、DW2△0A△abrB and DW2△0A/pHY-sad67 strains,which abrB was knocked out or its expression was repressed,the transcriptional levels of bacT and bacA were improved in different ranges,and the bacitracin yield on cell numbers were improved by 19.32%,25.60%and 24.15%respectively compared to that of DW2.In the strains which AbrB expressed in a high level(DW2△0A and DW2△abrB/pHY-abrB),the transcription of bacT and bacA were repressed.These results indicated that AbrB was a negative regulator of bacT and bacA.Then,EMS A confirmed that AbrB regulated bacitracin biosynthesis by binding directly to the promoter of bacT and bacA.Deletion of sigH in DW2△0A△abrB did not affect bacitracin biosynthesis and transcription of bacT and bacA suggested that SigH did not participate in the transcription of bacT and bacA directly.2.Regulation of AbrB-YvnA-YvmB system on pulcherriminic acid biosynthesisAfter abrB was knocked out,pulcherriminic acid yield was improved by 103.16%.The transcriptional levels of yvmC and yvnA were also improved,but yvmB decreased.In DW2/pHY-abrB,the transcription of yvmC,yvnA and pulcherriminic acid synthesis were repressed,but the transcription of yvmB was activated.Then,EMSA confirmed that AbrB regulated yvmC and yvnA by binding directly to the promoters which indicated that AbrB is a negative regulator of yvmC and yvnA.In DW2△yvmB,pulcherriminic acid yield was improved by 60.12%.yvmB deletion also caused the improment the transcriptions of yvmC and yvmA.In DW2/pHY-yvmB,the transcription of yvmC,yvmA and pulcherriminic acid synthesis were repressed.EMSA confirmed that YvmB regulated yvmC and yvmA by binding directly to their promoters and indicated that YvmB is a negative regulator of yvmC and yvmA.Both deletion and overexpression of yvnA repressed pulcherriminic acid synthesis completely.In DW2/pHY-yvnA,YvnA protein repressed transcription of yvmC and yvmB by binding directly to their promoters.In DW2△yvnA yvmB expressed in a high level as the remove of YvnA and enhanced the repression to yvmC.Knock out yvmB gene in DW2A△yvn4 could restore the synthesis of pulcherriminic acid.In summary,pulcherriminic acid synthesis operon yvmC-cypX is repressed directly by AbrB,YvnA and YvmB.While,AbrB is also the repressor of yvnA gene and YvnA is the repressor of yvmB.YvmA acted as an export protein of pulcherriminic acid,deletion of the yvmA gene led to intracellular accumulation of pulcherriminic acid.3.The self-resistance of pulcherriminic acid in B.licheniformisThe yvmC deletion control strains were constructed on the base of various strains,and the cell growth status at different concentrations of FeCl3 was investigated.In iron limiting conditions(3 mg/L-10 mg/L FeCl3),excessive pulcherriminic acid synthesis inhibited the growth of the strains by causing iron starvation.Further comparison of the transcriptional levels of yvmC,abrB;yvmB,yvnA in DW2,DW2△yvmB and DW2△yvnA△yvmB strains at 0 mg/L,5 mg/L,and 20 mg/L FeCl3 concentrations,we found that B.licheniformis changed yvnA expression level directly to control pulcherriminic acid yield and avoied iron depleting.