The Multi-pathway Regulatory Network Of DegU On Lichenysin Biosynthesis And The Construction Of Lichenysin High-production Strain In Bacillus Licheniformis

Cyclic lipopeptides,used as biosurfactants and antibiotics,can be applied in the fields of oil exploitation,environmental remediation,disease treatment and biological control due to their unique amphipathic molecular structure.Lichenysin is one of the important cyclic lipopeptide biosurfactants produced by Bacillus spp.,which has high surface activity,bacteriostatic activity and is stable at extremes of p H,temperature,and even in salt.However,the current production of lichenysin is not enough to support industrial production.Moreover,lichenysin has the complex anabolic pathway and unclear synthetic regulatory network,which limits the metabolic engineering of lichenysin high production strains.Transcription factor DegU was reported to play a significant role in multicellular behavior,such as biofilm formation,sporulation,flagellum formation and swarming motilit.In recent years,the studies about the regulation of DegU on production synthesis have drawn more attentions.Aiming at the unclear regulatory network of lichenin biosynthesis,this study systematically explored the regulatory network of transcription factor DegU on lichenysin synthesis in Bacillus licheniformis by the means of ¹³C metabolic flow analysis,transcriptional level analysis and electrophoretic mobility shift assays（EMSA）.In order to solve the problem of low yield of lichenysin,the breeding of lichenysin high-production strain was carried out by modular metabolic engineering method.The main results of this study are as follows:1.DegU has directly negative regulation on lichenysin synthase lch A operon.In our study,DegU was deleted in B.licheniformis WX02（WX02△degU）,in which the lichenysin yield was increased by 13.6 folds（2.18±0.03 g/L）compared with the wild-type strain WX02（0.15±0.01 g/L）.On the other side,overexpression of DegU had strong inhibition on lichenysin yield.Transcriptional level analysis indicated that the transcription of lch A operon（include genes lch AA,lch AB,lch AC and lch AD）in WX02△degU was increased by a large margin compared with the control strain.EMSA further proved that DegU can bind to the promoter region of lch A operon,which illustrated that transcription factor DegU was the key inhibitor of lichenysin biosynthesis by directly inhibiting the expression of lch A operon.2.DegU has regulation on glucose uptake,amino acid synthesis,fatty acid synthesis and acetoin synthesis.It was found in our study that lichenysin yield was still inhibited by overexpressing DegU in the promoter-substituted strain（WX02-PP43lch）,in which promoter of lch A operon cannot be controlled by DegU.Thus,through 13C-metabolic flux analysis,we found that deletion of degU also enhanced glucose uptake,branched chain amino acid synthesis,and fatty acid synthesis,while decrease acetoin synthesis.Our further explored the regulation of DegU on following modules:1)DegU has directly negative regulation on pts G gene.The glucose consumption rate of WX02△degU was higher than that of the control strain.The transcription of gene pts G was increased 3.74 folds in WX02△degU compared with the wild-type strain WX02,while the transcription levels of three genes（glc U,glc P,glc K）involved in NPTS had no significant difference between these two strains.EMSA proved that DegU can bind to the promoter region of gene pts G,which illustrated that DegU can negatively control glucose uptake by directly regulating the phosphotransferase system.2)DegU has directly negative regulation on aspB,ilvD gene and ilv-leu operon.The concentrations of four amino acids（Asp,Ile,Leu,Val）in the double deletion strain WX02△lch AC△degU were increased 56.83%,227.46%,42.96%and 152.42%respectively compared the control strain WX02△lch AC,which proved that the deletion of degU can inproved the synthesis of above amino acids.The transcriptional levels of asp B,ilv A,ilv D,and ilv-leu operon（including genes ilv B,ilv H,ilv C,leu A,leu B,leu C and leu D）were increased obviously in WX02△degU compared with WX02.EMSA showed that DegU can bind to the promoter regions of asp B,ilv D gene and ilv-leu operon,but have no binding activity on the promoter PilvA.Overall,the synthesis of aspartic acid and BCAAs was improved in degU deletion strain due to the directly negative regulations of DegU on asp B,ilv D,and ilv-leu.3)DegU has directly negative regulation on bkdR gene and acc DA,bkd operon.The result of GC-MSMS analysis showed that the concentrations of straight chain fatty acids（SCFAs）C14:0,C16:0 and C18:0 in WX02△degU were increased by 14.48,3.04 and 2.29folds respectively compared with WX02.The concentrations of branched chain fatty acids（BCFAs）i C14:0,i C15:0,a C15:0,i C16:0,i C17:0 and a C17:0 in the double deletion strain WX02△lch AC△degU were increased 71.80%,37.39%,45.58%,78.80%,43.28%and 34.76%respectively compared with WX02△lch AC.The transcriptional levels of the gene clusters acc DA and bkd were increased in degU deletion strain compared with WX02 and the transcriptional level of gene bkdR was increased by 5.44 folds,which is the positive regulator of bkd operon.EMSA showed that DegU had binding activity on probes Pacc DA,Pbkd and Pbkd R,which further proved that DegU can influent the synthesis of fatty acids by direct regulating the expression of above genes.4)DegU has directly negative regulation on alsSD operon.The concentration of acetoin and 2,3-butanediol were decreased 65.37%in WX02△degU compared with WX02.The transcriptional level of gene cluster als SD was decreased by about50%in WX02△degU compared with WX02.EMSA showed that DegU had binding activity on probes Pals SD,which illustrated that DegU positively regulates the synthesis of acetoin by directly binding to the promoter region of als SD.3.Metabolic engineering for lichenysin high production in Bacillus licheniformis.Bacillus licheniformis DW2 was selected as the chassis cell in this study.DW2Cas9n△bac ABC△hsd R△com I（Y1）was chosen as the starting strain,the lichenysin yield if which was20.14 mg/L.In this study,the promoter of the lch A operon was replaced by promoter P_P43UTR12and a non-coding sequence before the start codon of lch AB,lch AC and lch AD genes was replaced with the sequence of UTR12 to constructed the strain Y7,the lichenysin yield if which was 3.40 g/L.The transcription level of the lch A operon was improved by optimizing the promoter and the 5’-UTR regions.Then,the recombinant strain Y15 was constructed by knocking out the synthetic gene cluster of lichenysin competitive products such as pulcherriminc acid,polysaccharide and non-ribosomal peptide based on Y7 and the lichenysin yield of Y15 was increased to 6.85 g/L.Through the analysis of strain growth and metabolism,we found that Y15 significantly increased the biomass of bacteria and reduced the production of byproducts such as acetoin,2,3-butanediol and acetic acid.Next,the synthesis of lichenysin precursors was enhanced in strain Y15 via replacing the promoters of aspartic acid,branched amino acid and branched fatty acid synthesis pathway genes by P_P43UTR12 promoter to construct strain Y26.However,lichenysin yield and biomass in Y26 strain were significantly reduced compared with Y15 even though the results of RT-q PCR showed that the promoter modification in Y26 successfully improved the transcription level of related genes.We found that the growth of strain Y26 was restore in lichenysin fermentation medium with adding 5 g/L peptone and the lichenysin yield of Y26 was increased to 10.20 g/L.Finally,strain Y28 was constructed based on strain Y26 by enhancing lichenysin transport and glucose uptake and the lichenysin yield of Y28 in the optimized medium was 11.40 g/L,which was 570 times higher than that of the original strain Y1.Overall,this study confirmed the transcription factor DegU as a key inhibitor of lichenysin synthesis for the first time.Moreover,this study systematically demonstrated the regulatory network of DegU on carbon and nitrogen metabolic pathways including glucose uptake,amino acid synthesis,fatty acid synthesis and acetoin synthesis.On this basis,the strain with high yield of lichenysin was constructed through modular metabolic engineering including the optimization of promoter elements,elimination of competitive pathway and enhancement of precursor.This study finally obtained a lichenysin high production strain Y28 which can produce 11.40 g/L lichenysin.