Modification Of Bacillus Subtilis Strain, Promoter Optimization And High-level Expression Of Pullulanase

Bacillus subtilis is listed as generally recognized as safe(GRAS)bacterial by America food and drug administration,owns powerful protein secretion capability and has been widely applied in the expression of heterologous proteins.In our laboratory,a wild-type B.subtilis were screened previously that owning superior cell growth and protein secretion capability,which also has many undomesticated properties and is not suitable to be used as heterologous gene expression host directly.This study carried out a series of modifications on the wild-type strain on the basis of constructing B.subtilis CRISPR/Cas9 system,obtained a dual promoter expression system for this strain through promoter screening.The reporter enzymes used in this study contain β-cyclodextrin glycosyltransferase(β-CGTase)and α-cyclodextrin glycosyltransferase(α-CGTase),used in cyclodextrin production,and pullulanase,used in starch debranching,of which the expression of pullulanase was focused investigated.The main points of this study were listed as follows:(1)Constructing CRISPR/Cas9 system in B.subtilis and improving the fermentation characteristic of strain through gene disruption.Constructing CRISPR/Cas9 system in B.subtilis ATCC 6051 a,the knockout plasmid constructed contains sg RNA,cas9 gene,homologous repair template,E.coli replicon p15 A,temperature-sensitive replicon PE194,ampicillin resistance gene and tetracycline resistance gene.The system above were used to disrupt genes srf C,spo IIAC,npr E,apr E and amy E of B.subtilis ATCC 6051 a orderly,yielding strain BS1 that produced less foam,BS2 that owned lower sporulation efficiency,BS3 and BS4 that showed lower protease activity,BS5 that showed lower amylase activity successively.The system above were used to modify the wild-type strain screened in our laboratory by disrupting genes srf C,spo IIAC,amy E,npr E and apr E successively,yielding strain WS5.(2)Screening promoters using β-CGTase as reporter enzyme in stain WS5.Firstly,investigating the β-CGTase expression levels of promoters Pamy Q/Ba,Psrf,Pxyl,Pgsi B,Pxyl/Bm,PHpa II,Pamy Q,Papr E and Pnpr E through shake-flask cultivations.The highest expression promoter was Pamy Q with extracellular activity of 24.10 U·m L-1 at 48 h.Then,constructing six dual promoters Psrf-Pamy Q,Pxyl-Pamy Q,Pgsi B-Pamy Q,PHpa II-Pamy Q,Pamy Q-Pamy Q and Pnpr E-Pamy Q and getting the highest expression promoter PHpa II-Pamy Q through shake-flask cultivations,which showed extracellular activity of 30.51 U·m L-1 at 48 h.The real-time PCR showed that the promoting capability of promoter PHpa II-Pamy Q was higher than that of promoters Pamy Q and Pamy Q/Ba.Using dual promoter PHpa II-Pamy Q to express pullulanase and α-CGTase,the extracellular activities were 90.69 and 9.51 U·m L-1 after 48 h of shake flask cultivations,respectively.During the 3-L fermenter cultivation,dual promoter PHpa II-Pamy Q mediated pullulanase expression with a highest activity of 623.17 U·m L-1,while the SDS-PAGE analysis showed that the degradation of pullulanase by extracellular proteases were severe.(3)Disruption of six extracellular proteases and optimizing the fermentation culture of pullulanase recombinant strain.Disrupting the rest six extracellular proteases in strain WS5,yielding strain WS11.During shake flask cultivations,the expressions of α-CGTase and β-CGTase in strains WS5 and WS11 showed no significant differences,while the expression of pullulanase in strain WS11 showed 26.2% less than strain WS5.During the 3-L fermenter cultivation,the highest activity of pullulanase was 174.47 U·m L-1,SDS-PAGE analysis showed that the degradation of pullulanase by extracellular proteases in host strain WS11 was much weaker than that of host strain WS5.Through shake flask and 3-L fermenter cultivations optimization,during 3-L fermenter cultivation,the highest activity of pullulanase recombinant strain WS11 PUL,using strain WS11 as the host strain,was 1047.58 U·m L-1,which was 9.48 folds of that of shake flask cultivations.(4)Investigating the influence of proteases on pullulanase expression and furtherly improving the expression through optimization of 3-L fermenter cultivation feeding solution.Investigating the enzyme activity of nine pullulanase recombinant strains WS3 PUL to WS11 PUL during shake flask cultivations,the recombinant strain WS5 PUL showed the highest extracellular activity.Through investigating the pullulanase expressions of recombinant strains WS5 PUL,WS9PUL,WS10 PUL and WS11 PUL during 3-L fermenter cultivations,the recombinant strain WS9 PUL showed the highest extracellular activity of 2449.62 U·m L-1,which was 1.17 folds of that of recombinant strain WS5PUL(2094.98 U·m L-1).The kinetic parameters and thermostability of recombinant strains WS5 PUL,WS9PUL,WS10 PUL and WS11 PUL during 3-L fermenter cultivations samples indicated that the folding states of pullulanase decreased with the increase of the number of protease disrupted.Finally,through optimizing of 3-L fermenter cultivation feeding solution,recombinant strain WS9 PUL achieved the highest pullulanase activity of 5951.84 U·m L-1 at 99 h with a carbon to organic nitrogen source(corn steep powder and yeast extract)ratio of 4:1.(5)Investigating the influence of chaperone on pullulanase expression.The results demonstrated that in host strain WS11,the disruption of gene hrc A in host strain WS11,the expression of chaperone genes prs A and csa A in episomal plasmid p BE-S194,and the expression of chaperone genes prs A,csa A,dna K and dna J in pullulanase expression plasmid p HYPULd4 showed no significant beneficial effect on the pullulanase expression,while the expression of chaperone gene grp E in pullulanase expression plasmid p HYPULd4 showed 13% beneficial effect on the pullulanase expression.Simutaneously,the expression of chaperone gene grp E in pullulanase expression plasmid p HYPULd4 showed 4.53% and 8.19% beneficial effects in host strains WS4 and WS5,respectively,while showed no beneficial effect in host strains WS9 and WS10.(6)Investigating the influence of disruption of D-alanine esterification regulatory gene dlt B and autolysin encoding gene lyt C on the expression of pullulanase,and improving pullulanase expression furtherly through signal peptide optimization.The disruption of gene dlt B in host strain WS9 yielded host strain WS9 D and improved the pullulanase expression by 48% during shake flask cultivations simultaneously.The disruption of gene lyt C in host strain WS9 improved the pullulanase expression by 24% during shake flask cultivations,while the disruption of gene lyt C in host strain WS9 D showed no significant influence on the pullulanase expression during shake flask cultivations.Through high-throughput screening method,two signal peptides were screened for the pullulanase expression,the genes ywt F and phr K signal peptides.During 3-L fermenter cultivation,the highest pullulanase activity mediated by gene ywt F signal peptide in host strain WS9 D was 8037.91 U·m L-1,which was the highest pullulanase fermentation value reported so far.