Optimization Of Regulatory Elements For Pullulanase Gene Expression In Recombinant Bacillus Subtilis

Pullulanase from Bacillus naganoensis can specifically hydrolyze the?-1,6-glycosidic linkages of amylopectin with desirable enzymatic properties,which can be applied to starch saccharification and valuable for industrial application.Bacillus subtilis expression system possesses many advantages,including generally recognized as safe,favorable protein secretion system and simple culture conditions.Therefore,overexpression of pullulanase in B.subtilis is important for both academic research and industrial application.In this study,the constitutive and inducible expression systems were recombined by optimization of regulatory elements and used in both B.subtilis WB600 and B.subtilis WB800.The effects of promoter tandem,addition of regulator genes and optimization of 5'-UTR sequence on the extracelluar expression of pullulanase were explored.After that,the induction conditions of recombinant strains with great potential were optimized.This study not only increased the extracellular pullulanase expression level in B.subtilis,but also provided ideas and options for recombination expression system of B.subtilis.The main contents are as follows:?1?For obtaining recombinant expression vector pMA0911-P₄₃-pul,pMA0911-P₄₃?D?-pul,pMA0911-P₄₃?T?-pul and pMA0911-P₄₃?F?-pul,sequences haboring 1 to 4 constitutive P₄₃3 promoters were constucted.The extracellular expression level of pullulanase in recombinant strain B.subtilis WB600 and B.subtilis WB800 were measured respectively.Pullulanase expressed in recombinant strain B.subtilis WB600/pMA0911-P₄₃-pul showed the highest enzymatic activity,which reached to 15.1 U·mL^-1.?2?The enhancer genes degQ,degU and degS were introduced into the vector pMA0911-P_sacB-pul,which haboured an inducible promoter P_sacB.In recombinant strain B.subtilis WB800,DegQ demonstrated positive regulatory effect on the expression of pullulanase,increasing the pullulanase activity from 4.5 U·mL^-1 to 7.2 U·mL^-1.When the degQ gene was inserted into the position that closed to promoter P_sacB,the pullulanase activity was further increased to 12.1 U·mL^-1,which was 2.7-fold higher than that from original strain B.subtilis WB800/pMA0911-P_sacB-pul.?3?To further improve inducible expression vector pMA0911-P_sacB-pul-degQ?N?,the5'-UTR of mRNA was designed rationally,resulting in 4 recombinant strains having different5'-UTR sequences.Among these recombinants,the pullulanase activity of pullulanase expressed in B.subtilis WB800/pMA0911-P_sacB-pul-R4 reached 16.8 U·mL^-1,which was1.4-fold than that of the control strain without optimizing 5'-UTR sequence and 3.7-fold than that from the original strain B.subtilis WB800/pMA0911-P_sacB-pul.?4?After inserting gene degQ and optimizing 5'-UTR sequence,the induction conditions of recombinant strain B.subtilis WB800/pMA0911-P_sacB-pul-R4 were optimized.After incubation for 9 h before induction,inducer sucrose was added to a final concentration of 5%.Finally,the pullulanase activity of pullulanase increased to 28.7 U·mL^-1,which was 3.7-fold than that from the original strain B.subtilis WB800/pMA0911-P_sacB-pul.