Regulation Strategy For Efficient Expression Of Pullulanase In Bacillus Subtilis

Pullulanase（EC3.2.1.41,Pul）can specifically hydrolyzeα-（1,6）glycosidic bonds in amylopectin,and is widely used in the production of high glucose syrup,ultra-high maltose syrup,beer and modified starch food.To improve the fermentation level of Pul,the researchers expressed it in hosts such as Escherichia coli,Bacillus subtilis,and Pichia.B.subtilis has many advantages,including a short fermentation period,strong protein secretion ability,and no pathogenicity.It is one of the Pul production hosts stipulated by National Food Safety Standard GB 2760-2014.However,the expression level of Pul in B.subtilis is low due to the lack of effective expression regulation strategies.In this study,the expression of B.deramificans Pul in B.subtilis WB600 was significantly increased through the rational design of the N-terminal coding sequence（NCS）,the selection of autoinduction expression patterns,expression element combination optimization,and the intelligent regulation of cell lysis inhibition.The main findings were as follows:（1）Rational design of the signal peptide NCS to enhance the translation efficiency of PulSynonymous mutations were conducted within the first ten amino acids of the N-terminal of super folded green fluorescent protein（sf GFP）,and 8598 NCS mutants with different expression levels were constructed.Their relative fluorescence intensity（RFI）was 2300 to127671.Mutants were sorted based on RFI,and 1～2 clones per 50 clones were selected for shake flask validation.A total of 172 representative NCS mutants were obtained.The effects of four types of gene sequences on sf GFP expression were analyzed,including nucleotide composition（GC/GC3/T3s/C3s/A3s/G3s）,codon usage(CAI/CBI/F_op),m RNA secondary structure（ΔG）and translation initiation rate（TIR）.A statistical model for the correlation of protein expression and NCS was established by stepwise multiple regression:P_{sf GFP=}274497.657-108717.401×GC3+4886.529×ΔG(ρ=0.675,ρ_s=0.657,P=3.5×10^-24).Four Bgls signal peptide NCSs with different expression levels were designed based on this model.The Pul activity analysis showed a high positive correlation between the Bgls signal peptide-mediated extracellular Pul activity and the predicted P_{sf GFP} value(ρ=0.675,ρ_s=0.657,P=3.5×10^-24.When the optimal NCS was used,the Apre signal peptide-mediated Pul activity reached 50.4 U/m L,5.15 times higher than before the NCS optimization.（2）Screening Pul autoinduction expression pattern to reduce cell growth stressBased on the quorum-sensing Com QXPA,a series of autoinduction expression systems with different expression intensities and induction initiation were constructed in B.subtilis.In order to enhance the expression strength,the-30,-15,and-10 regions of the QS promoter P_srfawere replaced with conserved sequences,and the P₁ promoter（only the-30 region was replaced）with an 80%higher expression strength was obtained.The spacer region between the-35 and-15 regions of the P₁ promoter was randomly mutated.The QS promoter P_S1Ewas obtained by flow cytometry,whose expression intensity was 8.22 times higher than that of P_srfa.Three autoinduction systems were obtained using different phase-dependent promoters to express the auto-inducible pheromone Com X and the transcription factor activator protein Com A.The autoinduction start and end times ranged from 4-14 h（type I）,1.5-20 h（type II）,and 8-16 h（type III）,respectively.The Pul activity expressed by P_S1E reached 80.2 U/m L when P_{sig W} and P_{xyn A} expressed Com X and Com A,respectively,the expression level of which was 36%higher than that of the most potent constitutive P₅₆₆promoter.The analysis of the expression process of Pul showed that the strength of the autoinduction expression system was lower than that of P₅₆₆in the cell growth stages.In the middle and late stages of fermentation,the expression intensity of the former was higher than that of the latter,and the autoinduction expression system showed a higher bacterial mass.The above results indicated that the autoinduction expression reduced host cell growth stress.（3）Optimizing the combination of expression elements to enhance the adaptability of Pul transcription and translationThe promoter,ribosome binding site（RBS）,and terminator libraries were constructed using sf GFP as the reporter.The-30 and-10 regions of the QS promoter P_{srf A}were randomly mutated and replaced by conservative sequences,and 12 promoter mutants with the most significant difference in RFI of 46 times were screened from 948 promoter variants.Based on the RBS Calculator,12,000 RBSs with different translation initiation rates were designed,and13 representative RBSs with a maximum difference of 57 times in RFI were obtained.The random mutation was performed between the SD sequence and ATG in R11 with the highest RFI,and a stronger R11E was obtained by flow cytometry sorting,making the maximum difference in RFI of the RBS library up to 102 times.According to the same free energy difference interval,11 terminators from 425 Rho-independent terminators were selected for expression verification.The neck loop region of the T8 terminator with the highest RFI value was randomly mutated,and the terminator library with a maximum RFI difference of 6 times was obtained by flow cytometry.The above promoter,RBS,and terminator libraries were combined using Gibson protocol,and 34 expression element modules with a maximum RFI difference of 627 times were obtained using flow cytometry.Regression analysis showed that the interaction of promoter,RBS,and terminator contributed 63%to the expression of sf GFP in B.subtilis.Under the strongest expression module H₃₃,the Pul activity reached 138.3 U/m L,which was 72%higher than before optimization.（4）Intelligent regulation of anti-lysis genes to enhance the degradation resistance of host cellsPrevious studies showed that the adaptor protein Ssp B could specifically bind to the ssr A-tagged protein and deliver the latter to the Clp XP protease for degradation in B.subtilis.To investigate the effect of the initiation time of inhibiting cell lysis on the cell growth and Pul synthesis,a xylose-induced degradation system was constructed to hydrolyze the autolysin N-acetylmuramyl-L-alanine amidase（Lyt C）and N-acetylglucosaminidase（Lyt D）.The ssr A tags were fused to the C-terminus of Lyt C and Lyt D,respectively,and Ssp B was expressed by the P_xyl promoter.The results showed that with the increase of xylose addition time,the Pul activity expressed by module H₃₃ first increased and then decreased,and the biomass generally showed a downward trend.When xylose was added at 20 h,the highest Pul activity of 181.4 U/m L was obtained.The above results indicate that inhibition of Lyt C and Lyt D contributes to cell growth and Pul synthesis.Transmission electron microscopy analysis showed that inhibition of Lyt C and Lyt D activities at the early stage of fermentation to thicken the cell wall may be the main reason for the lower extracellular yield of Pul.In order to avoid adding the inducer,the Lux IR quorum sensing was introduced to express Ssp B,which achieved the automatic degradation of Lyt C and Lyt D in about 20 h,and the Pul activity reached 184.8 U/m L.In order to eliminate the possible adverse effects of autolysin inhibition on the proliferation and protein secretion of fresh cells,an"AND"logic gate expression system composed of the Lux IR quorum sensing and growth stationary phase promoter P_{yqf D} was constructed to regulate the degradation of Lyt C and Lyt D.By screening the RBS of P_{yqf D} to adjust the autolysin inhibition time,the Pul activity in recombinant B.subtilis in shake flask and 5 L fermenter reached 274.8 U/m L and 644.8U/m L,respectively.