Development Of Heat-Resistant Protease For Feedstuff And Revealing The Mode Of Secretion Regulation In Bacillus Subtilis

Feed proteases are famous for their improving animal production performance,protecting intestinal health,reducing feed costs and pollutant emissions.However,the thermal stability of commercial proteases cannot meet the requirement for granulation of pelleted feed,so the development of heat-resistant feed protease is in urgent need.B.subtilis is a model organism that has unique advantages over other expression systems and is an ideal expression host for proteases.In this study,we explored the expression levels of different heat-resistant proteases from B.subtilis,and constructed the expression toolbox of B.subtilis,the signal peptide and strong promoter element were also optimized for further improvement of the extracellular enzyme activity for different heat-resistant proteases.Additionally,the heat resistance of protease with high enzymatic activity was improved based on rational design.Several feed proteases with improved heat resistance have been obtained,proposing the foundation for the development of heat-resistant feed protease products in the future.Finally,the metabolic pathway of which is the most closely related to the efficient expression of foreign proteins（proteases）in the fermentation process of B.subtilis was determined,and the key differentially expressed genes were identified via joint analysis of the transcriptome and translatome.The above findings provide a useful guideline and theoretical basis for the metabolic engineering and efficient expression of foreign proteins by B.subtilis using genetic engineering in the future.The specific results were shown as follows:（1）Based on screening the expression of 18 genes from heat-resistant proteases,the constitutive extracellular expression of 4 heat-resistant protease genes was realized,and their extracellular enzyme activities are 1059.95 U/m L,43.51 U/m L,14.54 U/m L,and 19.40 U/m L for proteases 1143,164,158,and K19,respectively.The enzymatic properties of these four recombinant heat-resistant proteases were further studied:protease 164 was neutral metalloproteinease,158 was weak alkaline serine protease,and 1143 and K19 were basophilic proteases.All these four proteases showed excellent chemical reagent stability.Among them,the heat tolerances of protease 164,158 and K19 were superior compared with 28 commercial protease products,indicating they have potential to become commercial products.Further studies on the thermostability of neutral metalloproteinase 164 showed that the metal ions Ca²⁺and Mg²⁺can improve its thermal stability,particularly Ca²⁺.The presence of 10 m M Ca²⁺and Mg²⁺synergistically improved the thermostability of protease 164,and the half-life(t_1/2)value was increased by 50.42-fold compared to that of the wild type（7.42 min）when treating at 80°C.In the presence of metal ions,protease 164 exhibits excellent stability at 95°C and was considerably superior to current commercially available protease products.To explore the reason for the improved thermostability,structural analysis revealed that the significant enhancement in thermostability of protease 164 in the presence of Ca²⁺and Mg²⁺may be due to the covalent docking and electrostatic interactions between amino acid residues and metal ions,which was conducive to improve the stability of secondary structure,thereby significantly improving its thermal stability.（2）To improve the extracellular activity of proteases 1143 and 164,a toolbox fragment library containing 244 signal peptides（SP）of B.subtilis was built,and a basic vector containing a strong promoter（p Shuttle-09）was constructed.A signal peptide expression libraries of B.subtilis were established.Signal peptide(SP_{Bsl B})was identified as the best expressing fragment from 2,000 clones for protease 1143 was achieved via high-throughput screening,the enzyme activity of protease produced by the strain containing the SP_{Bsl B}was 1.88-fold higher than that of the strain with the wild type SP,reaching 3548.05±80.39 U/m L.The best signal peptide(SP_{Xyn D})with high yields of protease 164 were identified from 5,000 clones based on the high-throughput assay.Specifically,the enzyme activity of protease produced by the strain（383.55±15.20 U/m L）containing the SP_{Xyn D}was 5.15-fold higher than that of the strain with the initial SP.The feasibility of the toolbox fragment library was verified via screening the optimal signaling peptides of two proteases.（3）To improve the thermal stability of protease 1143（highest extracellular activity）,eight mutation sites（N18,S97～S101,E110 and R143）were determined according to analysis of the B-factor values from crystal structure.Based on multiple sequence alignment between protease 1143 and 146 thermophilic proteases,21mutants were constructed and heterologous expression was achieved using the B.subtilis expression system.Fifteen mutants（N18L,S97A and S97H,S98R and S98E,S99L and S99A,G100E and G100A,S101G and S101V,E110L,R143V,R143L,and R143G）exhibited increased t_1/2values at 65°C（1.13～31.61 times compared to that of WT）.Based on the weighted values of enzyme activity and thermostability,six complex mutants were implemented.The t_1/2value of these six complex mutants was increased by 2.12～10.05 times compared with wild-type protease 1143 at 65°C.Among them,the thermal stability of the complex mutants N18L/R143L/S97A,N18L/R143L/S99L and N18L/R143L/G100A was increased by～4 times,which reveals application potential in industry.Structural analysis discovered that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and decreased flexibility of the structure.（4）Finally,to study the mechanism of secretion and expression regulation of heat-resistant protease in B.subtilis.First,the types of carbon and nitrogen sources and their dosages,liquid filling amount,inoculation amount,skimmed milk powder content and fermentation time for proteases 1143 and 164 in B.subtilis using a single-factor optimization assay.The results showed that the optimal shaker fermentation medium was lactose 1%,peptone 6%,NH₄Cl 1%,skimmed milk powder0.8%,Na Cl 0.5%,Mg SO₄·7H₂O 0.5%,and K₂HPO₄0.5%.Other optimized conditions included inoculum volume 5%,liquid filling volume 16%,and fermentation time>66 h.Under these conditions,the extracellular enzyme activities of protease 1143 and 164 reached 4687.93±191.03 and 468.38±18.20 U/m L,respectively.Regarding protease 1143 as the reporter strain,the results showed that there were41 differential genes which were significantly associated with the rapid enzyme-producing phase（12,48,and 60 h of fermentation）on the basis of joint analysis of transcriptome and translatome in different fermentation periods.Moreover,these 41 genes were verified by translatome sequencing to be differentially expressed at the translation level,and the pathway in the top 10 enrichment had 9 metabolism and a membrane transport pathway,involving 16 significantly different genes.Additionally,transcriptome analysis showed 160 differentially expressed genes associated with salience in the late stages of enzyme production（48 h,60 h,and 72 h of fermentation）,with 9 metabolism and an immune system pathway in the top 10significant enrichments,involving 28 significant differential genes.Moreover,joint analysis of the transcriptome and translatome at the same fermentation period showed that only 54.69%of the differentially expressed genes of B.subtilis were in consist.However,436 differential genes cannot be revealed by the transcriptome,despite their expression are differential at the translation level.The top 10 pathways with significant enrichment had 7 metabolism,2 cell motility,and a signal transduction pathway,involving 47 significantly different genes.In summary,the expression and regulation mode of B.subtilis was dominated by"the central metabolic pathway（carbon metabolism and nitrogen metabolism）and supplemented by other metabolic pathways（secondary metabolism,degradation of organic compounds,electron transport chain,two-component system and bacterial motility）.