Establishment And Application Of Bacillus Subtilis Protein Expression System With Expression Cassette Integrated In Chromosome

Bacillus subtilis is an important microbial cell factory for the production of valuable enzymes and chemicals in fermentation industry.As a system of recombinant protein expression,B.subtilis has the advantages of good safety,strong protein secretion ability and easy large-scale fermentation;on the other hand,it has some shortcomings,one of which is plasmid instability and antibiotic safety issue when using plasmids.A strategy to solve this problem is to integrate the expression cassette into chromosome without resistant marker left.However,due to limited copy number of expression cassette,this strategy usually resulted in low protein yield.To construct a safe,stable and efficient recombinant protein expression system,this work firstly developed a fast marker-free genome manipulation method and then,improved the yield of recombinant protein through optimizing transcription,translation,secretion and host.The main results and conclusions were summarized as follows.1 pheS~*and PCR based marker-free genome manipulation methodSince this study involves many genome manipulations,a fast and simple genetic tool is desired.Thus,to begin,we developed a CSM and PCR based marker-free genome manipulation method.We established a method for marker-free genome manipulation(gene deletion,gene insertion and site-direct mutation)based on PC cassette,which can be finished in three days with 100%positive rate.The method has been widely used in B.subtilis and B.amyloliquefaciens,receiving positive comments from researchers.In addition,to further enhance its affinity for p-Cl-Phe,PheS*was modified by Thr255Ser substitution,generating pheSTS*.Host cells harboring pheSTS*were sensitive to 1 mM p-Cl-Phe Taking together,the method provides a powerful tool for following work involving genetic manipulation.2 Improving the protein production in transcriptional levelProtein production is directly affected by mRNA abundance,which is mainly affected by promoter strength and mRNA stability.Therefore,this study optimized promoter strength and mRNA stability to increase the yield of the target protein.(1)Engineering of auto-inducible promoter PylbIn terms of promoters,we focus on strength and controllability(without using toxic or expensive inducers).To begin with,the upstream element(UP),-35,-22,-16,-10 region of auto-inducible promoter Pylb(start working in the late exponential growth phase)was rational designed.BgaB(encoded ?-galactosidase)and sfGFP(encoded super-folded Green fluorescent protein)were used as reporter genes to evaluate promoter strength.The results showed that the activity of Pylb was enhanced by changing-35,-10 and UP region to"TTGACA","TATAAT" and "TTAAAAATTTTTTTTAAAAAAA",respectively.Real-time PCR showed that the transcription level of PNBP3510 was enhanced by 340-fold compared with that of Pylb.The yield of BgaB and sfGFP under the control of strongest promoter PNBP3510 were 26-fold and 195-fold higher than these controlled by Pylb.BgaB and sfGFP accounted for 43%and 30%of intracellular proteins under the control of PNBP3510,respectively,both of which were higher than their best yield reported.Then,PNBP3510was employed to drive the extracellular production of Methyl Parathion Hydrolase(MPH)and Chlorothalonil hydrolytic dehalogenase(Chd)with SPaprE as signal peptide.The extracellular activity and yield of MPH was 144 U/ml and 0.3 g/L,which was higher than that of plasmid expression system reported(27.1 U/ml,53 mg/L).The extracellular activity and yield of Chd was 4.4 U/ml and 0.27 g/L,which was higher than that of expression system reported(14.5 U/L,5.65 mg/L).In addition,when PNBP3510 was used to intracellular expression of MPH and Chd,both of the expression level were much lower than that of BgaB and sfGFP.To solve this problem,we developed a strategy,which was that fusion of MPH to the C-domain of sfGFP.The results showed that the expression level of fusion protein was comparable to sfGFP.The strategy provided another choose for efficient intracellular expression of recombinant protein.(2)Engineering of xylose-inducible promoter PxylAAs an inducer,xylose is safe and inexpensive.However,the transcription of xylose promoter is relative low.Therefore,PxylA is an ideal promoter if the transcription intensity is high.According to the strategy in Pylb engineering,PxylA from B.subtilis 168 was rational designed.Under the control of the optimized PG3510xylA,a very low expression level of BgaB could be detected without addition of xylose and the highest intracellular BgaB activity remains unchanged.The expression level of BgaB was enhanced by 136-fold.The rigor of PG3510xylA was further examined by expressing the toxic protein MazF.Finally,PG3510xylA was used for extracellular expression of MPH and MPH activity reached to 22 U/ml,which was significantly lower than that of PNBP3510 mediated expression.(3)Construction of light-controllable promoterLight control has the advantages of good safety and low cost,which has potential application value in industrial production.In this word,we tried to develop a light-controllable promoter in B.subtilis based on PNBP3510.We concluded that the light-controllable promoter could control protein expression in spite of the low expression level and obvious background expression.It has reference significance for the transient regulation of genes in the field of metabolic engineering and the establishment of environmentally-friendly expression systems.(4)Improving the stability of mRNAIt has been reported that several mRNA stabilizer,such as STAB-SD and SP82 can improve the half-life of mRNA.To improve the abundance of mRNA,mRNA stabilizer STAB and SP82 were respectively introduced to the transcription initial site of PNBP3510,generating PNBP3510ST and PNBP3510SP.Under the control of sigle copy of PNBP3510ST and PNBP3510SP,MPH activity was 178 U/ml and 21 U/ml,respectively.The former MPH activity was enhanced by 24%compared with that of PNBP3510.The results showed that STAB-SD could improve mRNA stability.3 Optimization of protein expression at translation and secretion level(1)Optimization of protein expression at translation levelAt translational level,the efficiency of initiation and extension determines the efficiency of protein translation.It was considered that the initial efficiency is mainly affected by the secondary structure of mRNA near RBS,and the extension efficiency is mainly affected by mRNA secondary structure and codon.Two strategies(random mutation and rational design)can be used for improving the efficiency of initiation and extension.First,we developed a random mutation method to change the efficiency of the initiation and extension.A positive mutant E6 was screened with MPH activity 26%higher than the original strain.This method is also applicable to construction of RBS libraries and directed evolution of enzymes.In addition,we try to improve the translation rate through rational design.At the initial rate,the secondary structure of mRNA in the translation initiation region of mpd expression cassette was predicted.The stability of mRNA secondary structure was reduced by base substitution and the free energy of mRNA folding was increased from-8.70 kcal/mol to-4.10 kcal/mol.MPH production was increased by 21%.At the extension level,the mpd codon was optimized,but the expression of MPH was not improved.(2)Optimization of protein expression at secretion levelAt secretion level,Lipoteichoic acid(LTA)is a component of gram-positive bacteria cell wall and it can absorb Mg2+ to support the activities of several synthetases on the cell membrane.D-alanylation of LTA is mediated by dlt operon and deletion of dlt operon will benefit to protein folding.PrsA is a lipoprotein on the outer surface of the plasma membrane of B.subtilis,which function as a molecular chaperone.Deletion of the dlt operon and the overexpression of the chaperone PrsA increased the MPH activity by 14%and 16%,respectively.(3)Comprehensive optimization of MPH expressionThe host used in this paper is B.subtilis PD8 constructed in our laboratory.The host was constructed by continuously marker-free deletion of eight extracellular proteases in B.subtilis 168 using PC cassette.The deleted proteases are identical to B.subtilis WB800,except that no resistant marker left.We combined the optimized condition to strain PD8 and added an additional copy of mpd cassette at the aprE locus.The final MPH activity was enhanced to 300 U/ml,which was 11-fold than that of plasmid pP43NMK expression system reported.The yield of MPH was 0.6 g/L.In summary,this study constructed a highly efficient counter-selectable marker pheS~*.It has been used for marker-free genome manipulation in B subtilis and B.amyloliquefaciens.The method provided technical support for marker-free integration of expression cassettes to chromosome.By modification of the auto-inducible promoter Pylb and introduction of mRNA stabilizer,the recombinant protein was expressed efficiently,Based on the optimized promoter,a light-controllable expression system was constructed in B subtilis.It has reference significance for the transient regulation of genes in the field of metabolic engineering and the establishment of environmentally friendly induced expression systems.By optimization of the inducible promoter PxylA,a rigorous induction expression system was established,which is suitable for non-industrial production of recombinant proteins.At the level of translation and secretion,MPH production was finally increased to 300 U/ml by modifying the secondary structure of the mRNA in the translation initiation region,codon optimization,deletion of the dlt operon,overexpression of the molecular chaperone PrsA and increasing the copy number.The MPH activity was enhanced to 11-fold compared with that of plasmid pP43NMK expression system reported.The yield of MPH reached to 0.6 g/L.In addition,this study established a method for random mutation of expression cassette in B subtilis,which can be used to regulate the expression level of recombinant proteins and directed evolution of enzymes.