| Bacillus amyloliquefaciens has been used as a "cell factory" to produce important metabolites and the expression of heterologous proteins,and it has a good application prospect in industrial enzyme fermentation production.In the process of heterologous protein production,the autolysis drawback of bacteria in the fermentation process,which may significantly decrease the total biomass,fermentation yields and limit the ability of B.amyloliquefaciens to continuously express heterologous proteins.In this study,B.amyloliquefaciens TCCC111018(BA TCCC111018)with the lack of spores was taken as a model chassis,due to the lack of spores,the strain had a physiological defect of large area autolysis at the late fermentation stage.This study took TCCC111018 as the research cell chassis and the autolysis-related mechanism of B.amyloliquefaciens was explored,and the chassis cells with delayed autolysis and efficient production of heterologous proteins were constructed,which laid the foundation for the efficient expression of heterologous proteins.The main research results are as follows:(1)The physiological characteristics of the strain BA TCCC111018 were analyzed,and provided gene targets for the BA TCCC111018 chassis modification through multiomics analysis.BA TCCC111018 did not produce spores during the culture of LB medium,MM medium and supernatant fermentation medium.Among them,the bacteria showed autolysis occurred in LB medium for 12 h,and autolysis in MM medium and supernatant fermentation medium for 24 h.In the fermentation medium,the α-amylase activity of BA TCCC111018 reached 678.54 U/m L,and the alkaline protease activity reached 13074.34U/m L.Heterologous expression of alkaline protease and exogenous addition of alkaline protease both accelerated the autolysis rate of BA TCCC111018.By comparative genomics analysis,the possible key genes that BA TCCC111018 do not produce spores were identified: sig K,cox A,yjc Z,bcl B,ger BA and cot D.Through comparative transcriptome analysis,the differential genes at different culture times were identified,and it was determined that in the apoptosis phase,cell components including cell wall related genes were significantly up-regulated,and quorum-sensing pathway genes related to cell lysis were significantly enriched.(2)The effects of different peptidoglycan hydrolases and transcriptional regulators on the autolysis of BA TCCC111018 were investigated.Based on the comparative transcriptome and KEGG functional annotations,the autolysis-related peptidoglycan hydrolases Lyt C,Lyt D,Lyt E,Lyt G and the transcriptional regulator Sig D were identified.Through single knockout validation,the number of viable cells of its mutants Δlyt D,Δlyt E and Δsig D increased by 8.8%,9.2% and 9.0%,respectively,and the production of acidstable Bacillus licheniformis alpha-amylase was increased by 106.58%,106.29% and123.37%,respectively.After culturing for 12 h,the results showed that the viable count of the strain BA ΔSDE reached 9.75 Log/cfu/m L,which was 20.8% higher than that of the control strain BA Δupp,and the acid-stable Bacillus licheniformis alpha-amylase production was increased by 146%.In the verification of batch fermentation and fed-batch fermentation in 2L quadruple fermentor,the viable count and acid-stable Bacillus licheniformis alpha-amylase yield of mutant strain BA ΔSDE were significantly higher than those of BA Δupp.During the fed-batch fermentation,the enzyme activity of mutant strain BA ΔSDE reached a maximum of 7851.53 U/ m L at 78 h,which was significantly higher than BA Δupp,by 1.48-fold.(3)The effect of D,L-endopeptidase on cell lysis was studied.The overexpression of D,L-endopeptidase lyt E,lyt F,cwl S and cwl O accelerated the rate of cell lysis,which further confirmed the overexpression of D,L-endopeptidase inhibits the industrial properties of B.amyloliquefaciens.By rationally knocking out D,L-endopeptidase,the optimal D,Lendopeptidase-deficient host BA Δlyt F-cwl S was obtained.Compared with the control strain BA ΔU,the number of viable cells of BA Δlyt F-cwl S was increased by 11.90%,the cell length and cell wall thickness increased by 2.31 μm and 7.03 nm,respectively.Compared with the control strain BA ΔU,the intracellular soluble peptidoglycan content of BA Δlyt F-cwl S was increased by 1.24 mg/g(DCW).Through transcriptome analysis,compared with the control strain BA ΔU,the mutant strain BA Δlyt F-cwl S peptidoglycan synthesis-related genes mur C,mur D,mur E,mra Y,mur F,mur G,dac F,pbp F and spo VD of transcription levels were significantly up-regulated,and proteomic analysis further confirmed that the expression of cell wall-associated synthetic proteins Dat,Gca D,Gl MM,Div IVA,Spo VID and Mur E in mutant strain BA Δlyt F-cwl S was significantly up-regulated.Using the heterologous proteins acid-stable Bacillus licheniformis alpha-amylase,PLD and e GFP as reporter genes,the effects of D,L-endopeptidase engineered strains on the yield of heterologous proteins were analyzed.Compared with the control strain BA ΔU,the acidstable Bacillus licheniformis alpha-amylase,PLD and e GPF yield of BA Δlyt F-cwl S were increased by 50.00%,55.89% and 113%,respectively.(4)The effect of prophages on cell lysis was studied,and a high-efficiency gene editing method of the CRISPR/Cas9 n dual-plasmid system was first established.Using upp as the targeting gene,the xylose-induced Cas9 n expression plasmid p HY300-PLK-Cas9 n and the upp-targeting sg RNA plasmid p WH-T2-sg RNA-Haupp were efficiently knocked out in BA TCCC111018,and the knockout efficiency was greater than 95%.By using CRISPR/Cas9 n,holin protein was integrated into the genome and co-expressed with the endolysin gene on the plasmid.The co-expression of Holin protein-endolysin gene in prophage P1,P2,P3 accelerated the autolysis of the strain,which proved that Holin protein-endolysin in three prophages was a functional module potentially leading to programmed cell death.Single or tandem knockout of five prophages P1,P2,P3,P4 and P5 using the CRISPR/Cas9 n dual plasmid editing system showed that the single deletion of the prophage was beneficial to reduce the autolysis rate of B.amyloliquefaciens,especially the number of viable cells with the deletion of prophage P1 increased by 53.55%.Based on prophage P1,by tandem deletion of P2,P3 and P4,the number of viable cells of the prophage deletion strain was increased by 117.68%,which effectively slowed down the cell lysis.Using acid-stable Bacillus licheniformis alpha-amylase as a reporter gene,the enzyme activity reached 128258.64 U/m L in a 5-L fed-batch fermenter,which reached the peak of yield 10 h earlier than the control strain BAΔU.(5)The effects of alkaline protease on cell lysis under the control of different fermentation temperatures were studied.In order to achieve high-density fermentation,on the basis of the prophage mutant strain BA ΔP12345,the tandem deletion of the peptidoglycan hydrolase Lyt D,Lyt E and the transcriptional regulator Sig D significantly reduced the cell lysis.The number of viable cells of BA ΔP12345-SDE increased by209.4% compared with the control strain,and the activity of heterologous protein acidstable Bacillus licheniformis alpha-amylase,alkaline protease increased by 43.13% and11.63% after 48 h.By staged variable temperature fermentation,the temperature is controlled to 30 °C by 12 h and 16 h fermentation,which effectively slowed down the cell lysis and increased the yield of alkaline protease,especially after 16 h of fermentation,the temperature is controlled to 30 °C,the alkaline protease yield reached 14755.5 U/m L,which was 36.9% higher than the control at the end of fermentation.Under the condition of adding alkaline protease with different amounts of enzyme,the autolysis rate was slowed down to different degrees by the staged variable temperature fermentation,especially in the environment of medium and low enzyme activity(12000 U/m L and 8000 U/m L),the autolysis rate was significantly decreased at the temperature controlled to 24 °C and 30 °C.It is proved that the staged temperature regulation is beneficial to slow down the autolysis effect of alkaline protease on B.amyloliquefaciens in the later stage of fermentation. |
