Effects Of Bacillus Amyloliquefaciens Genome Reduction On The Expression Of Alkaline Protease

Microbial genome reduction is an important method to construct chassis cells,which can effectively remove redundant intracellular regulatory and metabolic networks,reduce genome complexity,reduce the interference of non-essential metabolic pathways,and improve the utilization efficiency of substrates and energy by strains.In this study,Bacillus amyloliquefaciens TCCC19030 was taken as the research object,non-essential genes were mined by bioinformatics,and a series of genome simplified strains were constructed by continuously simplifying the genome.The effects of different degrees of genome reduction operations on the growth of strains were analyzed through related experiments,and the value and potential of genome reduction strains as chassis cells for producing alkaline protease were explored.Firstly,the genome and transcriptome data of the original strain B.amyloliquefaciens TCCC111018 were combined with NCBI,Uni Prot,anti SMASH,Islandviewer 4,Subti Wiki,BSub Cyc and other databases for bioinformatics analysis,and five types of non-essential gene types were summarized,including secondary metabolism Product,extracellular sticky substance,genome island,spore formation-related genes,flagella formation-related genes.The length of these gene sequences reached 873 kb,and the genome accounted for more than 21%.Subsequently,based on the strain B.amyloliquefaciens TCCC19030 with six extracellular protease genes deleted,homologous recombination technology was used to delete the above-mentioned nonessential gene clusters,including riboflavin,bacitracin,bacteriocin,bacteriocin,surfactin,6 gene clusters for the synthesis of secondary metabolites such as polyketides,3 gene clusters for the synthesis of viscous substances such as exopolysaccharide,polyglutamic acid,and fructan,1 gene cluster for flagella synthesis and 1 gene cluster for phage synthesis,and finally obtained a series of condensed strains,among which the most condensed strain was TCCC112270,with a total condensed 179.14 kb,and the genome accounted for 4.5%.Physiological phenotype results of the simplified genome strains showed that most of the strains lacking single-function gene clusters were not significantly different from the starting strains,but the strains lacking gene clusters related to bacteriocin,bacteriocin and flagella synthesis showed an accelerated decline rate in the later stage of growth.However,among the bacteria with different degrees of simplification,the strain with more simplification of the genome showed the trend of faster decline in the later stage of growth.The expression ability of alkaline protease of strains with different genome reduction degrees was investigated,and it was found that the strains with reduced fructan-related gene clusters had a greater impact on the expression of alkaline proteases,that is,the reduction of extracellular polysaccharides,polyglutamine After the introduction of the alkaline protease expression cassette in the strain TCCC112255 of the acid and fructan synthesis-related gene clusters,the enzyme activity reached 22523.73 U/m L in the shaker flask,and the 7 L fermenter was used to scale up the culture.The highest was 96783.17U/m L,which was about 20% higher than that of the control strain,while the expression of alkaline protease of the remaining reduced strains had no significant change.Based on the strain TCCC112255,the repressor protein encoding gene hrcA and the phosphatidylserine synthase pssA gene of the intracellular molecular chaperone were further deleted,aiming to further enhance the secretion of alkaline protease by the strain.The analysis of growth characteristics showed that the knockout strains had prolonged lag phase,decreased biomass,and accelerated autolysis rate of the strains in the decline phase.The results of fermentation verification showed that the relative enzyme activity of alkaline protease of B.amyloliquefaciens TCCC19046 carrying the hrcA and pssA double-knockout strains carrying the p LY-2 expression vector was increased by 8.71 % compared with that of B.amyloliquefaciens TCCC112212.The detection of the transcription level proved that the deletion of hrcA gene leads to the overexpression of Dna K and Gro EL;The detection of the transcription level proved that the double deletion of hrcA and pssA could increase the transcription level of the alkaline protease apr E,indicating that it is feasible to construct a cell factory with high-yielding alkaline protease by overexpressing intracellular molecular chaperones.