Construction Of A T7 Expression System In Bacillus Subtilis

Bacillus Subtilis is a safe gram-positive bacterium with rapid growth ability and strong protein-secretion ability and is widely used in the industrial fermentation production of proteins such as?-amylase,protease,lipase and so on.However,because its original expression system of generally has some weakpoints such as low genetic transformation efficiency and low expression level of heterologous proteins,B.subtilis cannot meet the increasing demand of scientific research and actual production.The T7 expression system is one of the most effective systems for the expression of heterologous proteins in prokaryotic microorganisms,which pocesses advantages of simple gene manipulation,strict gene-expression regulation,and high protein-expression efficiency.This project intends to combine the advantages of B.subtilis and T7 expression system to develop a high-efficiency expression system of heterologous protein.This system consists of two parts:One is the T7 host strain suitable for high-density fermentation.the strain SCK8 was obtained after upp gene was knocked out from B.subtilis SCK6 and a traceless genetic operating system was established;Then,in order to reduce the sporulation rate and foaming rate during the fermentation process,the spo IIAC gene and the sfr AC gene were one-by-one knocked out to obtain the strain SCK9 and SCK10 respectively.finally,the T7 RNA polymerase encoding gene was integrated into the genome amy E site of SCK10 to obtain strain SCK22 as the host of the T7 expression system;Another is the vector plasmid that T7 promoter regulates and expresses the target gene.Using the green fluorescent protein(GFP)gene gfp as the reporter gene,the episomal expression vectors p HT01-T7-GFP and p HT7-GFP,and the genome-integrated expression vectors p SS-T7-GFP and p SS-T7lac-GFP were designed.The plasmids were transformed into SCK22 or integrated into the genome of SCK22 to obtain recombinant strains SCK22/p HT01-T7-GFP,SCK22/p HT7-GFP,SCK24 and SCK25.Under the same culture conditions,the recombinant strain SCK22/p HT7-GFP had the highest intracellular GFP expression.Therefore,in this paper,SCK22 was used as the host strain and p HT7 was used as the target gene expression vector to construct the T7expression system.In order to improve the expression level of heterologous protein,the culture time and IPTG concentration of the inducer of the recombinant strain SCK22/p HT7-GFP were optimized.Results showed that under the fixed culture conditions(inoculation OD₆₀₀ 0.05,culture temperature 37°C,shake flask speed 250 rpm,and IPTG concentration 1.00 mmol/L),the density of cells reached the maximum after 7.0 h culture,and the intracellular expression of GFP reached the highest of 0.15 g/L,accounting for 22.24%of total intracellular protein content.The intracellular expression of GFP in high-density fermentation in a 5.0 L fermenter was 2.94 g/L,accounting for 15.70%of total intracellular protein content.In order to verify the applicability of the T7 system,six heterologous proteins(i.e.,isoamylase,?-glucan phosphorylase,phosphoglucomutase,inositol 1-phosphate synthase,inositol monophosphatase and 4-?-glucan transferase)of starch inositol production catalyzed by multiple enzymes in vitro were expressed in this B.subtilis T7 system.SDS-PAGE electrophoresis photo showed that expression levels of these six heterologous proteins accounted for 10-40%of the total intracellular protein content,which could be comparable to the efficiency of the T7 system in E.coli,and no inclusion bodies were found basically.The recombinant strain SCK22/p HT7-IMP could produce 4.78 g/L inositol monophosphatase in high-density fermentation in a 5 L fermenter,accounting for 27.20%of the total intracellular protein.The obtained T7 expression system in strain SCK22,which could realize high-density fermentation,was able to stably express these six enzymes that consisted of in vitro multi-enzymes involed in producing inositol from starch.