Construction And Optimization Of Recombinant Bacillus Subtilis Expressing Trehalose Synthase Efficiently

Trehalose is widely used in food,medicine and cosmetics because of its unique biological characteristics and the function of protecting the characteristics of biological macromolecules.At present,the single enzyme method is widely accepted as the preparation technology of trehalose.Single enzymatic method directly converts maltose into trehalose under the action of trehalose synthase?TreS?.This method is simple and easy to operate,but it still has some shortcomings.First,TreS is mainly induced by recombinant Escherichia coli.Second,intracellular expression requires high pressure homogeneous fragmentation,high energy consumption,large equipment investment,and easy to retain endotoxin in products.?1?Density-inducible promoters P_srfA,constitutivepromoters P₄₃3 and four phase-specific promoters P_abrB,P_spoVG,P_LytRytR and P_mmgAmgA were selected as expression elements.The four phase-specific promoters were expressed in logarithmic phase,pre-stable phase,stable phase and death phase of cell growth respectively.A new promoter P_{abrB-spoVG-LytR-mmgA}brB-spoVG-LytR-mmgA was constructed by tandem construction.The expression vector was pHT01 plasmid.The recombinant Bacillus subtilis expressing trehalose synthase was induced and fermented in shaking flask and fermenter respectively.The recombinant B.subtilis WB800n/pHT01-P₄₃-treS with high activity of trehalose synthase was obtained.The intracellular activity of the recombinant B.subtilis reached3937 U/g.?2?Using promoters P_srfA,P₄₃3 and P_{abrB-spoVG-LytR-mmgA}brB-spoVG-LytR-mmgA as expression elements,the two most typical signal peptides PhoD and YwbN in Tat secretion pathway signal peptide of Bacillus subtilis were selected.The recombinant B.subtilis WB800n/pHT01-P_srfA-PhoD/YwbN-treS?B.subtilis WB800n/pHT01-P_{abrB-spoVG-LytR-mmgA}-PhoD/YwbN-treS and B.subtilis WB800n/pHT01-P₄₃-PhoD/YwbN-treS were constructed by using pHT01 plasmid as expression vector.Through fermentation in 5 L fermentor,the recombinant strain B.subtilis WB800n/pHT01-P₄₃-PhoD-treS with relatively high total enzyme activity and highest extracellular enzyme activity was obtained.The extracellular enzyme activity reached 7954.58 U/L,the total enzyme activity reached26341.39 U/L,and the extracellular enzyme activity accounted for more than 35%of the total enzyme activity.?3?The fermentation medium and fermentation conditions of recombinantB.subtilisWB800n/pHT01-P₄₃-PhoD-treS were optimized.The effects of carbon source,carbon source concentration,pH,temperature and feeding mode on TreS extracellular secretion were analyzed.The high-density fermentation of recombinant bacteria was carried out on the optimized medium.The growth and metabolism rate of cells was controlled by adding sucrose and controlling temperature in the later stage.The proportion of enzyme activity and extracellular enzyme activity in total enzyme activity was increased.The fermentation results showed that the highest extracellular enzyme activity was 15868 U/L,accounting for more than 42%of total enzyme activity.