The Heterologous Expression Of Trehalose Synthase, Regulatory Element Adaptation And Fermentation Conditions Optimization

At present,we have put a high value on food safety,so that trehalose synthase?tres?which is the production strain of trehalose,must meet standards of food-grade microorganisms.Compared with E.coli,B.subtilis can serve as an efficient and safe host for recombinant protein secretion.However,the production of most heterologous proteins which have been cloned and expressed in B.subtilis,is quite low.Theoretically,every step of protein from production to transport,may be the reason of low production.In this study,promoter and signal peptide optimization and optimized culture conditions,were applied to improve the production of foreign proteins.In this work,we got the signal peptide screening vectors of Pseudomonas stutzeri Qlu3,and to screen a matched signal peptide of B.subtilis for trehalose,we used a semi-rational approach.Six signal peptides SP_AprE,SP_amyE,SP_wapA,SP_SacB,SP_vpr and SP_{yvg O} were screened from nineteen candidate signal peptides by comparing their compositions.The results revealed that the recombinant strain containing SP_NprE signal peptide showed the highest secretion level of PsTS,the activity of PsTS is 14.5U/mL.Promoter is an vital impact factor on gene transcription.Therefore,we chose widely used strong promoter P_aprE to promote tres expression,and the result indicated that the activity of PsTS is 24.8U/mL.Further,the culture conditions was optimized in shaking flasks for extracellular fermentation of the recombinant PsTS with B.subtilis.The results showed that the initial medium was terrific broth?TB?in pH 7.0,after incubation at 37?for 48 h,the activity of PsTS was 25.8 U/mL.Further optimized the carbon and nitrogen source of the TB medium,replacements the glycerinum in TB medium with 20 g/L solubility corn starch and 20 g/L yeast extract as the nitrogen source led the extracellular activity to 29.8 U/mL.And 1mmol/L Mg²⁺could furtherly enhance the activity to 30.7 U/mL.