Heterologous Expression Of Trehalose Synthase In B.subtilis W800n And Its Application In P_glv Regulation

Due to the biological characteristics of trehalose,it is widely used in food,medicine,fodder etc.Trehalose is also increasing demand.The current accepted trehalose preparation process is trehalose synthase producing trehalose by one-step conversion method,however,access and security are limited to a certain extent of trehalose,resulting in the preparation of high purity high cost of trehalose.In this paper,through the comparison of the BLAST genome of Pseudomonas putida KT2440 submitted in NCBI gene bank,trehalose synthase gene was obtained by PCR amplification.The fragment was constructed on the expression plasmid pHT01,and maltose synthase was used to induce the expression of trehalose synthase in Bacillus subtilis in order to achieve the purpose of producing trehalose efficiently.?1?The expression of trehalose synthase can be regulated by the use of Bacillus subtilis maltose as the inducible regulatory element,and by changing the carbon metabolic regulation sequence of cre binding protein CcpA of maltose promoter,which weakens the feedback inhibition of glucose metabolism.The Pglv gene and trehalose synthase gene?treS?fragments were optimized by PCR amplification and overlapping PCR technique,and through enzyme digestion and ligation,successfully constructed into shuttle vector pHT01 of Escherichia coli and Bacillus subtilis.The recombinant plasmid P_glv-pHT01-treS transformed into B.subtilis WB800n and the expression of trehalose synthase in Bacillus subtilis was realized under the condition of maltose induction,and the expression was as high as 2708.4 U/g.?2?In order to prevent the induction of maltose induced by the extracellular?-amylase in the late stage of induction,the effect of reducing the transcription of the maltose promoter was induced.This experiment through single crossover method of knockout of Bacillus subtilis WB800n?-amylase gene amyE and obtain recombinant B.subtilis WB800n??amyE?.Then the constructed plasmid Pglv-pHT01-treS was transformed into B.subtilis WB800n??amyE?and obtain the recombinant strain B.subtilis WB800n??amyE,Pglv-pHT01-tre S?.The results of HPLC analysis showed that the initial expression was increased to 4228.5 U/g,and the efficiency of maltose promoter was improved.?3?Under the condition of optimization of culture medium,the initial fermentation of recombinant bacteria B.subtilis WB800n??amyE,Pglv-pHT01-treS?was verified,and the lysozyme broken cell wall concentration and maltose induced concentration,induction time,induction temperature,induction time do optimization.The results showed that when the seed culture medium was amplified and the concentration of OD₆₀₀ reached 1.6,and then added to the final concentration of 40g/L maltose to be continuous induced for 20h in 37?.The cell was collected and the lysozyme was added with the final concentration of 4mg/L in order to broke cell wall for one hour.Under these conditions,the trehalose synthase activity was the highest,reaching 9213.5U/g.The application of maltose as inducer and the efficient expression of trehalose synthase in Bacillus subtilis.A new preparation method of trehalose was obtained,which provided the basis for industrial production.