| Maltooligosyl trehalose synthase（MTSase）and maltooligosyl trehalose trehalohydrolase（MTHase）are the key enzymes in the double enzymatic synthesis of trehalose.In the existing reports,the enzyme system mainly uses the E.coli expression system as the host,but the E.coli will secrete endotoxin during the fermentation process,which affects the application of its products in the fields of food and the like.Bacillus licheniformis is a recognized generally regarded as safe（GRAS）strain,and its extracellular protein secretion can reach more than 20g·L-1.It has been used in the industrial production of protease and amylase for a long time.However,compared with model strains such as E.coli,the development of the Bacillus licheniformis expression system needs to be improved.Most of the expression elements in the Bacillus licheniformis expression system are Bacillus universal expression elements derived from Bacillus subtilis,which limits the system.expression level.In this study,MTSase and MTHase were functionally expressed in the Bacillus licheniformis expression system using promoters from different sources,and the promoter with the highest expression activity was screened by comparing the enzyme activities.On this basis,a series of tandem promoters and synthetic promoters were constructed by promoter engineering to further improve the expression levels of MTSase and MTHase.Finally,through fermentation optimization,the culture conditions suitable for the expression system were established,and the high-efficiency expression of MTSase and MTHase in Bacillus licheniformis was achieved.The main research contents and results of this study are as follows:（1）In this study,Pmtl A,PP2,PHpa II,and PSpov G were selected to construct a recombinant expression system in Bacillus licheniformis to express MTSase and MTHase..The expression systems of different natural promoters were compared,and the results showed that compared with other promoters,the expression of MTSase and MTHase was the highest under the mediation of PP2,and the maximum enzyme activities were 370.0 U·m L-1 and 83.8 U·m L-1,respectively.（2）On the basis of the screening results of different natural promoters,Pmtl A,PSpov G and PP2 were concatenated to the upstream of PP2 to obtain concatenated promoters Pmtl A-P2,PSpov G-P2 and PP2-P2.At the same time,5 upstream element（UPE）derived from PP2 were connected in series to the upstream of PP2 by gene synthesis to obtain a synthetic promoter P5-P2,which can obtain synthetic promoters P1-P2,P3-P2,P5-P2.By comparing the effects of the above promoters on the expression of MTSase and MTHase in Bacillus licheniformis,the enzyme activity results showed that compared with PP2,the maximum enzyme activities of MTSase and MTHase mediated by P1-P2 increased by 37%and 10%,respectively,which was better than other promoters.son.（3）In order to further improve the expression of trehalose synthase in Bacillus licheniformis,the effects of carbon source,nitrogen source,trace elements,fermentation temperature,initial p H,carbon source concentration and nitrogen source concentration on the growth and enzyme production of recombinant bacteria were investigated.The results showed that the optimal culture conditions of recombinant bacteria were:cottonseed protein 30 g·L-1,sucrose 75 g·L-1,K2HPO4·3H2O 9.12 g·L-1,KH2PO4 1.36 g·L-1,Fe Cl3 0.5 g·L-1,（NH4）2H2O410 g·L-1,fermentation temperature 42°C,initial p H 7.5.Under this condition,the enzyme activities of recombinant bacteria BLSup1 and BLHup1 reached 6586.7 U·m L-1 and 1246.8U·m L-1,respectively,and the expression levels of heterologous proteins reached 0.419 g·L-1and 0.057 g·L-1,respectively.（4）The recombinant bacteria were amplified and cultured under fermenter conditions,and the carbon source feeding process was optimized.The results showed that under the condition of maltodextrin feeding.,the enzyme activities of recombinant bacteria BLSup1 and BLHup1 reached 8006.4 U·m L-1 and 84.4 U·m L-1,respectively. |
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