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Selfinduced Secretion Expression of Trehalose Synthase in Bacillus subtilis WB800n
Xihui WANG1，2#， Hongling LIU1，2，3#， Songsen SUI4， Shaojie YANG1，2， Tengfei WANG1，2*
1. State Key Laboratory of Biobased Material and Green Papermaking （LBMP）， Qilu University of Technology， Jinan 250353， China; 2. Shandong Key Laboratory of Microbial Engineering， Qilu University of Technology， Jinan 250353， China; 3. Key Laboratory of Industrial Fermentation Microbiology （Tianjin University of Science & Technology）， Ministry of Education and Tianjin Key Lab of Industrial Microbiology， Tianjin University of Science & Technology， Tianjin 300457， China; 4. Zhucheng Dongxiao Biotechnology Co.， Ltd.， Weifang 261000， China
Abstract Bacillus subtilis is a nonpathogenic Grampositive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose， a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products， can be prepared by trehalose synthase （TreS）. The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study， the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800N using pHT01 as expression vector. For avoiding the use of inducer， promoter PsrfA was used to replace the promoter Pgrac in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800N under different phosphate concentrations， carbon sources， carbon source concentrations， nitrogen sources and pH. The results showed that the PsrfA promoter had a good regulation effect under pH 8.0 condition， and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide， TreS was effectively secreted， and the extracellular enzyme activity reached 2 100 U/L， accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions， the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor， and the proportion reached 48%. The pHT01PsrfAPhoDtreS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.
Key words Trehalose synthase; Bacillus subtilis W800N; PsrfA promoter; Selfinduction; PhoD signal peptide
Trehalose is stable with special biological function[1]. At present， the use of organisms to produce trehalose synthase （TreS） to catalyze the production of trehalose by maltose is a relatively simple method[2]. Due to the low substrate cost， simple reaction and high conversion rate， this route is beneficial to the production of trehalose[3]. TreS is a bacterial intracellular enzyme that can be produced by a variety of bacterial strains[4]. Therefore， many treS genes have been cloned from different strains and expressed in E. coli. However， due to the presence of endotoxin， the use of enzyme production in E. coli is severely limited[5]. Therefore， it is necessary to select a food grade expression host. Bacillus subtilis is a recognized safe （GRAS） Grampositive soil bacterium approved by the US Food and Drug Administration （FDA） for longterm production of multiple enzymes and overexpression of large quantities of pharmaceutical and industrial recombinant proteins[6]. Compared with other protein expression hosts， B. subtilis is characterized by high cell density growth and well established genetic manipulation methods， and is suitable for large scale industrial production[7]. In the past years， many enzymes have been expressed in B. subtilis by inducible expression system used inducer specific promoters. Recently， the autoinducible expression systems have been constructed ， and entered a new stage[8]. In B. subtilis， the well known sigma factors such as ┮A， ┮B， ┮H， ┮D and so on that associate with RNA polymerase core enzyme for promoter recognition have been characterized[9]. Compared with the chemical and specific environment condition inducible systems， the phase dependent autoinducible expression systems regulated by sigma factors attracted much more attention because of its practical applications in flexible and dynamic regulation of pathway genes.
Among B. subtilis， the most studied and clearest secreted protein translocation pathways are the Sec pathway and the Tat pathway[10]. Although the Sec pathway is the primary strategy for commercial enzyme production， unfortunately， heterologous proteins secreted by this pathway are often unsuccessful， primarily due to insufficient translocation efficiency， misfolding， and protein degradation[11]. Based on the Tat pathway， which secretes fully folded proteins， the development of the Tat pathway to secrete recombinant proteins is a significant and potentially valuable recombinant protein cell plant challenge.
Materials and Methods
Strains， plasmids and materials
The bacterial strains and plasmids that were used in this study are listed in Table 1. E. coli DH5 was used for plasmid construction. B. subtilis W800N was used as the expression host. Secretion of trehalose synthase using the selfinducible promoter PsrfA and the Tat pathway signal peptide PhoD. The media used in this study were LuriaBertani medium （LB）， and Terrific Broth （TB） medium. B. subtilis growth medium was supplemented with chloramphenicol （25 g/ml） and E. coli growth medium with ampicillin （100 g/ml）. PZEROBlunt null background flat end fast connection kit， 2≠Phanta Max Master Mix and 2≠Taq PCR Master Mix were purchased from Vazyme Biotechnology Co.， Ltd. T4 DNA ligase and restriction enzymes were purchased from Thermo company. SanPrep column DNA gel recovery kit and DNA purification kit were purchased from Shanghai Sangon Biotech Co.， Ltd.
Recombinant DNA techniques
Plasmid construction was performed in E. coli and DNA extraction was performed by following standard procedure as previously described. Recombinant plasmids were transformed into B. subtilis W800N as previously described. Enzymes were obtained from TOYOBO （OSaka， Japan）， TaKaRa （Dalian， China）， or NEW ENGLAND BioLabs （Beijing， China） and were used according to the manufacturers protocols. The primers used in this study are listed in Table 1. PCR was performed using KOD DNA polymerase （Osaka， Japan）. All of the recombinant plasmids constructed in this work were confirmed by DNA sequencing （Shanghai Sangon Biotech Co.， Ltd.，）. Construction of selfinduced secretory expression plasmid
The DNA fragments containing promoter PsrfA and signal peptide PhoD gene from B. subtilis 168 were prepared with bacteria genome DNA extracting kit. The DNA fragments containing treS gene from Pseudomonas putida KT2440 were prepared with bacteria genome DNA extracting kit. Sequence analysis showed that the treS gene does not contain signal peptide. The promoter PsrfA and signal peptide PhoD were amplified from the B. subtilis 168 chromosomal DNA using primers SrfAF/srfAPhoDR， PhoDsrfAF/PhoDtreSR， add a cutting site BamHI at the 5′terminal of primer SrfAF. The treS gene were amplified from the P. putida KT2440 chromosomal DNA using primers treSPhoDF/TreSR.， add a cutting site AatII at the 3′terminal of primer TreSR. Three gene sequences were ligated using overlapping PCR to obtain the PsrfAPhoDtreS gene sequence. The PCR products was digested by BamHI and AatII and ligated into pHT01 by using T4 DNA ligase to generate the recombinant vector pHT01PsrfAPhoDtreS. The recombinant plasmid was transformed into the host strain B. subtilis W800N by electroporation to obtain the recombinant strain B. subtilis W800N（pHT01PsrfAPhoDtreS）.
TB medium （12 g/L peptone， 24 g/L yeast extract， 4 g/L glycerol， 2.4 g/L KH2PO4 and 16.5 g/L K2HPO4） was used for flask cultures. The recombinant B. subtilis W800N （pHT01PsrfAPhoDtreS）， stored at -80， was revived in 250 ml Erlenmeyer flasks containing 50 ml of LB medium with 25 g of chloramphenicol and grew at 37 and 200 rpm on rotary shakers for 12 h. Primary seed culture （1 ml） was used to inoculate two parts of 100 ml of secondary TB medium in a rotary shaker， respectively. To further improve the TreS productivity and achieve the maximal bioactivity， shake flask cultivation was performed in different culture conditions， the effects of phosphate concentration， carbon source， sucrose concentration and pH on TreS activity were investigated. Each condition was measured in triplicate， and each experiment was investigated three times. The results are shown as the mean ÷ SD from three independent experiments.
Expanding fermentation with 5 L fermenter
A single colony of the recombinant B. subtilis W800N （pHT01PsrfAPhoDtreS） strain selected from LB agar plate was inoculated into 50 ml of LB liquid medium and cultivated overnight for 12 h， next， 1.0 ml of preculture was inoculated into 500 ml shake flasks containing 100 ml of LB liquid medium. After 8 h of cultivation with shaking， the entire culture were transferred into the fermenter. Dissolved oxygen （DO） was maintained above 30% saturation by controlling both the inlet air and the agitation rate between 200 and 800 rpm. Foaming was controlled by the addition of an antifoaming agent. Agitation was regulated between 50 and 500 rpm to keep the dissolved oxygen level in an appropriate level. Additionally， pH was maintained at 8.0 by automatic pH control with the addition of 5% HCl. The temperature was controlled by a heating sleeve and an integrated cooling system to 37→ throughout the fermentation process. Enzymatic assay and SDSPAGE analysis
Cells were separated from the growth medium by centrifugation and were resuspended in 10 ml lysis buffer （20 mM phosphate buffer， pH 8.0）. After， cells were disrupted by sonication. Then the cellfree extract was recovered by centrifugation. Both the culture supernatant and cellfree extract were used for the enzymatic assay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis （SDSPAGE） analysis.
The TreS activity was determined by using maltose as substrate. Samples （5 ml） were added into 5 l of maltose solution （600 g/L in 20 mM phosphate buffer， pH 8.0）. After being incubated for 4 h at 25， the mixture was boiled for 10 min to terminate the reaction.
One unit （U） of trehalsoe synthase was defined as the amount of enzyme required to produce 1 mol of trehalose per hour under the specified conditions.
Results and Discussion
Expression of recombinant TreS
The recombinant expression vector pHT01PsrfAPhoDTreS was transformed into B. subtilis W800N strain. The activity of trehalose synthase in the intracellular and supernatant of the recombinant bacteria in TB medium was verified. As shown in Fig. 1， enzyme activity was detected in both intracellular and supernatant， while the activity in the supernatant was only about 35%. It is necessary to further increase the secretion of trehalose synthase by optimizing culture conditions. In addition， SDSPAGE analysis was carried out to confirm the TreS expression. As shown in Fig. 2， distinct bands with a molecular mass of about 76 kDa were observed， which is in good agreement with the predicted value.
Enzyme activity comparison in different phosphate concentration
Phosphate concentration has a large effect on protease secretion. Therefore， changing the phosphate concentration in the medium verifies the effect on enzyme activity. The concentration of phosphate in the medium was adjusted to 100% （TB6）， 75% （TB5）， 50% （TB4）， 37.5% （TB3）， 25% （TB2） and 0% （TB1） of the initial phosphate concentration in the original TB medium， respectively， and analysis of the dry weight of the cells and the ratio of unit enzyme activity and extracellular enzyme activity to total enzyme activity was performed， as shown in Fig. 3.
The results showed that when the phosphate concentration in the medium was 37.5% of the original TB medium， the enzyme activity was highest in the supernatant and the highest proportion of total enzyme activity. Enzyme activity comparison in different carbon source
The TB medium optimized with phosphate concentration was used as the basic medium， the carbon source components in the TB medium were changed， and the recombinant bacteria were subjected to fermentation enzyme activity analysis in 500 ml of TB medium containing 100 ml of different carbon sources. The flask was fermented for 24 h at 37 and 200 rpm， and the results are shown in the Fig. 4 and Fig. 5.
When sucrose was used as the carbon source， the extracellular enzyme activity， total enzyme activity and extracellular enzyme activity ratio were the highest， the extracellular enzyme activity reached 1 700 U/L， the total enzyme activity reached 4 500 U/L， and the extracellular enzyme activity accounted for the total 38% of enzyme activity.
When the sucrose concentration was 12 g/L， the extracellular enzyme activity， total enzyme activity and extracellular enzyme activity ratio were expressed. A good advantage is that the extracellular enzyme activity reaches 2 200 U/L， the total enzyme activity reaches 5 200 U/L， and the extracellular enzyme activity accounts for 43% of the total enzyme activity.
Enzyme activity comparison in different pH
In addition to the above conditions， the effect of pH on enzyme activity was also investigated. Therefore， five pH levels （6.5， 7.0， 7.5， 8.0 and 8.5） were adjusted by phosphate buffer and used to detect trehalose synthase activity. The results are shown in Fig. 6.
The total enzyme activity was relatively high at pH 7.0-8.0. At pH 8.0， the total enzyme activity was the highest， and the dry weight of the cells also had obvious advantages， but the extracellular enzyme activity was highest at pH 7.0. Moreover， the extracellular enzyme activity accounts for a higher proportion of total enzyme activity， so at pH 7.0-8.0， the enzyme activity has better expression characteristics and secretion characteristics.
Fermentation in 5 L fermentor
To obtain the optimal growth of B. subtilis W800N/pHT01PsrfAPhoDtreS resulting in high yields of active TreS. The recombinant bacteria were fermented in a 5 L fermenter containing 2.7 L of optimized TB medium. The total volume of the fermenter was 3 L， the stirring speed was controlled at 500 r/min， the ventilation was 0.3 vvm， the temperature was 37， and the pH was 7.0 before the first 8 h of fermentation， followed by maintaining pH 8.0 by dropwise addition of HCl. The results are shown in Fig. 7. Extracellular enzyme activity accounts for 48% of total enzyme activity.
Conclusion
By using a selfinducible promoter and a Tat pathway signal peptide， and using B. subtilis WB800n as an expression strain， a selfinducing secretory expression recombinant strain was constructed， and trehalose synthase was successfully secreted extracellularly. The proportion of extracellular enzyme activity was increased by optimizing the fermentation conditions. The expression system has great potential for the production of foodgrade trehalose， which simplifies the conversion process and saves costs compared to intracellular expression of trehalose synthase， and provides experience for largescale production of trehalose.
References
[1]OHTAKE S， WANG YJ. Trehalose： current use and future applications[J]. Journal of Pharmaceutical Sciences， 2011， 100（6）： 2020-2053.
[2]PAN YT， CARROLL JD， ASANO N， et al. Trehalose synthase converts glycogen to trehalose[J]. Febs journal， 2008， 275（13）： 3408-3420
[3]ZUBER P， HEALY JM， LOSICK R. Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis[J]. Journal of Bacteriology， 1987， 169（2）： 461.
[4]PANAHI R， VASHEGHANI FE， SHOJAOSADATI SA， et al. Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation[J]. Annals of Microbiology， 2014， 64（2）： 879-882.
[5]GUAN C， CUI W， CHENG J， et al. Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J]. Microbial Cell Factories， 2016， 15（1）： 66.
[6]YANG S， DU G， CHEN J， et al. Characterization and application of endogenous phasedependent promoters in Bacillus subtilis[J]. Applied Microbiology and Biotechnology， 2017， 101（10）： 1-11.
[7]KENNEY TJ， YORK K， YOUNGMAN P， et al. Genetic evidence that RNA polymerase associated with ┮ A factor uses a sporulationspecific promoter in Bacillus subtilis[J]. Proceedings of the National Academy of Sciences of the United States of America， 1989， 86（23）： 9109.
[8]HAMOEN LW， KAUSCHE D， MARAHIEL MA， et al. The Bacillus subtilis transition state regulator AbrB binds to the 35 promoter region of comK[J]. Fems Microbiology Letters， 2003， 218（2）： 299-304.
[9]LEE SJ， PAN JG， PARK SH， et al. Development of a stationary phasespecific autoinducible expression system in Bacillus subtilis[J]. Journal of Biotechnology， 2010， 149（1）： 16-20.
[10]ROSHANI PATEL， SARAH M SMITH， COLIN ROBINSON. Protein transport by the bacterial Tat pathway[J]. Biochimica et Biophysica Acta，1843 （2014） 1620-1628.
[11]GUAN CR， CUI WJ， CHENG JT， et al. Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis[J]. New Biotechnology， 2016， 33（3）： 372-379.
Xihui WANG1，2#， Hongling LIU1，2，3#， Songsen SUI4， Shaojie YANG1，2， Tengfei WANG1，2*
1. State Key Laboratory of Biobased Material and Green Papermaking （LBMP）， Qilu University of Technology， Jinan 250353， China; 2. Shandong Key Laboratory of Microbial Engineering， Qilu University of Technology， Jinan 250353， China; 3. Key Laboratory of Industrial Fermentation Microbiology （Tianjin University of Science & Technology）， Ministry of Education and Tianjin Key Lab of Industrial Microbiology， Tianjin University of Science & Technology， Tianjin 300457， China; 4. Zhucheng Dongxiao Biotechnology Co.， Ltd.， Weifang 261000， China
Abstract Bacillus subtilis is a nonpathogenic Grampositive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose， a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products， can be prepared by trehalose synthase （TreS）. The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study， the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800N using pHT01 as expression vector. For avoiding the use of inducer， promoter PsrfA was used to replace the promoter Pgrac in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800N under different phosphate concentrations， carbon sources， carbon source concentrations， nitrogen sources and pH. The results showed that the PsrfA promoter had a good regulation effect under pH 8.0 condition， and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide， TreS was effectively secreted， and the extracellular enzyme activity reached 2 100 U/L， accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions， the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor， and the proportion reached 48%. The pHT01PsrfAPhoDtreS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.
Key words Trehalose synthase; Bacillus subtilis W800N; PsrfA promoter; Selfinduction; PhoD signal peptide
Trehalose is stable with special biological function[1]. At present， the use of organisms to produce trehalose synthase （TreS） to catalyze the production of trehalose by maltose is a relatively simple method[2]. Due to the low substrate cost， simple reaction and high conversion rate， this route is beneficial to the production of trehalose[3]. TreS is a bacterial intracellular enzyme that can be produced by a variety of bacterial strains[4]. Therefore， many treS genes have been cloned from different strains and expressed in E. coli. However， due to the presence of endotoxin， the use of enzyme production in E. coli is severely limited[5]. Therefore， it is necessary to select a food grade expression host. Bacillus subtilis is a recognized safe （GRAS） Grampositive soil bacterium approved by the US Food and Drug Administration （FDA） for longterm production of multiple enzymes and overexpression of large quantities of pharmaceutical and industrial recombinant proteins[6]. Compared with other protein expression hosts， B. subtilis is characterized by high cell density growth and well established genetic manipulation methods， and is suitable for large scale industrial production[7]. In the past years， many enzymes have been expressed in B. subtilis by inducible expression system used inducer specific promoters. Recently， the autoinducible expression systems have been constructed ， and entered a new stage[8]. In B. subtilis， the well known sigma factors such as ┮A， ┮B， ┮H， ┮D and so on that associate with RNA polymerase core enzyme for promoter recognition have been characterized[9]. Compared with the chemical and specific environment condition inducible systems， the phase dependent autoinducible expression systems regulated by sigma factors attracted much more attention because of its practical applications in flexible and dynamic regulation of pathway genes.
Among B. subtilis， the most studied and clearest secreted protein translocation pathways are the Sec pathway and the Tat pathway[10]. Although the Sec pathway is the primary strategy for commercial enzyme production， unfortunately， heterologous proteins secreted by this pathway are often unsuccessful， primarily due to insufficient translocation efficiency， misfolding， and protein degradation[11]. Based on the Tat pathway， which secretes fully folded proteins， the development of the Tat pathway to secrete recombinant proteins is a significant and potentially valuable recombinant protein cell plant challenge.
Materials and Methods
Strains， plasmids and materials
The bacterial strains and plasmids that were used in this study are listed in Table 1. E. coli DH5 was used for plasmid construction. B. subtilis W800N was used as the expression host. Secretion of trehalose synthase using the selfinducible promoter PsrfA and the Tat pathway signal peptide PhoD. The media used in this study were LuriaBertani medium （LB）， and Terrific Broth （TB） medium. B. subtilis growth medium was supplemented with chloramphenicol （25 g/ml） and E. coli growth medium with ampicillin （100 g/ml）. PZEROBlunt null background flat end fast connection kit， 2≠Phanta Max Master Mix and 2≠Taq PCR Master Mix were purchased from Vazyme Biotechnology Co.， Ltd. T4 DNA ligase and restriction enzymes were purchased from Thermo company. SanPrep column DNA gel recovery kit and DNA purification kit were purchased from Shanghai Sangon Biotech Co.， Ltd.
Recombinant DNA techniques
Plasmid construction was performed in E. coli and DNA extraction was performed by following standard procedure as previously described. Recombinant plasmids were transformed into B. subtilis W800N as previously described. Enzymes were obtained from TOYOBO （OSaka， Japan）， TaKaRa （Dalian， China）， or NEW ENGLAND BioLabs （Beijing， China） and were used according to the manufacturers protocols. The primers used in this study are listed in Table 1. PCR was performed using KOD DNA polymerase （Osaka， Japan）. All of the recombinant plasmids constructed in this work were confirmed by DNA sequencing （Shanghai Sangon Biotech Co.， Ltd.，）. Construction of selfinduced secretory expression plasmid
The DNA fragments containing promoter PsrfA and signal peptide PhoD gene from B. subtilis 168 were prepared with bacteria genome DNA extracting kit. The DNA fragments containing treS gene from Pseudomonas putida KT2440 were prepared with bacteria genome DNA extracting kit. Sequence analysis showed that the treS gene does not contain signal peptide. The promoter PsrfA and signal peptide PhoD were amplified from the B. subtilis 168 chromosomal DNA using primers SrfAF/srfAPhoDR， PhoDsrfAF/PhoDtreSR， add a cutting site BamHI at the 5′terminal of primer SrfAF. The treS gene were amplified from the P. putida KT2440 chromosomal DNA using primers treSPhoDF/TreSR.， add a cutting site AatII at the 3′terminal of primer TreSR. Three gene sequences were ligated using overlapping PCR to obtain the PsrfAPhoDtreS gene sequence. The PCR products was digested by BamHI and AatII and ligated into pHT01 by using T4 DNA ligase to generate the recombinant vector pHT01PsrfAPhoDtreS. The recombinant plasmid was transformed into the host strain B. subtilis W800N by electroporation to obtain the recombinant strain B. subtilis W800N（pHT01PsrfAPhoDtreS）.
TB medium （12 g/L peptone， 24 g/L yeast extract， 4 g/L glycerol， 2.4 g/L KH2PO4 and 16.5 g/L K2HPO4） was used for flask cultures. The recombinant B. subtilis W800N （pHT01PsrfAPhoDtreS）， stored at -80， was revived in 250 ml Erlenmeyer flasks containing 50 ml of LB medium with 25 g of chloramphenicol and grew at 37 and 200 rpm on rotary shakers for 12 h. Primary seed culture （1 ml） was used to inoculate two parts of 100 ml of secondary TB medium in a rotary shaker， respectively. To further improve the TreS productivity and achieve the maximal bioactivity， shake flask cultivation was performed in different culture conditions， the effects of phosphate concentration， carbon source， sucrose concentration and pH on TreS activity were investigated. Each condition was measured in triplicate， and each experiment was investigated three times. The results are shown as the mean ÷ SD from three independent experiments.
Expanding fermentation with 5 L fermenter
A single colony of the recombinant B. subtilis W800N （pHT01PsrfAPhoDtreS） strain selected from LB agar plate was inoculated into 50 ml of LB liquid medium and cultivated overnight for 12 h， next， 1.0 ml of preculture was inoculated into 500 ml shake flasks containing 100 ml of LB liquid medium. After 8 h of cultivation with shaking， the entire culture were transferred into the fermenter. Dissolved oxygen （DO） was maintained above 30% saturation by controlling both the inlet air and the agitation rate between 200 and 800 rpm. Foaming was controlled by the addition of an antifoaming agent. Agitation was regulated between 50 and 500 rpm to keep the dissolved oxygen level in an appropriate level. Additionally， pH was maintained at 8.0 by automatic pH control with the addition of 5% HCl. The temperature was controlled by a heating sleeve and an integrated cooling system to 37→ throughout the fermentation process. Enzymatic assay and SDSPAGE analysis
Cells were separated from the growth medium by centrifugation and were resuspended in 10 ml lysis buffer （20 mM phosphate buffer， pH 8.0）. After， cells were disrupted by sonication. Then the cellfree extract was recovered by centrifugation. Both the culture supernatant and cellfree extract were used for the enzymatic assay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis （SDSPAGE） analysis.
The TreS activity was determined by using maltose as substrate. Samples （5 ml） were added into 5 l of maltose solution （600 g/L in 20 mM phosphate buffer， pH 8.0）. After being incubated for 4 h at 25， the mixture was boiled for 10 min to terminate the reaction.
One unit （U） of trehalsoe synthase was defined as the amount of enzyme required to produce 1 mol of trehalose per hour under the specified conditions.
Results and Discussion
Expression of recombinant TreS
The recombinant expression vector pHT01PsrfAPhoDTreS was transformed into B. subtilis W800N strain. The activity of trehalose synthase in the intracellular and supernatant of the recombinant bacteria in TB medium was verified. As shown in Fig. 1， enzyme activity was detected in both intracellular and supernatant， while the activity in the supernatant was only about 35%. It is necessary to further increase the secretion of trehalose synthase by optimizing culture conditions. In addition， SDSPAGE analysis was carried out to confirm the TreS expression. As shown in Fig. 2， distinct bands with a molecular mass of about 76 kDa were observed， which is in good agreement with the predicted value.
Enzyme activity comparison in different phosphate concentration
Phosphate concentration has a large effect on protease secretion. Therefore， changing the phosphate concentration in the medium verifies the effect on enzyme activity. The concentration of phosphate in the medium was adjusted to 100% （TB6）， 75% （TB5）， 50% （TB4）， 37.5% （TB3）， 25% （TB2） and 0% （TB1） of the initial phosphate concentration in the original TB medium， respectively， and analysis of the dry weight of the cells and the ratio of unit enzyme activity and extracellular enzyme activity to total enzyme activity was performed， as shown in Fig. 3.
The results showed that when the phosphate concentration in the medium was 37.5% of the original TB medium， the enzyme activity was highest in the supernatant and the highest proportion of total enzyme activity. Enzyme activity comparison in different carbon source
The TB medium optimized with phosphate concentration was used as the basic medium， the carbon source components in the TB medium were changed， and the recombinant bacteria were subjected to fermentation enzyme activity analysis in 500 ml of TB medium containing 100 ml of different carbon sources. The flask was fermented for 24 h at 37 and 200 rpm， and the results are shown in the Fig. 4 and Fig. 5.
When sucrose was used as the carbon source， the extracellular enzyme activity， total enzyme activity and extracellular enzyme activity ratio were the highest， the extracellular enzyme activity reached 1 700 U/L， the total enzyme activity reached 4 500 U/L， and the extracellular enzyme activity accounted for the total 38% of enzyme activity.
When the sucrose concentration was 12 g/L， the extracellular enzyme activity， total enzyme activity and extracellular enzyme activity ratio were expressed. A good advantage is that the extracellular enzyme activity reaches 2 200 U/L， the total enzyme activity reaches 5 200 U/L， and the extracellular enzyme activity accounts for 43% of the total enzyme activity.
Enzyme activity comparison in different pH
In addition to the above conditions， the effect of pH on enzyme activity was also investigated. Therefore， five pH levels （6.5， 7.0， 7.5， 8.0 and 8.5） were adjusted by phosphate buffer and used to detect trehalose synthase activity. The results are shown in Fig. 6.
The total enzyme activity was relatively high at pH 7.0-8.0. At pH 8.0， the total enzyme activity was the highest， and the dry weight of the cells also had obvious advantages， but the extracellular enzyme activity was highest at pH 7.0. Moreover， the extracellular enzyme activity accounts for a higher proportion of total enzyme activity， so at pH 7.0-8.0， the enzyme activity has better expression characteristics and secretion characteristics.
Fermentation in 5 L fermentor
To obtain the optimal growth of B. subtilis W800N/pHT01PsrfAPhoDtreS resulting in high yields of active TreS. The recombinant bacteria were fermented in a 5 L fermenter containing 2.7 L of optimized TB medium. The total volume of the fermenter was 3 L， the stirring speed was controlled at 500 r/min， the ventilation was 0.3 vvm， the temperature was 37， and the pH was 7.0 before the first 8 h of fermentation， followed by maintaining pH 8.0 by dropwise addition of HCl. The results are shown in Fig. 7. Extracellular enzyme activity accounts for 48% of total enzyme activity.
Conclusion
By using a selfinducible promoter and a Tat pathway signal peptide， and using B. subtilis WB800n as an expression strain， a selfinducing secretory expression recombinant strain was constructed， and trehalose synthase was successfully secreted extracellularly. The proportion of extracellular enzyme activity was increased by optimizing the fermentation conditions. The expression system has great potential for the production of foodgrade trehalose， which simplifies the conversion process and saves costs compared to intracellular expression of trehalose synthase， and provides experience for largescale production of trehalose.
References
[1]OHTAKE S， WANG YJ. Trehalose： current use and future applications[J]. Journal of Pharmaceutical Sciences， 2011， 100（6）： 2020-2053.
[2]PAN YT， CARROLL JD， ASANO N， et al. Trehalose synthase converts glycogen to trehalose[J]. Febs journal， 2008， 275（13）： 3408-3420
[3]ZUBER P， HEALY JM， LOSICK R. Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis[J]. Journal of Bacteriology， 1987， 169（2）： 461.
[4]PANAHI R， VASHEGHANI FE， SHOJAOSADATI SA， et al. Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation[J]. Annals of Microbiology， 2014， 64（2）： 879-882.
[5]GUAN C， CUI W， CHENG J， et al. Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J]. Microbial Cell Factories， 2016， 15（1）： 66.
[6]YANG S， DU G， CHEN J， et al. Characterization and application of endogenous phasedependent promoters in Bacillus subtilis[J]. Applied Microbiology and Biotechnology， 2017， 101（10）： 1-11.
[7]KENNEY TJ， YORK K， YOUNGMAN P， et al. Genetic evidence that RNA polymerase associated with ┮ A factor uses a sporulationspecific promoter in Bacillus subtilis[J]. Proceedings of the National Academy of Sciences of the United States of America， 1989， 86（23）： 9109.
[8]HAMOEN LW， KAUSCHE D， MARAHIEL MA， et al. The Bacillus subtilis transition state regulator AbrB binds to the 35 promoter region of comK[J]. Fems Microbiology Letters， 2003， 218（2）： 299-304.
[9]LEE SJ， PAN JG， PARK SH， et al. Development of a stationary phasespecific autoinducible expression system in Bacillus subtilis[J]. Journal of Biotechnology， 2010， 149（1）： 16-20.
[10]ROSHANI PATEL， SARAH M SMITH， COLIN ROBINSON. Protein transport by the bacterial Tat pathway[J]. Biochimica et Biophysica Acta，1843 （2014） 1620-1628.
[11]GUAN CR， CUI WJ， CHENG JT， et al. Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis[J]. New Biotechnology， 2016， 33（3）： 372-379.