title: High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.
abstract: BACKGROUND
  We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, PamyQfrom Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536.
  
  
  RESULTS
  Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the PamyQ'promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter PHpaII-PamyQ'produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter PHpaII-PamyQ'also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter PHpaII-PamyQ'system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications.
  
  
  CONCLUSIONS
  The dual-promoter PHpaII-PamyQ'system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Kang
authors.initials: K
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Lingqia
authors.initials: L
authors.lastname: Su
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. jingwu@jiangnan.edu.cn.|School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China. jingwu@jiangnan.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000069977:Bacillus amyloliquefaciens / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase
keywords: Bacillus subtilis; General applicability; High-level expression; Promoter optimization; Scale-up production