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Background  Secretory expression of valuable proteins by  B. subtilis  and its related species has attracted intensive work over the past three decades. Although very high yields can be achieved with homologous proteins, production of heterologous proteins by  B. subtilis  is unfortunately not the straight forward. The Sec pathway is the major route for protein secretion in  B. subtilis . Therefore, the aim of this work was to identify the bottlenecks of the Sec pathway and improve the secretion of heterologous proteins by molecular genetic techniques.    Results  Two α-amylases (AmyL and AmyS) both under the control of the P  HpaII   promoter and equipped with their native signal peptides SP  amyl   and SP  amyS   were successfully secreted with significantly different expression levels. To improve the secretion efficiency, 23 main genes or gene operons involved in or closely related to the Sec pathway were overexpressed singly by increasing an additional copy on the chromosome, and the overexpression of  prsA  enhanced the production of α-amylases (AmyL and AmyS) by 3.2- and 5.5-fold, respectively. With the induction by xylose of different concentrations,  prsA  overexpression level was optimized and the secretion efficiency of α-amylase was further improved. Moreover, combinatorial overexpression of  prsA  and nine screened genes or gene operons, respectively, was performed, and the overexpression of  prsA  combined with partial  dnaK  operon improved the α-amylase activity of AmyL and AmyS by 160 and 173%, respectively, compared with the overexpression of  prsA  singly. Finally, the performance of the recombinant  B. subtilis  1A237 was evaluated with the fed-batch fermentation in 7.5 L fermentor, and the level of secreted AmyL and AmyS reached 1,352 and 2,300 U/mL with the productivity of 16.1 U/mL h and 27.4 U/mL h, respectively.    Conclusions  Our systematic gene overexpression approach was designed to investigate the bottleneck of Sec pathway in  B. subtilis . The deficiency of PrsA lipoprotein and chaperones of DnaK series was main rate-limiting factors for heterologous proteins secretion. Systematic and deep insight into how components of Sec pathway interact with each other may be the key to improving the yield of heterologous proteins thoroughly.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-015-0282-9) contains supplementary material, which is available to authorized users.

microbial cell factories

Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression