BackgroundSecretory 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.ResultsTwo α-amylases (AmyL and AmyS) both under the control of the PHpaII promoter and equipped with their native signal peptides SPamyl and SPamyS 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.ConclusionsOur 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 materialThe online version of this article (doi:10.1186/s12934-015-0282-9) contains supplementary material, which is available to authorized users.

α-Amylase was used as a heterologous model protein to investigate the effects of promoters, signal peptides and over-expression of an extra-cytoplasmic molecular chaperone, PrsA lipoprotein, on enhancing the secretion of α-amylase in Bacillus subtilis. Four promoters and six signal peptides were compared, successively, and the highest yield of α-amylase was achieved under the promotion mediated by PAprE, a strong constitutive promoter, and secretion by SPnprE, a signal peptide from B. subtilis. Moreover, under conditions of overexpressed PrsA lipoprotein, the secretion production and activity of α-amylase increased to 2.5-fold. The performance of the recombinant B. subtilis 1A751PL31 was evaluated with a fed-batch fermentation in a 7.5 l fermentor. Optimization of regulatory elements and over-expression of PrsA lipoprotein had a significant effect on enhancing the production of α-amylase in B. subtilis.

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