The Heterologous Expression Of ?-amylase,Regulatory Element Optimization And Identification Of Secretion Bottleneck

Bacillus subtilis is an important producer of industrial enzymes due to its huge capacity to secrete proteins directly into the growth medium. A great number of foreign protein genes from different organisms have been cloned and expressed in B. subtilis, but the low yield of the secreted proteins has not been solved. To enable B. subtilis to be an efficient secretion host for heterologous proteins, here several strategies, i.e. gene codon optimization, directed evolution, promoter and signal peptide optimization and identification of Sec secretion pathway, were applied to improve the production of heterologous proteins.Two kinds of ?-amylases, AmyL and AmyS encoding by amyl and amys, are from Bacillus licheniformis and Bacillus stearothermophilus, respectively. The gene amys was synthesized with codon optimization. We transformed the expression plasmids pMA5 L and pMA5 S into B. subtilis, and the ?-amylases AmyL and AmyS were successfully expressed. Furthermore, directed evolution was performed on AmyL, and we reduced the optimal reaction pH and improved the thermostability to some extent by Leu134?Arg mutation.Promoter is an vital impact factor on gene transcription. Therefore, we chose widely used P_HpaII?P43?P_aprE and P_amyl to promote amylM2 expression, and the result indicated that P_aprE was the strongest promoter. To screen a matched signal peptide for AmyLM2, we used a semi-rational approach. Six signal peptides were screened from fifteen candidate signal peptides by comparing their compositions, and SP_nprE was proved to be the optimal for AmyLM2. Furthermore, we tested the effect of P_aprE and SP_nprE on AmyS expression.In B. subtilis, Sec pathway was the major secretion pathway. By single overexpression of 23 genes or gene operons of Sec pathway, we found that prsA overexpression enhanced AmyLM2 and AmyS secretion by 3.2-fold and 5.5 fold, respectively. By the optimization of prsA overexpression level and stage, the production of AmyLM2 and AmyS was further improved. We also performed the combinational overexpresion of prsA and 9 screened genes or gene operons. Simultaneous overexpression of prsA and dnaK operon enhanced the secretion of AmyLM2 and AmyS by 160% and 173%, respectively. Based on the results above, we can see that the deficiency of PrsA and Dnak series is one bottleneck of heterologous protein secretion in B. subtilis.Comparing the characterization of parental and engineered strains, it was found that the secretion of AmyLM2 and AmyS eventually was improved by 9.1-fold and 11.7-fold, respectively. At last, the engineered strains 1A237 L and 1A237 S were evaluated by fed-batch fermentation in 7.5 L fermentator, the activity of AmyLM2 and AmyS reached 1352 U/m L and 2300 U/mL at 84 h, respectively.