Development Of Bacillus Subtilis Efficient Expression System And Development Research Of Feed Enzyme Preparation

Bacillus subtilis is a spore-forming and well-known Bacillus as a model for Gram-positive bacteria, which has widely applied in the production of industrial, food, medical and feed industrial recombinant proteins. In this study, we achieved the secretory expression of feed enzymes preparation and explored the secretion mechanism of AIO6BS protein. To construct high efficiency expression system, we deleted the intracellular proteinase quality control system and developed a suite of engineered strains for high efficiency production of recombinant proteins. The present study had three experiments.The experiment 1, For the secretion of heterologous proteins commonly depend on the guidance of B. subtilis signal peptide itself, and studies have showed that there is suitability between the signal peptide and heterologous protein. So screening of signal peptides for optimizing the export of target protein in B. subtilis might be necessary. The results revealed that the recombinant strain containing YlqB signal peptide showed the highest secretion level of CBP21.The experiment 2, In this study, we achieved secretory expression of the non-classical export pathway dependent protein AIO6 and AIO6BS and explored its secretion mechanism. The results demonstrated that AIO6BS without the signal peptide was successfully expressed and secreted into the cell culture. Expression analysis of AIO6BS in the single or double mutant of the lytC and lytD genes for cell autolysis in B. subtilis 1A751 and cell autolysis-resistant engineered strain LM2531 derived from the wild type 168 indicated that the release of the heterologous protein AIO6BS was not simply mediated by cell lysis. Expression level of AIO6BS did not change in the mutants of B. subtilis that harbored mutations in the secA, tat AC, or ecsA genes compared with that in the parent wild type strain. These results suggested the AIO6BS protein was likely secreted via a non-classical secretion pathway. Saturation mutation results for positions 267 showed the amino acids Cys267 is important for synthesis and secretion of the quenching AIO6BS. In addition, we evaluated the ability of AIOBS to act as a signal to export recombinant proteins into the culture medium. Five proteins, including homologous proteins, heterologous proteins and molecular chaperones from the bacterium B. subtilis, were fused to AIOBS linked by a flexible 21-bp linker to keep a distance between two single proteins. The results indicated all of the fusions failed to be exported into the medium with the direction of the non-classically secreted protein AIOBS.The experiment 3, we deleted the intracellular proteinase quality control system and developed a suite of engineered strains for high efficiency production of recombinant proteins. One of the bottlenecks that affect secretion of recombinant protein is known as the quality control system of protease. Extracellular protease, the cell membrane and cell wall protease Bacillus subtilis had been detailed research, but study on intracellular protein quality control system is less, especially the intracellular proteases effect on the secretion of recombinant protein expression are less. In this study based on the principle of homologous recombination method, we constructed the intracellular proteinase knockout carrier, obtained the correct knockout strains BSAAlbF, BS?AmpS, BS?AprX, BS?ClpE, BS?ClpP, BS?IspA, BS?MlpA, BS?TepA, BS?YlbL, BS?YmfH, BS?YrrN, BS?YwpA, BS?YwpE via identification of molecular biology method.In this sdudy We used unconventional secretory protein AIOBS and Sec-dependent secretory protein CBP21 as report genes, respectively analyzes the intracellular proteinase knockout strains of two different types of secretory expression of recombinant proteins. Compared with wild-type strains, CBP21 in BS?YlbL and BS? MlpA knock-out strains can't secretory express, and the amount of the secretory expression in other mutant strains have different degrees of increase. AIO6BS in BS ?AlbF strains can't secretory express, the amount of the secretory expression in other mutant strains have various degree of increase.