Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis.

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.

[The significance of IFN-gamma measurement in middle ear effusion of secretory otitis media].

OBJECTIVE: To explore the clinical significance of IFN-gamma in secretory otitis media (SOM) by measuring and analyzing and the level of INF-gamma in the effusion of middle ear. METHOD: A commercially available enzyme-linked immunosorbent assay (ELISA) incorporating a biotinylated monoclonal antibody probe was utilized for the quantitative measurement of IFN-gamma in the middle ear effusion and serum samples of the patients and that of the normal persons. RESULT: The level of IFN-gamma in the middle ear effusion was much higher than that in the serum samples. The difference of the level of IFN-gamma in the serum samples is not significant between the test group and the control group. The level of IFN-gamma in chronic period was much higher that in acute period. The level of IFN-gamma in the middle ear effusion has no significant difference between the group undergoing tympanocentesis for the first time and the group undergoing tympanocentesis for the second time (P > 0.05). The levels of IFN-gamma in the middle ear effusion in the group undergoing tympanocentesis for three or more times arose obviously (P < 0.01). CONCLUSION: The IFN-gamma in the middle ear effusion may be produced by local tympanum,but not exuded simply from blood. The high expression of IFN-gamma in the middle ear effusion may be a reference of SOM tending to chronic course.