Beneficial regulation of vitamin D3-rich extract from the processing by-products of Penaeus sinensis on preosteoblastic MC3T3-E1 cells and improvement of bone health in VD-deficient mice.

Vitamin D (VD) plays an important role in preventing osteoporosis. However, knowledge of the osteogenic effect of VD3 from shrimp processing by-products is limited. In this study, a VD3-rich extract from Penaeus sinensis processing by-products was prepared by saponification and liquid-liquid extraction combined with solid phase extraction for purification. The activity of purified VD3-rich extract (PPs-VD3) on MC3T3-E1, a preosteoblastic cell line, was determined. Furthermore, the improvement effect of PPs-VD3 on bone health of VD-deficient mice was investigated. PPs-VD3 stimulated the proliferation and differentiation of MC3T3-E1 cells. Compared to the same concentration of the VD3 standard, mineralization of MC3T3-E1 cells increased after 14 d or 21 d of PPs-VD3 treatment. Western blotting showed that PPs-VD3 significantly upregulated the protein levels of bone morphogenetic protein 2 and runt-related transcription factor 2 compared to the VD3 standard. Furthermore, PPs-VD3 treatment activated the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway in MC3T3-E1 cells, especially increased OPG expression was detected at day 3 to day 14 compared to the VD3 standard treatment. More than ten medium and long chain fatty acids were detected in PPs-VD3, of which n-3 polyunsaturated fatty acids (PUFA) constituted 38.83 ± 8.61%, and the n-3/n-6 PUFA ratio in PPs-VD3 was 2.84 ± 0.23. The femoral trabeculae number and thickness of VD-deficient mice increased after 3 weeks of PPs-VD3 treatment. The changes of parameters associated with bone resorption including parathyroid hormone, bone mineral density and tartrate resistant acid phosphatase revealed the contribution of PPs-VD3 treatment in improving bone remodeling in VD-deficient mice. Our results suggest that PPs-VD3 could have potential prospects in alleviating osteoporosis or promotion of bone health.

[An efficient marker-free genome editing method for Aspergillus niger].

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.