One-pot biosynthesis of 7ß-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum.

BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.

Butein inhibits the migration and invasion of SK-HEP-1 human hepatocarcinoma cells through suppressing the ERK, JNK, p38, and uPA signaling multiple pathways.

Liver cancer is one of the most commonly diagnosed cancers and the leading cause of death in human populations. Butein, a tetrahydroxychalcone, has been shown to induce apoptosis in many human cancer cells, but the effects of butein on the migration and invasion of human liver cancer cells are not reported. Herein, we found that butein is effective in the suppression of migration and invasion in SK-HEP-1 human hepatocarcinoma cells by using the Matrigel cell migration assay and invasion system. The gelatin zymography assay indicated that butein inhibited the activity of matrix metalloproteinases 2 (MMP-2) and MMP-9. Western blotting analysis indicated that butein decreased the levels of MMP-2, -7, and -9, uPA, Ras, Rho A, ROCK1, ERK1/2, JNK1/2, p-p38, and p-c-Jun in SK-HEP-1 cells. Furthermore, butein inhibited the NF-κB binding activity in SK-HEP-1 cells by electrophoretic mobility shift assay. We also found that butein decreased the ERK, JNK, and p38 in SK-HEP-1 cells by in vitro kinase assay. In conclusion, this is the first study to demonstrate that butein might be a novel anticancer agent for the treatment of hepatocarcinoma through inhibiting migration and invasion.