RESUMO
10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor. Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell. Then, we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatin III. We found that baccatin III could be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+ under alkaline condition. Thus, we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatin III. The fermentation conditions were optimized and the baccatin III titers reached 2, 3 and 4.6 g/L, respectively, in a 3-L bioreactor culture when 2, 3 and 6 g/L of 10-DAB were supplied. Our study provides an environment-friendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis. The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.
RESUMO
OBJECTIVE: To evaluate the effects of laser in situ keratomileusis (LASIK) on the patients' visual field. METHODS: 92 eyes of 46 patients with mild, moderate, and high degrees of myopia were undergone LASIK. Perimetry was performed before and 1 day, 1 month, 3 months, 6 months after LASIK surgery by Humphrey II-750 visual field analysis. The central 30 - 2 fast threshold program was used to examine the change in central 30 degree. Mean defect (MD) and pattern standard deviation (PSD) were record. All operations were performed by an experienced surgeon. Statistical analyses were performed by SPSS using the randomized complete block design ANOVA (two factors analysis of variance). Differences among variables were evaluated by q-test (Student-Newman-Kueuls method). RESULTS: 93.5% patients obtained estimated best correct visual acuity or better at six months after LASIK. No complications were found in the subjects. No difference was found in fundus examinations before and after surgery. Intraocular pressure was normal at all preoperative and postoperative examinations. MD was significant differences among preoperative and postoperative values (P < 0.05, for all comparisons) and was correlated with visual acuity post-LASIK significantly (P < 0.05). While PSD did not show significant differences before and after LASIK (P > 0.05) and was not correlated with visual acuity post-LASIK. The indexes of visual field were not correlated with the degree of myopia and the other surgical parameters (P > 0.05). CONCLUSION: In current research, no significant visual field change was demonstrated pre-LASIK and post-LASIK after 6 months follow-up. The study suggests that LASIK does not affect the retinal physiological functions.
