Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis / 生物工程学报

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.

Biological treatment of high-pH and high-concentration black liquor of cotton pulp by an immediate aerobic-anaerobic-aerobic process.

In this study, an immediate aerobic-anaerobic-aerobic (O/A/O) biological process was established for the treatment of black liquor of cotton pulp and was tested by both laboratory-scale batch experiment and pilot-scale continuous experiment. The effects of the hydraulic retention time (HRT) were studied, as were the alkaliphilic bacteria number, the culturing temperature and the concentration of black liquor on COD (chemical oxygen demand) removal. The total COD (COD(tot)) removal rate of the novel O/A/O process, for a black liquor with influent COD(tot) over 8,000 mg/L and pH above 12.8, was 68.7+/-4% which is similar with that of the traditional acidic-anaerobic-aerobic process (64.9+/-3%). The first aerobic stage based on alkaliphilic bacteria was the crucial part of the process, which was responsible for decreasing the influent pH from above 12 to an acceptable level for the following treatment unit. The average generation time of the alkaliphilic bacteria in the black liquor was about 36 minutes at 40 degrees C in a batch aerobic activated sludge system. The efficiency of the first aerobic stage was affected greatly by the temperature. The COD(tot) removal at 55 degrees C was much lower in comparison with the COD(tot) removal at 45 degrees C or 50 degrees C. Both the laboratory-scale batch experiments and the pilot-scale continuous experiment showed that the COD(tot) removal rate could reach about 65% for original black liquor with a pH of about 13.0 and a COD of 18,000-22,000 mg/L by the immediate O/A/O process. The first aerobic stage gave an average COD(tot) removal of 45.5% at 35 degrees C (HRT = 72 h) at a volume loading rate of 3.4 kg COD m(-3) d(-1).