期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 -, 期 -, 页码 -出版社
SPRINGER
DOI: 10.1007/s00253-023-12784-x
关键词
Paclitaxel (Taxol (R)); Taxane-10 beta-hydroxylase; De novo biosynthesis; Protein engineering; Taxadiene-5 alpha-yl-acetoxy-10 beta-ol
In this study, the taxane-10 beta-hydroxylase (T10 beta H) from Taxus cuspidata was expressed and engineered in Escherichia coli. Through protein engineering, the expression and activity of T10 beta H were improved. The de novo biosynthesis of T10OH was successfully achieved in E. coli.
Paclitaxel (Taxol (R)) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10 beta-hydroxylase (T10 beta H) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5 alpha-yl-acetate (T5OAc) to taxadiene-5 alpha-yl-acetoxy-10 beta-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10 beta H, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. Key points The T10 beta H from T. cuspidata was expressed and engineered in E. coli unprecedentedly. The expression and activity of T10 beta H were improved through protein engineering. De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L.
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