Computational redesign of taxane-10ß-hydroxylase for de novo biosynthesis of a key paclitaxel intermediate.
Appl Microbiol Biotechnol
; 107(23): 7105-7117, 2023 Dec.
Article
em En
| MEDLINE
| ID: mdl-37736790
ABSTRACT
Paclitaxel (Taxol®) 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-10ß-hydroxylase (T10ßH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10ß-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10ßH, 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 T10ßH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. ⢠The expression and activity of T10ßH 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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MEDLINE
Assunto principal:
Paclitaxel
/
Taxus
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article