Abstract
Paclitaxel is a highly functionalized diterpenoid that is broadly used for the treatment of several cancer types. This valuable specialized metabolite naturally exists in the inner bark of Taxus species in low amounts. The limited-scale production of paclitaxel in Taxus cell cultures has necessitated the use of several elicitors. Recently, methyl-β-cyclodextrin (CD) and coronatine (COR) have been considered to be highly effective elicitors in producing plant specialized metabolites. Given the limited production of paclitaxel due to the rate limiting enzymes' function, bottleneck removal is conducive to the production of more significant amounts of paclitaxel. In the present study, the full length of DBTNBT coding sequence (CDS), as one of the paclitaxel pathway bottlenecks, was integrated downstream of the CaMV 35S promoter (pCAMBIA1304-DBTNBT) and transiently expressed in Taxus baccata leaves via Agrobacterium tumefaciens and vacuum infiltration method. Paclitaxel production and the expression level of several involved genes were evaluated through different treatments. The transient overexpression of the DBTNBT gene, associated with dual elicitation, resulted in 7.4-fold more paclitaxel production compared with the no-inoculation/no-elicitation control. These ratios were 2.1 and 1.8 in the CD + COR and pCAM treatments, respectively. Among T13αH, T14βH, DBAT, BAPT, DBTNBT, and ABC genes, the most increased expression level belonged to the DBTNBT gene, followed by ABC and BAPT genes. It seems as though in the near future, bottleneck removal could be used on a large scale in Taxus metabolic engineering, resulting in the relative removal of some other bottlenecks and an increase in the final paclitaxel production.
Key message
DBTNBT overexpression associated with CD and COR elicitation led to the much more paclitaxel production and the prevention of feedback repression on the upstream bottleneck genes such as DBAT and BAPT.
AbstractSection Graphical abstract
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Data Availability
Data generated or analyzed during this study was included in this paper.
Code availability
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Abbreviations
- MeJA:
-
Methyl jasmonate
- COR:
-
Coronatine
- CD:
-
Methyl-β-cyclodextrin
- GAPDH :
-
Glyceraldehyde-3-phosphate dehydrogenase
- TXS :
-
Taxadiene synthase
- T13αH :
-
Taxane 13α-hydroxylase
- T14βH :
-
Taxane 14β-hydroxylase
- TBT :
-
Taxane-2α-O-benzoyl transferase
- DBAT :
-
10-deacetylbaccatin III-10-O-acetyltransferase
- BAPT :
-
Baccatin III-3-amino 13-phenylpropanoyl- CoA transferase
- DBTNBT :
-
Debenzoyltaxol N-benzoyl transferase
- PAM :
-
Phenylalanine amino mutase
- ABC :
-
ATP-binding cassette
- ORF:
-
Open Reading Frame
- GFP :
-
Green fluorescent protein
- GUS :
-
β- glucuronidase
- OD:
-
Optical Density
- SE:
-
Standard Error
- CRD:
-
Completely Randomized Design
- LSD:
-
Least Significant Difference
- CDS:
-
Coding Sequence
- CaMV 35S Promoter:
-
Cauliflower Mosaic Virus Promoter
- NOS Terminator:
-
Nopaline Synthase Terminator
- cDNA:
-
Complementary DNA
- HPLC:
-
High- Performance Liquid Chromatography
- RP:
-
Reverse Phase
- F:
-
Forward Primer
- R:
-
Reverse Primer
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All authors contributed to the study’s conception and design. Kimia Kashani performed material preparation, data collection, and analysis. She wrote the first draft of the manuscript and the other authors (Mohammad Sadegh Sabet, Mokhtar Jalali Javaran, and Ahmad Moieni) commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Kashani, K., Sabet, M.S., Jalali Javaran, M. et al. Bottleneck removal of paclitaxel biosynthetic pathway by overexpression of DBTNBT gene under methyl-β-cyclodextrin and coronatine elicitation in Taxus baccata L.. Plant Cell Tiss Organ Cult 149, 485–495 (2022). https://doi.org/10.1007/s11240-022-02279-4
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DOI: https://doi.org/10.1007/s11240-022-02279-4