Abstract
Catharanthus roseus produces many pharmaceutically important terpenoid indole alkaloids (TIAs) such as vinblastine, vincristine, ajmalicine, and serpentine. Past metabolic engineering efforts have pointed to the tight regulation of the TIA pathway and to multiple rate-limiting reactions. Transcriptional regulator ORCA3 (octadecanoid responsive Catharanthus AP2-domain protein), activated by jasmonic acid, plays a central role in regulating the TIA pathway. In this study, overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter in C. roseus hairy roots resulted in no change in the total amount of TIAs measured. RT-qPCR results showed that ORCA3 overexpression triggered the upregulation of transcripts of most of the known TIA pathway genes. One notable exception was the decrease in strictosidine glucosidase (SGD) transcripts. These results corresponded to previously published results. In this study, ORCA3 and SGD were both engineered in hairy roots under the control of a glucocorticoid-inducible promoter. Co-overexpression of ORCA3 and SGD resulted in a significant (p < 0.05) increase in serpentine by 44 %, ajmalicine by 32 %, catharanthine by 38 %, tabersonine by 40 %, lochnericine by 60 % and hörhammericine by 56 % . The total alkaloid pool was increased significantly by 47 %. Thus, combining overexpression of a positive regulator and a pathway gene which is not controlled by this regulator provided a way to enhance alkaloid production.
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Abbreviations
- 7-DLGT:
-
7-deoxyloganetic acid-O-glucosyl transferase
- 7-DLH:
-
7-deoxyloganic acid hydroxylase
- ADH:
-
alcohol dehydrogenase
- ASα:
-
anthranilate synthase alpha subunit
- BPF:
-
box P-binding factor
- CPR:
-
cytochrome P450 reductase
- CRSDH4H:
-
desacetoxyvindoline 4-hydroxylase
- D4H:
-
desacetoxyvindoline 4-hydroxylase
- DAT:
-
deacetylvindoline acetyltransferase
- DMAPP:
-
dimethylallyl pyrophosphate
- DXR:
-
1-deoxy-d-xylulose-5-phosphate reductoisomerase
- DXS:
-
1-deoxy-d-xylulose 5-phosphate synthase
- G10H:
-
geraniol 10-hydroxylase
- GBF:
-
G-box binding factor
- GPPS:
-
geranyl diphosphate synthase
- IO:
-
iridoid oxidase
- IPP:
-
isopentenyl pyrophosphate
- IS:
-
iridoid synthase
- JA:
-
jasmonic acid
- LAMT:
-
loganic acid methyltransferase
- MAT:
-
minovincinine 19-hydroxy-O-acetyltransferase
- MEP:
-
2-C-Methyl-d-erythritol 4-phosphate
- MYC2:
-
MYC2 transcription factor
- ORCAs:
-
octadecanoid responsive Catharanthus AP2-domain proteins
- Prx1:
-
peroxidase
- SGD:
-
strictosidine beta-glucosidase
- SLS:
-
secologanin synthase
- STR:
-
strictosidine synthase
- T16H:
-
tabersonine 16-hydroxylase
- T19H:
-
tabersonine 19-hydroxylase
- TDC:
-
tryptophan decarboxylase
- TIA:
-
terpenoid indole alkaloid
- WRKY1:
-
WRKY transcription factor 1
- ZCT:
-
transcription factor IIIA-type zinc finger family
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Acknowledgments
The authors express our gratitude to Dr. Ka-Yiu San at Rice University for providing A. rhizogenes 15834 strain and plasmids pTA7002/ORCA3 and pUCGALA. The authors would like to thank Dr. Nam-Hai Chua at the Rockefeller University for providing the inducible promoter plasmid (pTA7002) and Dr. Jacqueline V Shanks at Iowa State University for providing the lochnericine and hörhammericine standards. This work was supported by Colorado State University.
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Sun, J., Peebles, C.A.M. Engineering overexpression of ORCA3 and strictosidine glucosidase in Catharanthus roseus hairy roots increases alkaloid production. Protoplasma 253, 1255–1264 (2016). https://doi.org/10.1007/s00709-015-0881-7
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DOI: https://doi.org/10.1007/s00709-015-0881-7