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Engineering overexpression of ORCA3 and strictosidine glucosidase in Catharanthus roseus hairy roots increases alkaloid production

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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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Correspondence to Christie A. M. Peebles.

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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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