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Sclareol production in the moss Physcomitrella patens and observations on growth and terpenoid biosynthesis

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Abstract

The moss Physcomitrella patens was engineered to produce the diterpenoid sclareol, an important precursor for the synthesis of ambergris substitutes for the perfume industry. The best total yield of sclareol was 2.84 mg/g dry weight (2.28 mg/l culture) obtained after 18 days of cultivation in liquid media (extracted from both media and cell pellet). The two active sclareol synthase genes were integrated in a random fashion, and linked with the ribosomal skip 2A under the control of the CaMV 35S promoter. We conclude that moss can produce sclareol and utilize the ribosomal skip 2A. In addition, we observed growth impairment in all our sclareol-producing lines and moss lines knocked out in the endogenous diterpene synthase (copalyl/kaurene synthase—PpCPS/KS). A RT-PCR study, with ubiquitin as the best reference gene, showed that there was a down-regulation of the transcription of the terpenoid biosynthetic genes in the PpCPS/KS knock out moss. This down-regulation was recovered by the introduction of the two sclareol synthases, suggesting that the regulation of the general terpenoid biosynthesis is very flexible and can be amended in future biotechnological engineering.

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Acknowledgments

The project was co-funded by the Danish Agency for Science, Technology and Innovation, and the Firmenich S. A with a PhD stipend to Xi-Wu Pan and the Danish Strategic Research Council Grant to H. T. Simonsen. The authors also wish thank Firmenich for providing the sclareol synthase genes and the authentic standards; Yong-Liang Tang and Dr. Lei Han are also thanked for the technical support on GC–MS and HPLC for metabolites analysis, and Dr. Brian C. King is thanked for the pBK3 vector for expression of terpene synthases under the PpCPS/KS promoter.

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Authors and Affiliations

  1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark

    Xi-Wu Pan & Henrik Toft Simonsen

  2. R&D Division, Firmenich Aromatics (China), Shanghai, China

    Lei Han, Yu-Hua Zhang & Dong-Fang Chen

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  1. Xi-Wu Pan
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  2. Lei Han
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  3. Yu-Hua Zhang
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Correspondence to Henrik Toft Simonsen.

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Pan, XW., Han, L., Zhang, YH. et al. Sclareol production in the moss Physcomitrella patens and observations on growth and terpenoid biosynthesis. Plant Biotechnol Rep 9, 149–159 (2015). https://doi.org/10.1007/s11816-015-0353-8

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