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

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.