Forskning ved Københavns Universitet - Københavns Universitet

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Metabolic Engineering of the Moss Physcomitrella patens as a Green Cell Factory to Produce Terpenoids

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Xin Zhan
The moss Physcomitrella patens, as an alternative production host to Escherichia coli and Saccharomyces cerevisiae, has been evaluated and developed to produce terpenoids in this thesis. First of all, an effective method to construct and characterize transgenic P. patens lines is being set-up and published in our lab. Based on this method, three new diterpenoid metabolites (ent-beyerene, ent-sandaracopimaradiene and ent-15-kaurene) were discovered besides ent-16-kaurene and 16α-hydroxykaurane which were previously described in the wild type P. patens. A PpCPS/KS KO (Ppcps/ks) mutant showed the absence of all the five diterpenoid metabolites, implicating that PpCPS/KS is responsible for the biosynthesis of all the five diterpenoid metabolites described here. It provides the experimental evidence to support the previously proposed cyclization mechanism of the diterpene synthase activity that the secondary carbocation intermediates ent-beyerenyl+ and ent-isopimaranyl+ carbocation were stable enough to be deprotonated during the reaction catalyzed by P. patens PpCPS/KS.

The main objective of this thesis, heterologous production of two valuable perfumery ingredients patchoulol and β-santalene were also achieved with the yields of 1.3 and 0.035 mg/g dry weight respectively, after several metabolic engineering strategies were tried, including HMGR overexpression, CPS/KS gene disruption and plastidic localization of the terpene synthases. In order to synthesize more valuable perfumery ingredient (Z)-β-santalol in P. patens, five different well-characterized cytochromes P450 from GenBank, CYP71A5 (geraniol-10-hydroxylase) from Nepeta racemosa, CYP76B6 (geraniol-8-oxidase) from Catharanthus roseus, CYP76C4 (geraniol 8-, 9-hydroxylase) from Arabidopsis thaliana, CYP71D20 (5-epi-aristolochene-1, 3-dihydroxylase) from Nicotiana tabacum and germacrene A oxidase (GAO) from Lactuca sativa, were also tested, but they showed no catalytic activity towards β-santalene based on the preliminary HS-SPME-GCMS analysis and further investigations such as liquid extraction by ethyl acetate are needed to draw a solid conclusion.
OriginalsprogDansk
ForlagDepartment of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
StatusUdgivet - 2014

ID: 122831362