View metadata,Downloaded citation and from similar orbit.dtu.dk papers on:at core.ac.uk Dec 20, 2017 brought to you by CORE provided by Online Research Database In Technology Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form Nielsen, Morten Thrane; Ranberg, Johan Andersen; Christensen, Ulla; Christensen, Hanne Bjerre; Harrison, Scott James; Olsen, Carl Erik; Hamberger, Björn; Møller, Birger Lindberg; Nørholm, Morten Published in: Applied and Environmental Microbiology Link to article, DOI: 10.1128/AEM.02301-14 Publication date: 2014 Document Version Early version, also known as pre-print Link back to DTU Orbit Citation (APA): Nielsen, M. T., Ranberg, J. A., Christensen, U., Christensen, H. B., Harrison, S. J., Olsen, C. E., ... Nørholm, M. (2014). Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form. Applied and Environmental Microbiology, 80(23), 7258–7265. DOI: 10.1128/AEM.02301-14 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. AEM Accepts, published online ahead of print on 19 September 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.02301-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved. 1 Microbial synthesis of the forskolin precursor manoyl oxide in 2 enantiomerically pure form 3 4 Morten T. Nielsen1,2, Johan Andersen Ranberg2, Ulla Christensen1, Hanne Bjerre Christensen1, 5 Scott J. Harrison1, Carl Erik Olsen2, Björn Hamberger2, Birger Lindberg Møller2 and Morten H. H. 6 Nørholm1,3 7 8 1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle 9 Allé 6, DK-2970 Hørsholm, Denmark 10 2 Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of 11 Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark 12 3Address correspondence to: MHHN [email protected]. Phone: +45 217-99184 Fax: +45- 13 353-33300 14 15 Keywords: Terpenes, cell factories, forskolin, Coleus forskohlii, medicinal natural products. 16 Total number of manuscript pages: 28 17 Total number of pages for Figures: 4 18 Total number of pages for Tables: 2 19 Total number of pages for supplemental material: 7 1 20 ABSTRACT 21 Forskolin is a promising medicinal compound belonging to the plethora of specialized plant 22 metabolites that constitutes a rich source of bioactive high-value compounds. A major obstacle for 23 exploitation of plant metabolites is that they often are produced in low amounts and in plants 24 difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and 25 high sales prices. Hence, substantial efforts and resources have been invested in developing 26 sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial 27 synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other 28 medically important labdane-type terpenoids. Process optimization enabled synthesis of 29 enantiomerically pure (13R)-manoyl oxide as the sole metabolite providing a pure compound in just 30 two steps with a yield of 10 mg/l. The work presented here demonstrates the value of a standardized 31 bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable 32 synthesis of complex biochemicals. 33 2 34 INTRODUCTION 35 Terpenoids represent a highly diverse class of natural products with a multitude of applications in 36 modern society ranging from renewable fuels to structurally complex medicinal compounds (1). For 37 example they constitute the active compounds in several medicinal plant species including 38 paclitaxel (Taxus brevifolia), forskolin (Coleus forskohlii) and artemisinin (Artemisia annua). 39 Although several terpenoid derived natural products are currently in or have passed clinical trial for 40 treatment of a number of conditions (2), sufficient supply of the molecules remains an obstacle. 41 Purification from naturally producing plants has proven infeasible in many cases due to low yield, 42 while establishment of heterologous production systems have been hampered by insufficient insight 43 into the biosynthetic routes. However, the advent and rapid development of whole genome and 44 transcriptome sequencing have paved the way for several recent breakthroughs. Particularly tissue 45 specific metabolomics combined with RNA deep sequencing is an exceptionally promising 46 approach for gene discovery in non-model organisms (3, 4). 47 Our interest is focused on the diterpene forskolin produced by C. forskohlii. Forskolin is a 48 promising pharmaceutical which has shown potential as treatment against a wide range of diseases 49 (5-9). Forskolin is currently approved for treatment of glaucoma and heart-failure, while clinical 50 trials against several forms of cancer are ongoing - the compound has been tested in over 800 51 independent assays and in 76 cases forskolin was reported to be potently active (< 2 micro molar) 52 including activity as D3 dopamine and relaxin receptor agonist, as allosteric activator of Mas 53 related G-protein coupled receptor X1 and as antagonist of the androgen receptor (PubChem, 54 accessed August 2014 http://pubchem.ncbi.nlm.nih.gov). Finally, forskolin is extensively used in 55 basic research as an inhibitor of protein kinases (more than 50,000 publications in Web of Science, 56 Thompson Reuters). Recently, the combined use of metabolomics and RNA deep sequencing were 57 successfully applied to C. forskohlii root cells resulting in identification of two diterpene synthases, 3 58 CfTPS2 and CfTPS3, that catalyze the formation of (13R)-manoyl oxide (13R-MO) from 59 geranylgeranyl diphosphate (GGPP) (10). 13R-MO is proposed to be the first dedicated 60 intermediate in forskolin biosynthesis (11) .With this first biosynthetic knowledge in hand, 61 establishment of a microbial system for further elucidation of the biosynthetic route as well as 62 production of forskolin comes within reach. 63 From a microbial engineering perspective, terpenoids constitute an attractive class of compounds as 64 they are all derived from a single precursor, isopentenyl pyrophosphate (IPP), and its isomer 65 dimethylallyl pyrophosphate (DMAPP). Substantial efforts have therefore been made in increasing 66 IPP-supply as a driver for efficient terpenoid synthesis in microbes (12). IPP can be synthesized 67 through two biochemical routes; the mevalonate (MEV), and the 1-deoxy-D-xylulose 5-phosphate 68 (DXP) pathways, with the MEV-pathway being prevalent in eukaryotes and archea, while the DXP- 69 pathway dominates in eubacteria. While both routes have been subjected to numerous engineering 70 efforts, the MEV-pathway has been most successfully applied in Escherichia coli (12, 13). One 71 consensus minimal set of MEV-pathway enzymes required for boosting IPP/DMAPP supply can be 72 formulated as mevalonate kinase (e.g. Saccharomyces cerevisiae [Sc] ERG12), phosphomevalonate 73 kinase (e.g. ScERG8), mevalonate pyrophosphate decarboxylase (e.g. ScMVD1) and IPP isomerase 74 (e.g. E. coli Idi2). Biosynthesis of diterpenes in E. coli faces the additional challenge that 75 conversion of IPP to the general diterpene precursor, GGPP, is very inefficient in wild type E. coli 76 strains and therefore requires introduction of a heterologous GGPP-synthase (14, 15). As previously 77 shown for the C15 sesquiterpene amorphadiene, a substantial increase in product yield can be 78 gained by combining a heterologous farnesyl diphosphate (FPP) synthase with the minimal 79 consensus set of MEV-pathway genes (16). Likewise, high-level supply of GGPP in E. coli requires 80 coordinated expression of a minimum of five genes: four for producing the precursor IPP/DMAPP 81 (for example S. cerevisiae ERG12, ERG8, MVD1 and E. coli idi2), and one for a GGPP synthase. 4 82 As this consensus set is independent of the target diterpene, assembling all five genes on a single 83 plasmid would substantially simplify experimental designs such as strain screenings. The Keasling 84 group have reported the construction and verification of a single plasmid for synthesis of farnesyl 85 diphosphate (13). Previous attempts of adaptation of this strategy for synthesis of GGPP 86 unfortunately resulted in a non-functional plasmid (16). We have recently described a set of design 87 principles for standardizing molecular cloning and formulation of a cloning pipeline to simplify 88 multi-gene expression in E. coli (17). Here, we take advantage of this bioengineering pipeline to 89 assemble and validate a single plasmid enabling GGPP synthesis from mevalonate, providing a 90 versatile platform for diterpene biosynthesis in E. coli. By utilization of the platform, we achieve in 91 vivo microbial synthesis of the proposed forskolin precursor 13R-MO.