Unlike animals, plants cannot move away from a herbivore or other threats. Instead, they have evolved to produce a vast array of chemical compounds to protect themselves. Some of these compounds are also important to humans, for example, as medicines or fragrances. Plants usually only produce small amounts of these compounds in mixtures with many other compounds, which makes it difficult to purify them. As a result, the methods of purifying the compounds may require huge amounts of plant material, or be expensive and not environmentally friendly. One solution to this would be to genetically engineer microbes like bacteria or yeast to produce the compounds instead. In order to do that, we need to understand exactly which enzymes the plant uses to make each compound and introduce them into suitable microbes.

A compound called forskolin has been used since ancient times in traditional Indian medicine to treat conditions like high blood pressure, asthma and heart complications. Forskolin is found exclusively in the root of a plant called Coleus forskohlii, which is native to India and south-east Asia. It is stored inside cells within the bark of the root in structures called oil bodies, which are similar to oil drops. However, it is not known where forskolin is made, or which enzymes are involved.

Pateraki, Andersen-Ranberg et al. set out to uncover how C. forskohlii produces this compound. The experiments show that forskolin is produced within the cells that contain the oil bodies. A technique called RNA sequencing was used to identify several genes that are highly active in these cells and encode enzymes that could potentially be involved in producing forskolin. Further experiments demonstrated that these enzymes drive a cascade of chemical reactions that convert a molecule called 13R-manoyl oxide into forskolin. Next, Pateraki, Andersen-Ranberg et al. inserted the genes into yeast cells that could already produce 13R-manoyl oxide, which allowed the yeast to produce relatively high amounts of forskolin.

These findings show that it is possible to identify the genes involved in the production of medicinal compounds in a relatively short amount of time. This knowledge will aid the development of a method that can be used to produce forskolin and other similar compounds on a large scale without needing to harvest C. forskohlii plants.

Plants synthesize an impressive diversity of specialized metabolites enabling them to communicate and adapt to environmental challenges (Mithöfer and Boland, 2012; Woldemariam et al., 2011). Throughout history, humans have benefited from the medicinal properties of many of these phytochemicals (Hardy et al., 2012). Specialized plant metabolites and direct derivatives thereof still constitute more than a third of approved pharmaceuticals (Cragg and Newman, 2013; David et al., 2015). With over 50,000 known structures according to the ‘Dictionary of natural products’ (http://dnp.chemnetbase.com/), terpenoids are the largest class of plant specialized metabolites and constitute a vast repository of bio-active natural products including many structurally complex compounds (Pateraki et al., 2015). Examples of widely used plant-derived terpenoid pharmaceuticals are the anticancer drug paclitaxel (taxol) (Liu and Khosla, 2010), the therapeutic ingenol mebutate (picato) that is used for treatment of actinic keratosis (King et al., 2016; Luo et al., 2016) and artemisinin which is the most efficient treatment against malaria caused by Plasmodium parasites (Graham et al., 2010; Paddon and Keasling, 2014). Traditional chemical synthesis of plant-derived diterpenoid pharmaceuticals remains economically challenging, despite recent examples of elegant strategies mimicking natural routes (Appendino, 2014; Kawamura et al., 2016; Yuan et al., 2016). Extraction from plant biomass and semisynthesis from biotechnologically produced intermediates have been approached as alternative strategies (Graham et al., 2010; Paddon et al., 2013; Roberts, 2007). In contrast to recent examples demonstrating complete pathway reconstruction and production of opiate alkaloids in yeast (Galanie et al., 2015; Nakagawa et al., 2016), engineered total biosynthesis of terpenoid therapeutics—including paclitaxel and ingenol esters—has not yet been achieved. Challenges on the way to achieving this goal include the identification of pathway enzymes in native systems, particularly for those belonging to multi-enzyme families catalyzing the biosynthesis of specialized metabolites in plants, engineering of poorly understood multi-step enzymatic pathways and difficulties encountered in heterologous expression of key enzymes catalyzing monooxygenations critical for diterpenoid biosynthesis (Pateraki et al., 2015; Renault et al., 2014).

The diterpenoid forskolin is the active hypotensive principle accumulating in the root cork of Coleus forskohlii (Pateraki et al., 2014), a perennial shrub of the Lamiaceae family, indigenous to India and Southeast Asia with numerous reported applications in traditional medicine (Alasbahi and Melzig, 2010b; Kavitha et al., 2010). The pharmaceutical properties of forskolin are based on its ability to directly activate the adenylate cyclase enzyme resulting in elevated levels of the second messenger cyclic adenosine monophosphate (cAMP) (Doseyici et al., 2014; Seamon et al., 1981). Approved applications of forskolin range from alleviation of glaucoma (OcuforsEye drop solutions, Sabinsa, India), treatment of hypertension and heart failure (Colforsin daropate hydrochloride, a water-soluble derivative of forskolin, Nippon Kayaku, Japan) to lipolysis and body weight control (Godard et al., 2005; Kikura et al., 2004; Toya et al., 1998; Wagh et al., 2012; Yoneyama et al., 2002). Therapeutic opportunities were also suggested in animal tests, where forskolin-induced pigmentation of the skin, increasing protection against UV-associated carcinogenesis (D'Orazio et al., 2006). The complex chemical structure of forskolin with a decalin core, characteristic of labdane-type diterpenoids, a tetrahydropyran ring, five oxidized positions and eight chiral centers (Figure 1A) represents a challenge for classical organic chemical synthesis, although a key intermediate for stereoselective total synthesis has been reported (Ye et al., 2009). Hence, commercially available forskolin is extracted from C. forskohlii roots and purified from a mixture of over 60 structurally related abietane and epoxylabdane diterpenoids with a forskolin content varying from 0.013% to 0.728% of root dry weight (Alasbahi and Melzig, 2010a; Asada et al., 2012; Srivastava et al., 2017). As the demand for forskolin grows, reliable and sustainable commercial production from C. forskholii will become unachievable due to low yields, susceptibility of this species to diseases, changing climatic conditions and the resource intensive extraction and purification procedure required to obtain pharmaceutical grade forskolin (Mora-Pale et al., 2014). Elucidation of the biosynthetic pathway to forskolin and subsequent engineering of the pathway into microbial hosts offers a more clear and stable alternative production system that will be better able to address future needs.

Figure 1

Download asset Open asset

Figure 1—source data 1

cDNAs identified in the C. forskohlii root cork transcriptome and cloned during this work, with the GeneBank accession numbers.

https://doi.org/10.7554/eLife.23001.004

Download elife-23001-fig1-data1-v5.docx

Figure 1—source data 2

Table of FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values of the first 20 most abundant cDNAs identified in the root cork transcriptome library.

cDNAs involved in terpenoid metabolism are marked in bold.

https://doi.org/10.7554/eLife.23001.005

Download elife-23001-fig1-data2-v5.docx

Figure 1—source data 3

Table of primers used in this study.

Construction of plasmids for expression of CfTPS2, CfTPS3, CfTPS1 is described in Andersen-Ranberg et al. (2016). U (uracil, marked in bold), represents the cleavage site, used in the USER cloning (Nour-Eldin et al., 2006).

https://doi.org/10.7554/eLife.23001.006

Download elife-23001-fig1-data3-v5.docx

Recently, we reported specific accumulation of forskolin and its diterpene scaffold 13R-manoyl oxide in the root cork cells of C. forskohlii. A pair of diterpene synthases (CfTPS2 and CfTPS3), exclusively present in the root cork, was found to catalyze cyclization of the C₂₀ diterpenoid precursor geranylgeranyl diphosphate (GGPP) into 13R-manoyl oxide, the diterpene scaffold of forskolin (Pateraki et al., 2014). As a proof of concept, biosynthesis of 13R-manoyl oxide in enantiomerically pure form but in low yields was achieved by expressing CfTPS2 and CfTPS3 in Saccharomyces cerevisiae, E. coli and Synechocystis sp. (Andersen-Ranberg et al., 2016; Englund et al., 2015; Nielsen et al., 2014). Taking into account the functionalization steps needed for conversion of 13R-manoyl oxide to forskolin (Figure 1A), involvement of enzymes from the families of cytochrome P450s (CYPs) and acetyltransferases was predicted.

Here, we report an integrated biochemical and functional genomics approach, including metabolomics, single-cell-type transcriptome studies and a synthetic biology modular approach involving transient combinatorial expression of candidate genes in Nicotiana benthamiana to identify the panel of enzymes catalyzing functionalization of the C. forskohlii diterpene backbones and more specifically the biosynthesis of forskolin. Pathway intermediates were identified using GC- or HPLC-HRMS-SPE-NMR. To demonstrate the downstream application of the present work regarding biotechnological production of forskolin, the entire forskolin biosynthetic pathway was reconstituted in engineered S. cerevisiae for fermentation-based production of forskolin from glucose. Forskolin is the first example of a pharmaceutical diterpenoid produced entirely in yeast at titers relevant for industrial consideration. The outlined combinatorial biochemistry approach paves the way for development of yeast-engineered platforms for biosynthesis of other known or new-to-nature diterpenoids

The terpenoid biosynthetic pathways active in the root of C. forskohlii produce an array of 13R-manoyl oxide and miltiradiene-derived diterpenoids, including forskolin. Forskolin is one of the most complex and highly oxygenated diterpenoids reported in C. forskohlii. In the current study, the genes encoding the entire biosynthetic pathway for forskolin were identified. Availability of the transcriptome from the root cork cells of C. forskohlii, where forskolin biosynthesis takes place, the option to achieve rapid functional characterization of the gene candidates in planta by transient expression in N. benthamiana and high-sensitivity techniques for structural characterization made the pathway elucidation possible. With the genes encoding the entire forskolin biosynthetic pathway in hand, de novo production of forskolin in engineered yeast was achieved.

Initially, a number of genes encoding CYP76AH subfamily members, expressed mainly in the root cork of C. forskohlii, was cloned and transiently expressed in N. benthamiana leaves being able to produce 13R-manoyl oxide. The products profiles obtained with these enzymes revealed that the identified CfCYP76AHs have discrete roles in forskolin biosynthesis. Efficient monooxygenation at C-11 is catalyzed mainly by CfCYP76AH15 (but also by CfCYP76AH8, CfCYP76AH17 and CfCYP76AH11). Monooxygenation at position C-9 is catalyzed exclusively by CfCYP76AH16 and monooxygenation at C-1, C-6 and C-7 mainly by CfCYP76AH11. Monooxygenation at C-1 was also observed using CfCYP76AH8 and CfCYP76AH17 (4c, Figure 3—figure supplement 1). Collectively, this set of data displays the multifunctional roles of these enzymes. Together, they could account for all the oxygenated positions in forskolin. Co-expression of CfCYP76AH15, CfCYP76AH11 and CfCYP76AH16 resulted in specific and efficient formation of the final intermediate, deacetylforskolin.

Despite the complementary multifunctionality of the CYP76AH enzymes, partial functional redundancy is possible, as demonstrated by the varied oxygenation patterns observed in experiments with single enzymes. Overlapping functionalities may contribute to a coordinated action for efficient conversion of 1 to 13b. Moreover, certain C. forskohlii CYP76AH enzymes seem able to accept oxygenated forms of 1 as substrates which results in the observed shifts in profile toward higher decorated products when they are co-expressed in N. benthamiana (Supplementary file 1).

Although co-expression of CfCYP76AH11 and CfCYP76AH16 with either one of the three CfCYP76AH8, CfCYP76AH17 or CfCYP76AH15 in the engineered system resulted in biosynthesis of deacetylforskolin (13b) (Figure 5), the precise sequence of in planta 13R-manoyl oxide oxygenations cannot be deduced from the experimental results partly because all identified CfCYP76AHs accept 1 as substrate. The co-expression of the CfCYP76AH encoding genes in the root cork of C. forskohlii and their partial functional redundancy or complementarity may in vivo constitute the basis for the chemical diversity of labdane terpenoids present in the root cork of C. forskohlii. In planta, the forskolin biosynthetic pathway would thus appear to be entangled within a metabolic grid offering simultaneous production of a multitude of other diterpenoids.

Recently, CYP76AH enzymes accepting miltiradiene (a non-epoxylabdane, abietane diterpenoid, which is also present in C. forskohlii roots) as substrate were reported from other Lamiaceae species (Božić et al., 2015; Guo et al., 2016; Ignea et al., 2016a; Zi and Peters, 2013). This prompted us to examine whether the promiscuous and multifunctional CfCYP76AH could accept miltiradiene as substrate, and vice versa, e.g. if different Lamiaceae CYP76AHs can catalyze oxygenations of 13R-manoyl oxide (Figures 6 and 7). According to our results CfCYP76AH15 was found to have very similar catalytic activities compared to the rosemary CYP76AH4, an enzyme with a suggested role in the oxygenation of miltiradiene toward the synthesis of ferruginol (Zi and Peters, 2013). Efficient formation of ferruginol as well as 11-oxo-13R-manoyl oxide, by both enzymes indicates that they may represent orthologues. Two additional ferruginol synthases of the CYP76AH subfamily, one from Salvia fruticosa (SfFS) and one from Rosemary officinalis (RoFS), were found to catalyze the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide and 11-hydroxy-manoyl oxide when expressed in N. benthamiana leaves (Figures 6 and 7). These findings are not reflected in the phylogenetic analysis of the known CYP76AHs. All C. forskohlii CYP76AHs able to produce 11-oxo-13R-manoyl oxide are clustered together, while CYP76AHs from Salvia spp. and R. officinalis that can catalyze the formation of the same compound, as well as those CYPs able to accept miltiradiene as substrate, form a different cluster when analyzed with currently known CYP76AHs. Thus, it is likely that the ability of CYP76AHs to catalyze 11-oxo-13R-manoyl oxide has evolved convergently in these plants (Figure 11).

Figure 11

Download asset Open asset

These data highlight the functional versatility of the CYP76 family. The enzymes can exhibit broad substrate specificity which may advance metabolic evolution as they provide metabolic plasticity and flexibility affording synthesis of new diterpenoids. This facilitates the expansion of the number of possible diterpenoids produced in nature and potentially serves to diversify and augment the phytochemical defense of plants. The promiscuity of the CYP76AHs also provides potentials for their use in combinatorial approaches for synthesizing a range of diterpenoids with pharmaceutical relevance. The exclusive presence of the CYP76AH subfamily in Lamiaceae species may reflect gene duplications in a Lamiaceae ancestral species followed by expansion and neofunctionalization after speciation (Figure 11).

The identification of CfACT1-8 as an ACT catalyzing regiospecific acetylation of deacetylforskolin (13b) to afford forskolin completed the entire biosynthetic pathway for forskolin from its precursor, GGPP. Interestingly, the only currently identified acyltransferases in diterpenoid biosynthesis are those involved in the biosynthesis of paclitaxel. Those acetyltransferases show substantial regioselective promiscuity (Ondari and Walker, 2008; Walker and Croteau, 2000) and belong to Clade V (D'Auria, 2006; Tuominen et al., 2011), whereas the majority of the ACTs identified in the root cork transcriptome of C. forskohlii, including ACT1-6 and ACT1-8, belong to Clade III (Figure 12).

Figure 12

Download asset Open asset

With all forskolin biosynthetic pathway genes identified, we moved to the generation of a stable forskolin producing S. cerevisiae strain. To engineer a stable microbial production platform, we proceeded to integrate the minimum required set of functional parts into the S. cerevisiae genome. Specific de novo production of the highly functionalized diterpenoid at titers above 40 mg/L was achieved through a pathway consisting of a total of 10 enzymatic steps catalyzed by eight heterologously co-expressed enzymes. This high forskolin titer, achieved with no optimization steps, highlights the potential to develop a microbial manufacturing platform for efficient and stereospecific production of forskolin with further fine-tuning of the biosynthetic pathway. Currently, it is not possible to accurately estimate the forskolin titers necessary for industrially profitable production, as the exact commercial applications, market size and price as well as the production cost including downstream processing cannot be determined. Given the knowledge gained in our present study and experiences with other compounds (Paddon and Keasling, 2014) we find it realistic to aim for yields ranging from a single to double digits of gram per liter of yeast culture. To achieve higher forskolin yields, it is important upfront to ascertain a proper flux toward GGPP through the mevalonate pathway (Kampranis and Makris, 2012). Specifically for forskolin pathway, it seems clear that there is a limitation in flux through one or several of the P450s involved as we encounter accumulation of the intermediates, 300 mg/L and 500 mg/L of compounds 1 and 3a respectively, compared to forskolin (40 mg/L). Accumulation of only minute amounts of deacetylforskolin shows that CfACT1-8 is not a limiting step in the pathway. Accumulation of 1 and 3a intermediates was not observed in planta (Figure 10—figure supplement 1). This likely signifies that the expression levels of the heterologous CYPs expressed in yeast are not properly balanced or their efficiency and activity can be affected negatively after incoorporation into the yeast membrane, while in the native plant host, the pathway exhibits optimized carbon flux and enzymatic efficiency. P450s are notorious difficult to express in high amounts in yeast and recognized as exhibiting rather low K_cat values (Jung et al., 2011; Renault et al., 2014). Hence, to increase forskolin production in the yeast system, efforts should obviously be focused on optimizing CfCYP76AHs expression and enzyme kinetics, specificity and catalytic efficiency as well as pathway scaffolding to facilitate formation of a metabolon which will result in improved pathway flux and efficiency and reduced accumulation of pathway intermediates (Laursen et al., 2016). The high forskolin titers already obtained though in the engineered yeast strain highlights the potential to develop a microbial manufacturing platform for efficient and stereospecific production of forskolin and other labdane terpenoids by fine tuning the biosynthetic pathways.

A yeast-based production platform constitutes a sustainable alternative to traditional crop-based production but the gains always need to be compared to yield improvements obtained by classical or molecular breeding of the traditional host plant (Graham et al., 2010). The model-example from the literature and industry toward production of a pharmaceutically relevant terpenoid in S. cerevisiae is the sesquiterpenoid artemisinic acid, a pathway intermediate to the antimalarial compound artemisinin (Paddon et al., 2013). However, this prominent model example is dependent on a costly organic chemical synthesis component to chemically convert artemisinic acid to artemisinin (Peplow, 2016). Recent approaches of engineered de novo production of structurally complex diterpenoids, triterpenoids or alkaloids in microbial systems have also been limited to proof-of-concept studies, expression of partial pathways and sub-milligram yields, highlighting the challenges in synthetic biology to offer an economically realistic and sustainable alternative to isolation of the desired medicinal compounds from medicinal plants bred to produce elevated levels. The constraints to achieve high yields are connected to expression of multiple CYPs and reconstruction of pathways with multiple functionally divergent steps (Brown et al., 2015; Li and Smolke, 2016; Zhou et al., 2015). Strategies addressing these issues are for example the development of synthetic microbial consortia of S. cerevisiae and E. coli, optimization of CYPS for functional expression in E. coli, optimization of interactions between the CYPs and their reductase partner and N-terminal modifications (Biggs et al., 2016; Laursen et al., 2016; Vazquez-Albacete et al., 2017; Zhou et al., 2015).

Our current study demonstrates that mining for additional members of the CYP76AH family has the potential to facilitate the assembly of further optimized panels of mixed-species P450s for the biosynthesis of bioactive diterpenoids. This shows the great promise that combinatorial assembly including CYPs outside the CYP76AH subfamily may offer and the opportunity to design production systems for diterpenoids that are currently inaccessible due to their exclusive presence in rare or red-listed plants and to further expand the chemical diversity of diterpenoids to production of compounds currently not known in nature.

1. 1. Alasbahi RH
   2. Melzig MF

   (2010a) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology - Part 1

   Planta Medica 76:653–661.

   https://doi.org/10.1055/s-0029-1240898

   • PubMed
   • Google Scholar
2. 1. Alasbahi RH
   2. Melzig MF

   (2010b) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology - part 2

   Planta Medica 76:753–765.

   https://doi.org/10.1055/s-0029-1240919

   • PubMed
   • Google Scholar
3. 1. Andersen-Ranberg J
   2. Kongstad KT
   3. Nielsen MT
   4. Jensen NB
   5. Pateraki I
   6. Bach SS
   7. Hamberger B
   8. Zerbe P
   9. Staerk D
   10. Bohlmann J
   11. Møller BL
   12. Hamberger B

   (2016) Expanding the landscape of diterpene structural diversity through stereochemically controlled combinatorial biosynthesis

   Angewandte Chemie International Edition 55:2142–2146.

   https://doi.org/10.1002/anie.201510650

   • PubMed
   • Google Scholar
4. 1. Appendino G

   (2014) Omnia praeclara rara. the quest for ingenol heats up

   Angewandte Chemie International Edition 53:927–929.

   https://doi.org/10.1002/anie.201308281

   • PubMed
   • Google Scholar
5. 1. Asada Y
   2. Li W
   3. Terada T
   4. Kuang X
   5. Li Q
   6. Yoshikawa T
   7. Hamaguchi S
   8. Namekata I
   9. Tanaka H
   10. Koike K

   (2012) Labdane-type diterpenoids from hairy root cultures of coleus forskohlii, possible intermediates in the biosynthesis of forskolin

   Phytochemistry 79:141–146.

   https://doi.org/10.1016/j.phytochem.2012.03.007

   • PubMed
   • Google Scholar
6. 1. Bach SS
   2. Bassard JÉ
   3. Andersen-Ranberg J
   4. Møldrup ME
   5. Simonsen HT
   6. Hamberger B

   (2014) High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

   Methods in Molecular Biology 1153:245–255.

   https://doi.org/10.1007/978-1-4939-0606-2_18

   • PubMed
   • Google Scholar
7. 1. Biggs BW
   2. Lim CG
   3. Sagliani K
   4. Shankar S
   5. Stephanopoulos G
   6. De Mey M
   7. Ajikumar PK

   (2016) Overcoming heterologous protein interdependency to optimize P450-mediated taxol precursor synthesis in Escherichia coli

   PNAS 113:3209–3214.

   https://doi.org/10.1073/pnas.1515826113

   • PubMed
   • Google Scholar
8. 1. Božić D
   2. Papaefthimiou D
   3. Brückner K
   4. de Vos RC
   5. Tsoleridis CA
   6. Katsarou D
   7. Papanikolaou A
   8. Pateraki I
   9. Chatzopoulou FM
   10. Dimitriadou E
   11. Kostas S
   12. Manzano D
   13. Scheler U
   14. Ferrer A
   15. Tissier A
   16. Makris AM
   17. Kampranis SC
   18. Kanellis AK

   (2015) Towards elucidating carnosic acid biosynthesis in Lamiaceae: functional characterization of the three first steps of the pathway in salvia fruticosa and rosmarinus officinalis

   PLoS One 10:e0124106.

   https://doi.org/10.1371/journal.pone.0124106

   • PubMed
   • Google Scholar
9. 1. Brochado AR
   2. Matos C
   3. Møller BL
   4. Hansen J
   5. Mortensen UH
   6. Patil KR

   (2010) Improved vanillin production in Baker's yeast through in silico design

   Microbial Cell Factories 9:84–15.

   https://doi.org/10.1186/1475-2859-9-84

   • PubMed
   • Google Scholar
10. 1. Brown S
    2. Clastre M
    3. Courdavault V
    4. O'Connor SE

    (2015) De novo production of the plant-derived alkaloid strictosidine in yeast

    PNAS 112:3205–3210.

    https://doi.org/10.1073/pnas.1423555112

    • PubMed
    • Google Scholar
11. 1. Cragg GM
    2. Newman DJ

    (2013) Natural products: a continuing source of novel drug leads

    Biochimica Et Biophysica Acta (BBA) - General Subjects 1830:3670–3695.

    https://doi.org/10.1016/j.bbagen.2013.02.008

    • PubMed
    • Google Scholar
12. 1. D'Auria JC

    (2006) Acyltransferases in plants: a good time to be BAHD

    Current Opinion in Plant Biology 9:331–340.

    https://doi.org/10.1016/j.pbi.2006.03.016

    • PubMed
    • Google Scholar
13. 1. D'Orazio JA
    2. Nobuhisa T
    3. Cui R
    4. Arya M
    5. Spry M
    6. Wakamatsu K
    7. Igras V
    8. Kunisada T
    9. Granter SR
    10. Nishimura EK
    11. Ito S
    12. Fisher DE

    (2006) Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning

    Nature 443:340–344.

    https://doi.org/10.1038/nature05098

    • PubMed
    • Google Scholar
14. 1. David B
    2. Wolfender J-L
    3. Dias DA

    (2015) The pharmaceutical industry and natural products: historical status and new trends

    Phytochemistry Reviews 14:299–315.

    https://doi.org/10.1007/s11101-014-9367-z

    • Google Scholar
15. 1. Doseyici S
    2. Mehmetoglu I
    3. Toker A
    4. Yerlikaya FH
    5. Erbay E

    (2014) The effects of forskolin and rolipram on cAMP, cGMP and free fatty acid levels in diet induced obesity

    Biotechnic & Histochemistry 89:388–392.

    https://doi.org/10.3109/10520295.2014.883463

    • PubMed
    • Google Scholar
16. 1. Englund E
    2. Andersen-Ranberg J
    3. Miao R
    4. Hamberger B
    5. Lindberg P

    (2015) Metabolic engineering of synechocystis sp. PCC 6803 for production of the plant diterpenoid manoyl oxide

    ACS Synthetic Biology 4:1270–1278.

    https://doi.org/10.1021/acssynbio.5b00070

    • PubMed
    • Google Scholar
17. 1. Gabetta B
    2. Zini G
    3. Danieli B

    (1989) Minor diterpenoids of coleus forskohlii

    Phytochemistry 28:859–862.

    https://doi.org/10.1016/0031-9422(89)80130-X

    • Google Scholar
18. 1. Galanie S
    2. Thodey K
    3. Trenchard IJ
    4. Filsinger Interrante M
    5. Smolke CD

    (2015) Complete biosynthesis of opioids in yeast

    Science 349:1095–1100.

    https://doi.org/10.1126/science.aac9373

    • PubMed
    • Google Scholar
19. 1. Godard MP
    2. Johnson BA
    3. Richmond SR

    (2005) Body composition and hormonal adaptations associated with forskolin consumption in overweight and obese men

    Obesity Research 13:1335–1343.

    https://doi.org/10.1038/oby.2005.162

    • PubMed
    • Google Scholar
20. 1. Graham IA
    2. Besser K
    3. Blumer S
    4. Branigan CA
    5. Czechowski T
    6. Elias L
    7. Guterman I
    8. Harvey D
    9. Isaac PG
    10. Khan AM
    11. Larson TR
    12. Li Y
    13. Pawson T
    14. Penfield T
    15. Rae AM
    16. Rathbone DA
    17. Reid S
    18. Ross J
    19. Smallwood MF
    20. Segura V
    21. Townsend T
    22. Vyas D
    23. Winzer T
    24. Bowles D

    (2010) The genetic map of artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin

    Science 327:328–331.

    https://doi.org/10.1126/science.1182612

    • PubMed
    • Google Scholar
21. 1. Guo J
    2. Zhou YJ
    3. Hillwig ML
    4. Shen Y
    5. Yang L
    6. Wang Y
    7. Zhang X
    8. Liu W
    9. Peters RJ
    10. Chen X
    11. Zhao ZK
    12. Huang L

    (2013) CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts

    PNAS 110:12108–12113.

    https://doi.org/10.1073/pnas.1218061110

    • PubMed
    • Google Scholar
22. 1. Guo J
    2. Ma X
    3. Cai Y
    4. Ma Y
    5. Zhan Z
    6. Zhou YJ
    7. Liu W
    8. Guan M
    9. Yang J
    10. Cui G
    11. Kang L
    12. Yang L
    13. Shen Y
    14. Tang J
    15. Lin H
    16. Ma X
    17. Jin B
    18. Liu Z
    19. Peters RJ
    20. Zhao ZK
    21. Huang L

    (2016) Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones

    New Phytologist 210:525–534.

    https://doi.org/10.1111/nph.13790

    • PubMed
    • Google Scholar
23. 1. Hansen EH
    2. Møller BL
    3. Kock GR
    4. Bünner CM
    5. Kristensen C
    6. Jensen OR
    7. Okkels FT
    8. Olsen CE
    9. Motawia MS
    10. Hansen J

    (2009) De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and Baker's yeast (Saccharomyces cerevisiae)

    Applied and Environmental Microbiology 75:2765–2774.

    https://doi.org/10.1128/AEM.02681-08

    • PubMed
    • Google Scholar
24. 1. Hardy K
    2. Buckley S
    3. Collins MJ
    4. Estalrrich A
    5. Brothwell D
    6. Copeland L
    7. García-Tabernero A
    8. García-Vargas S
    9. de la Rasilla M
    10. Lalueza-Fox C
    11. Huguet R
    12. Bastir M
    13. Santamaría D
    14. Madella M
    15. Wilson J
    16. Cortés AF
    17. Rosas A

    (2012) Neanderthal medics? evidence for food, cooking, and medicinal plants entrapped in dental calculus

    Naturwissenschaften 99:617–626.

    https://doi.org/10.1007/s00114-012-0942-0

    • PubMed
    • Google Scholar
25. 1. Ignea C
    2. Athanasakoglou A
    3. Ioannou E
    4. Georgantea P
    5. Trikka FA
    6. Loupassaki S
    7. Roussis V
    8. Makris AM
    9. Kampranis SC

    (2016a) Carnosic acid biosynthesis elucidated by a synthetic biology platform

    PNAS 113:3681–3686.

    https://doi.org/10.1073/pnas.1523787113

    • PubMed
    • Google Scholar
26. 1. Ignea C
    2. Ioannou E
    3. Georgantea P
    4. Trikka FA
    5. Athanasakoglou A
    6. Loupassaki S
    7. Roussis V
    8. Makris AM
    9. Kampranis SC

    (2016b) Production of the forskolin precursor 11β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities

    Microbial Cell Factories 15:1–11.

    https://doi.org/10.1186/s12934-016-0440-8

    • PubMed
    • Google Scholar
27. 1. Jeandet P
    2. Delaunois B
    3. Aziz A
    4. Donnez D
    5. Vasserot Y
    6. Cordelier S
    7. Courot E

    (2012) Metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol

    Journal of Biomedicine and Biotechnology 2012:1–14.

    https://doi.org/10.1155/2012/579089

    • PubMed
    • Google Scholar
28. 1. Jensen NB
    2. Strucko T
    3. Kildegaard KR
    4. David F
    5. Maury J
    6. Mortensen UH
    7. Forster J
    8. Nielsen J
    9. Borodina I

    (2014) EasyClone: method for iterative chromosomal integration of multiple genes in saccharomyces cerevisiae

    FEMS Yeast Research 14:238–248.

    https://doi.org/10.1111/1567-1364.12118

    • PubMed
    • Google Scholar
29. 1. Jia M
    2. Potter KC
    3. Peters RJ

    (2016) Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis

    Metabolic Engineering 37:24–34.

    https://doi.org/10.1016/j.ymben.2016.04.001

    • PubMed
    • Google Scholar
30. 1. Jung ST
    2. Lauchli R
    3. Arnold FH

    (2011) Cytochrome P450: taming a wild type enzyme

    Current Opinion in Biotechnology 22:809–817.

    https://doi.org/10.1016/j.copbio.2011.02.008

    • PubMed
    • Google Scholar
31. 1. Kampranis SC
    2. Makris AM

    (2012) Developing a yeast cell factory for the production of terpenoids

    Computational and Structural Biotechnology Journal 3:e201210006.

    https://doi.org/10.5936/csbj.201210006

    • PubMed
    • Google Scholar
32. 1. Kavitha C
    2. Rajamani K
    3. Vadivel E

    (2010)

    Coleus forskohlii – A comprehensive review on morphology, phytochemistry and pharmacological aspects

    Journal of Medicinal Plants Research 4:278–285.

    • Google Scholar
33. 1. Kawamura S
    2. Chu H
    3. Felding J
    4. Baran PS

    (2016) Nineteen-step total synthesis of (+)-phorbol

    Nature 532:90–93.

    https://doi.org/10.1038/nature17153

    • PubMed
    • Google Scholar
34. 1. Kikura M
    2. Morita K
    3. Sato S

    (2004) Pharmacokinetics and a simulation model of colforsin daropate, new forskolin derivative inotropic vasodilator, in patients undergoing coronary artery bypass grafting

    Pharmacological Research 49:275–281.

    https://doi.org/10.1016/j.phrs.2003.09.012

    • PubMed
    • Google Scholar
35. 1. King AJ
    2. Brown GD
    3. Gilday AD
    4. Forestier E
    5. Larson TR
    6. Graham IA

    (2016) A cytochrome P450-Mediated intramolecular Carbon-Carbon ring closure in the biosynthesis of Multidrug-Resistance-Reversing lathyrane diterpenoids

    ChemBioChem 17:1593–1597.

    https://doi.org/10.1002/cbic.201600316

    • PubMed
    • Google Scholar
36. 1. Kosley RW
    2. Cherill RJ

    (1989) Regioselective acylations of 7-desacetylforskolin

    The Journal of Organic Chemistry 54:2972–2975.

    https://doi.org/10.1021/jo00273a039

    • Google Scholar
37. 1. Laursen T
    2. Borch J
    3. Knudsen C
    4. Bavishi K
    5. Torta F
    6. Martens HJ
    7. Silvestro D
    8. Hatzakis NS
    9. Wenk MR
    10. Dafforn TR
    11. Olsen CE
    12. Motawia MS
    13. Hamberger B
    14. Møller BL
    15. Bassard JE

    (2016) Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum

    Science 354:890–893.

    https://doi.org/10.1126/science.aag2347

    • PubMed
    • Google Scholar
38. 1. Li Y
    2. Smolke CD

    (2016) Engineering biosynthesis of the anticancer alkaloid noscapine in yeast

    Nature Communications 7:12137.

    https://doi.org/10.1038/ncomms12137

    • PubMed
    • Google Scholar
39. 1. Liu T
    2. Khosla C

    (2010) Chemistry. A balancing act for taxol precursor pathways in E. coli

    Science 330:44–45.

    https://doi.org/10.1126/science.1195014

    • PubMed
    • Google Scholar
40. 1. Luo D
    2. Callari R
    3. Hamberger B
    4. Wubshet SG
    5. Nielsen MT
    6. Andersen-Ranberg J
    7. Hallström BM
    8. Cozzi F
    9. Heider H
    10. Lindberg Møller B
    11. Staerk D
    12. Hamberger B

    (2016) Oxidation and cyclization of casbene in the biosynthesis of euphorbia factors from mature seeds of euphorbia lathyris L

    PNAS 113:E5082–E5089.

    https://doi.org/10.1073/pnas.1607504113

    • PubMed
    • Google Scholar
41. 1. Mikkelsen MD
    2. Buron LD
    3. Salomonsen B
    4. Olsen CE
    5. Hansen BG
    6. Mortensen UH
    7. Halkier BA

    (2012) Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform

    Metabolic Engineering 14:104–111.

    https://doi.org/10.1016/j.ymben.2012.01.006

    • PubMed
    • Google Scholar
42. 1. Mithöfer A
    2. Boland W

    (2012) Plant defense against herbivores: chemical aspects

    Annual Review of Plant Biology 63:431–450.

    https://doi.org/10.1146/annurev-arplant-042110-103854

    • PubMed
    • Google Scholar
43. 1. Mora-Pale M
    2. Sanchez-Rodriguez SP
    3. Linhardt RJ
    4. Dordick JS
    5. Koffas MA

    (2014) Biochemical strategies for enhancing the in vivo production of natural products with pharmaceutical potential

    Current Opinion in Biotechnology 25:86–94.

    https://doi.org/10.1016/j.copbio.2013.09.009

    • PubMed
    • Google Scholar
44. 1. Nakagawa A
    2. Matsumura E
    3. Koyanagi T
    4. Katayama T
    5. Kawano N
    6. Yoshimatsu K
    7. Yamamoto K
    8. Kumagai H
    9. Sato F
    10. Minami H

    (2016) Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli

    Nature Communications 7:10390.

    https://doi.org/10.1038/ncomms10390

    • PubMed
    • Google Scholar
45. 1. Nelson DR

    (2009)

    The cytochrome p450 homepage

    Human Genomics 4:59–65.

    • PubMed
    • Google Scholar
46. 1. Nelson D
    2. Werck-Reichhart D

    (2011) A P450-centric view of plant evolution

    The Plant Journal 66:194–211.

    https://doi.org/10.1111/j.1365-313X.2011.04529.x

    • PubMed
    • Google Scholar
47. 1. Nelson DR

    (2013) A world of cytochrome P450s

    Philosophical Transactions of the Royal Society B: Biological Sciences 368:20120430.

    https://doi.org/10.1098/rstb.2012.0430

    • PubMed
    • Google Scholar
48. 1. Nielsen MT
    2. Ranberg JA
    3. Christensen U
    4. Christensen HB
    5. Harrison SJ
    6. Olsen CE
    7. Hamberger B
    8. Møller BL
    9. Nørholm MH

    (2014) Microbial synthesis of the forskolin precursor manoyl oxide in an enantiomerically pure form

    Applied and Environmental Microbiology 80:7258–7265.

    https://doi.org/10.1128/AEM.02301-14

    • PubMed
    • Google Scholar
49. 1. Nour-Eldin HH
    2. Hansen BG
    3. Nørholm MH
    4. Jensen JK
    5. Halkier BA

    (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    Nucleic Acids Research 34:e122.

    https://doi.org/10.1093/nar/gkl635

    • PubMed
    • Google Scholar
50. 1. Nour-Eldin HH
    2. Geu-Flores F
    3. Halkier BA

    (2010) USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories

    Methods in Molecular Biology 643:185–200.

    https://doi.org/10.1007/978-1-60761-723-5_13

    • PubMed
    • Google Scholar
51. 1. Ondari ME
    2. Walker KD

    (2008) The taxol pathway 10-O-acetyltransferase shows regioselective promiscuity with the oxetane hydroxyl of 4-deacetyltaxanes

    Journal of the American Chemical Society 130:17187–17194.

    https://doi.org/10.1021/ja8067534

    • PubMed
    • Google Scholar
52. 1. Paddon CJ
    2. Westfall PJ
    3. Pitera DJ
    4. Benjamin K
    5. Fisher K
    6. McPhee D
    7. Leavell MD
    8. Tai A
    9. Main A
    10. Eng D
    11. Polichuk DR
    12. Teoh KH
    13. Reed DW
    14. Treynor T
    15. Lenihan J
    16. Fleck M
    17. Bajad S
    18. Dang G
    19. Dengrove D
    20. Diola D
    21. Dorin G
    22. Ellens KW
    23. Fickes S
    24. Galazzo J
    25. Gaucher SP
    26. Geistlinger T
    27. Henry R
    28. Hepp M
    29. Horning T
    30. Iqbal T
    31. Jiang H
    32. Kizer L
    33. Lieu B
    34. Melis D
    35. Moss N
    36. Regentin R
    37. Secrest S
    38. Tsuruta H
    39. Vazquez R
    40. Westblade LF
    41. Xu L
    42. Yu M
    43. Zhang Y
    44. Zhao L
    45. Lievense J
    46. Covello PS
    47. Keasling JD
    48. Reiling KK
    49. Renninger NS
    50. Newman JD

    (2013) High-level semi-synthetic production of the potent antimalarial artemisinin

    Nature 496:528–532.

    https://doi.org/10.1038/nature12051

    • PubMed
    • Google Scholar
53. 1. Paddon CJ
    2. Keasling JD

    (2014) Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development

    Nature Reviews Microbiology 12:355–367.

    https://doi.org/10.1038/nrmicro3240

    • PubMed
    • Google Scholar
54. 1. Pateraki I
    2. Andersen-Ranberg J
    3. Hamberger B
    4. Heskes AM
    5. Martens HJ
    6. Zerbe P
    7. Bach SS
    8. Møller BL
    9. Bohlmann J
    10. Hamberger B

    (2014) Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in coleus forskohlii

    Plant Physiology 164:1222–1236.

    https://doi.org/10.1104/pp.113.228429

    • PubMed
    • Google Scholar
55. 1. Pateraki I
    2. Heskes AM
    3. Hamberger B

    (2015) Cytochromes P450 for terpene functionalisation and metabolic engineering

    Advances in Biochemical Engineering/biotechnology 148:107–139.

    https://doi.org/10.1007/10_2014_301

    • PubMed
    • Google Scholar
56. 1. Peplow M

    (2016) Synthetic biology's first malaria drug meets market resistance

    Nature 530:389–390.

    https://doi.org/10.1038/530390a

    • PubMed
    • Google Scholar
57. 1. Renault H
    2. Bassard JE
    3. Hamberger B
    4. Werck-Reichhart D

    (2014) Cytochrome P450-mediated metabolic engineering: current progress and future challenges

    Current Opinion in Plant Biology 19:27–34.

    https://doi.org/10.1016/j.pbi.2014.03.004

    • PubMed
    • Google Scholar
58. 1. Roberts SC

    (2007) Production and engineering of terpenoids in plant cell culture

    Nature Chemical Biology 3:387–395.

    https://doi.org/10.1038/nchembio.2007.8

    • PubMed
    • Google Scholar
59. 1. Schwarz R
    2. Dayhoff M

    (1979)

    Atlas of Protein Sequences

    Matrices for detecting distant relationships, Atlas of Protein Sequences, National Biomedical Research Foundation.

    • Google Scholar
60. 1. Seamon KB
    2. Padgett W
    3. Daly JW

    (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells

    PNAS 78:3363–3367.

    https://doi.org/10.1073/pnas.78.6.3363

    • PubMed
    • Google Scholar
61. 1. Srivastava S
    2. Misra A
    3. Mishra P
    4. Shukla P
    5. Kumar M
    6. Sundaresan V
    7. Negi KS
    8. Agrawal PK
    9. Singh Rawat AK

    (2017) Molecular and chemotypic variability of forskolin in coleus forskohlii Briq., a high value industrial crop collected from western himalayas (India)

    RSC Adv. 7:8843–8851.

    https://doi.org/10.1039/C6RA26190F

    • Google Scholar
62. 1. Tamura K
    2. Stecher G
    3. Peterson D
    4. Filipski A
    5. Kumar S

    (2013) MEGA6: molecular evolutionary genetics analysis version 6.0

    Molecular Biology and Evolution 30:2725–2729.

    https://doi.org/10.1093/molbev/mst197

    • PubMed
    • Google Scholar
63. 1. Toya Y
    2. Schwencke C
    3. Ishikawa Y

    (1998) Forskolin derivatives with increased selectivity for cardiac adenylyl cyclase

    Journal of Molecular and Cellular Cardiology 30:97–108.

    https://doi.org/10.1006/jmcc.1997.0575

    • PubMed
    • Google Scholar
64. 1. Tuominen LK
    2. Johnson VE
    3. Tsai CJ

    (2011) Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among populus paralogues

    BMC Genomics 12:236.

    https://doi.org/10.1186/1471-2164-12-236

    • PubMed
    • Google Scholar
65. 1. Vazquez-Albacete D
    2. Cavaleiro AM
    3. Christensen U
    4. Seppälä S
    5. Møller BL
    6. Nørholm MH

    (2017) An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

    Biotechnology and Bioengineering 114:751–760.

    https://doi.org/10.1002/bit.26203

    • PubMed
    • Google Scholar
66. 1. Wagh VD
    2. Patil PN
    3. Surana SJ
    4. Wagh KV

    (2012) Forskolin: upcoming antiglaucoma molecule

    Journal of Postgraduate Medicine 58:199–202.

    https://doi.org/10.4103/0022-3859.101396

    • PubMed
    • Google Scholar
67. 1. Walker K
    2. Croteau R

    (2000) Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from taxus and functional expression in Escherichia coli

    PNAS 97:583–587.

    https://doi.org/10.1073/pnas.97.2.583

    • PubMed
    • Google Scholar
68. 1. Werck-Reichhart D
    2. Feyereisen R

    (2000) Cytochromes P450: a success story

    Genome Biology 1:reviews 3003.3001.

    https://doi.org/10.1186/gb-2000-1-6-reviews3003

    • PubMed
    • Google Scholar
69. 1. Woldemariam MG
    2. Baldwin IT
    3. Galis I

    (2011) Transcriptional regulation of plant inducible defenses against herbivores: a mini-review

    Journal of Plant Interactions 6:113–119.

    https://doi.org/10.1080/17429145.2010.544779

    • Google Scholar
70. 1. Ye H
    2. Deng G
    3. Liu J
    4. Qiu FG

    (2009) Expedient construction of the Ziegler intermediate useful for the synthesis of forskolin via consecutive rearrangements

    Organic Letters 11:5442–5444.

    https://doi.org/10.1021/ol902133q

    • PubMed
    • Google Scholar
71. 1. Yoneyama M
    2. Sugiyama A
    3. Satoh Y
    4. Takahara A
    5. Nakamura Y
    6. Hashimoto K

    (2002) Cardiovascular and adenylate cyclase stimulating effects of colforsin daropate, a water-soluble forskolin derivative, compared with those of isoproterenol, dopamine and dobutamine

    Circulation Journal 66:1150–1154.

    https://doi.org/10.1253/circj.66.1150

    • PubMed
    • Google Scholar
72. 1. Yuan C
    2. Jin Y
    3. Wilde NC
    4. Baran PS

    (2016) Short, enantioselective total synthesis of highly oxidized taxanes

    Angewandte Chemie International Edition 55:8280–8284.

    https://doi.org/10.1002/anie.201602235

    • PubMed
    • Google Scholar
73. 1. Zerbe P
    2. Hamberger B
    3. Yuen MM
    4. Chiang A
    5. Sandhu HK
    6. Madilao LL
    7. Nguyen A
    8. Hamberger B
    9. Bach SS
    10. Bohlmann J

    (2013) Gene discovery of modular diterpene metabolism in nonmodel systems

    Plant Physiology 162:1073–1091.

    https://doi.org/10.1104/pp.113.218347

    • PubMed
    • Google Scholar
74. 1. Zhang W-W
    2. Luo J-G
    3. Wang J-S
    4. Lu Y-Y
    5. Kong L-Y

    (2009) LC–DAD–ESI-MS-MS for Characterization and Quantitative Analysis of Diterpenoids from Coleus forskohlii

    Chromatographia 70:1635–1643.

    https://doi.org/10.1365/s10337-009-1370-y

    • Google Scholar
75. 1. Zhou K
    2. Qiao K
    3. Edgar S
    4. Stephanopoulos G

    (2015) Distributing a metabolic pathway among a microbial consortium enhances production of natural products

    Nature Biotechnology 33:377–383.

    https://doi.org/10.1038/nbt.3095

    • PubMed
    • Google Scholar
76. 1. Zi J
    2. Peters RJ

    (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the lamiaceae

    Organic & Biomolecular Chemistry 11:7650–7652.

    https://doi.org/10.1039/c3ob41885e

    • PubMed
    • Google Scholar

Author details

1. Irini Pateraki

   1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
   2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

   Contribution

   IP, Conceptualization, Data curation, Investigation, Methodology, Writing—original draft, Project administration

   Contributed equally with

   Johan Andersen-Ranberg

   For correspondence

   eipa@plen.ku.dk

   Competing interests

   IP: Filed international patent 600 applications (PCT/DK2015/050020) covering 'Biosynthesis of forskolin and related compounds

   "This ORCID iD identifies the author of this article:" 0000-0002-7526-2334

2. Johan Andersen-Ranberg

   1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
   2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

   Present address

   Department of Plant & Microbial Biology, University of California, Berkeley, United States

   Contribution

   JA-R, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Irini Pateraki

   Competing interests

   JA-R: Filed international patent 600 applications (PCT/DK2015/050020) covering 'Biosynthesis of forskolin and related compounds

3. Niels Bjerg Jensen

   Evolva A/S, Copenhagen, Denmark

   Contribution

   NBJ, Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   NBJ: Filed international patent 600 applications (PCT/DK2015/050020) covering 'Biosynthesis of forskolin and related compounds Employee of Evolva SA

4. Sileshi Gizachew Wubshet

   Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

   Present address

   Nofima AS, Osloveien, Tromsø, Norway

   Contribution

   SGW, Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared.

5. Allison Maree Heskes

   1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
   2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

   Contribution

   AMH, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-2732-5185

6. Victor Forman

   Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark

   Contribution

   VF, Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared.

7. Björn Hallström

   Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden

   Contribution

   BHal, Formal analysis, Methodology

   Competing interests

   No competing interests declared.

8. Britta Hamberger

   1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
   2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

   Present address

   Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, United States

   Contribution

   BHam, Formal analysis

   Competing interests

   No competing interests declared.

9. Mohammed Saddik Motawia

   1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
   2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

   Contribution

   MSM, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared.

10. Carl Erik Olsen

    1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
    2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

    Contribution

    CEO, Data curation

    Competing interests

    No competing interests declared.

11. Dan Staerk

    Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Contribution

    DS, Data curation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared.

12. Jørgen Hansen

    Evolva A/S, Copenhagen, Denmark

    Contribution

    JH, Supervision, Methodology

    Competing interests

    JH: Employee of Evolva SA

13. Birger Lindberg Møller

    1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
    2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

    Contribution

    BLM, Funding acquisition, Writing—original draft, Writing—review and editing

    Competing interests

    BLM: Filed international patent 600 applications (PCT/DK2015/050020) covering 'Biosynthesis of forskolin and related compounds

14. Björn Hamberger

    1. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark
    2. Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark

    Present address

    Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, United States

    Contribution

    BH, Conceptualization, Writing—review and editing

    Competing interests

    BH: Filed international patent 600 applications (PCT/DK2015/050020) covering 'Biosynthesis of forskolin and related compounds'

    "This ORCID iD identifies the author of this article:" 0000-0003-1249-1807

• 5,101

  views

• 810

  downloads

• 98

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.