DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS, ALIMENTARIAE, BIOLOGICAE. UNIVERSITATIS HELSINKIENSIS 2/2017
JUHA KONTTURI Type III Polyketide Synthases from Gerbera hybrida
DEPARTMENT OF AGRICULTURAL SCIENCES FACULTY OF AGRICULTURE AND FORESTRY DOCTORAL PROGRAMME IN PLANT SCIENCES UNIVERSITY OF HELSINKI
Type III Polyketide Synthases from Gerbera Hybrida
Juha Kontturi Biotechnology Doctoral school in Plant Sciences
Department of Agricultural Sciences Faculty of Agriculture and Forestry University of Helsinki
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in the lecture room B6 (Metsätieteiden talo, Latokartanonkaari 7) on 10th of February 2017 at 12 o’clock. Supervisor: Professor Teemu Teeri Department of Agricultural Sciences University of Helsinki
Follow-up group: Docent Jussi Joensuu VTT Technical Research Centre of Finland
Doctor Milla Pietiäinen University of Helsinki
Reviewers: Docent/Principal Scientist Anneli Ritala VTT Technical Research Centre Finland
Professor Rob Veerporte University of Leiden
Opponent: Doctor Günter Brader Austrian Institute of Technology
Custos: Professor Teemu Teeri Department of Agricultural Sciences University of Helsinki, Finland
ISSN 2342-5423 ISBN 978-951-51-2949-9 (paperback) ISBN 978-951-51-2950-5 (PDF) ethesis.helsinki.fi
Kiriprintti Oy 2017
2 CONTENTS
ABBREVIATIONS ...... 5 LIST OF ORIGINAL PUBLICATIONS ...... 6 ABSTRACT ...... 7 1. INTRODUCTION...... 8 1.1 Type III Polyketide synthases (PKS) ...... 8
1.1.1 Chalcone synthase (CHS) ...... 10 1.1.2 Stilbene synthase (STS) ...... 12 1.1.3 2-pyrone synthase (2PS) ...... 12 1.1.4 Pentaketide chromonesynthase (PCS) and Octaketidesynthase (OKS) ...... 12 1.1.5 Anther specific chalcone synthases (ASCLs) ...... 13 1.2 Type III PKS structure and function ...... 14
1.2.1 Engineering plant polyketides ...... 15 1.2.2 Combinatorial biosynthesis...... 16 1.3 Type III PKSs in the biosynthesis of plant secondary metabolites ...... 16
1.3.1 The biosynthesis of 4-hydroxy-5-methylcoumarin (HMC)...... 16 1.3.2 The biosynthesis of sporopollenin ...... 18 1.4 Glycosylation of plant metabolites ...... 20
1.4.1 The function of GTs ...... 20 1.4.2 The physiological role of GTs ...... 21 1.4.3 Structure of GTs ...... 21 1.4.4 Protein engineering of GTs and biotechnological applications...... 22 2. AIMS OF THE STUDY ...... 23 3. MATERIALS AND METHODS ...... 23 4. RESULTS AND DISCUSSION ...... 24 4.1 Characterization of new 2PS enzymes, G2PS2 and G2PS3 from Gerbera hybrida ...... 24
4.1.1 In vitro enzymatic activity of G2PS2 and G2PS3 ...... 24 4.1.2 G2PS2 and G2PS3 expression in planta ...... 25 4.1.3 Active site of G2PS2 and G2PS3 ...... 25 4.1.4 Enzyme activity of mutated G2PS1 and G2PS2 ...... 26 4.1.5 Expression patterns of G2PS2 and G2PS3...... 26
3 4.2 Functional characterization of GASCL1 and GASCL2, two ASCL enzymes from Gerbera hybrida ...... 27
4.2.1 In vitro enzyme activity of GASCL1 and GASCL2 ...... 27 4.2.2 Expression of GASCL1 and GASCL2 ...... 29 4.3 Characterization of gerbera GTs ...... 30
4.3.1 In vitro activity of gerbera GTs ...... 30 4.3.2 Virus induced gene silencing of glucosyltransferases ...... 30 5. CONCLUSION AND FUTURE PROSPECTS ...... 32 6. ACKNOWLEDGEMENTS ...... 33 7. REFERENCES ...... 34
4 ABBREVIATIONS
2PS 2-pyrone synthase ASCL Anther specific chalcone synthase ALS Aloesone synthase ANS Anthocyanidin synthase Asn Asparagine AT Acyl transferase BAS Benzalacetone synthase BIS Biphenyl synthase cDNA Complementary DNA CHI Chalcone isomerase CHS Chalcone synthase CoA Coenzyme A CUR Curcuminoid synthase Cys Cysteine DFR Dihydroflavonol reductase ESI-MS Electron spray ionization mass spectrometry FAS Fatty acid synthase F3H Flavanone 3ɴ-hydroxylase F3´H Flavanoid 3’-hydroxylase FLS Flavonol synthase FNS Flavone synthase GT Glycosyltransferase Gly Glysine TAL Triacetolactone His Histidine Ile Isoleucine HMC 4-hydroxy-5-methylcoumarin HPLC High performance liquid chromatography HR Hypersensitive response KAS Ketoacyl synthase LAR Leucoanthocyanidin reductase Leu Leucine OAC Olivetolic acid cyclase OARS Oxoalkylresorcinol synthase OKS Octaketide synthase PCR Polymerase chain reaction PCS Pentaketide chromone synthase Phe Phenylalanine PKR Polyketide reductase PKS Polyketide synthase SA Salicylic acid Ser Serine STCS Stilbenecarboxylate synthase STS Stillbene synthase TAL Triacetolactone Thr Threonine TLC Thin-layer chromatography TKPR Tetraketide-2-pyrone reductase TMV Tobacco Mosaic Virus Tyr Tyrosine UDP Uridine-diphosphate UPLC Ultra performance liquid chromatography VIGS Virus Induced Gene Silencing
5 LIST OF ORIGINAL PUBLICATIONS
The thesis is based on the following publications:
I *Pietiäinen M., *Kontturi J., Paasela., Deng X., Ainasoja M., Nyberg P., Hotti H., Teeri T. 2016. Two polyketide synthases are necessary for 4-hydroxy-5-methylcoumarin biosynthesis in Gerbera hybrida. Plant Journal, 87: 548-558. II Kontturi J., Osama R., Deng X., Bashandy H., Albert V.A., Teeri T.H. 2017. Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida. Phytochemistry, 134: 38-45. III Kontturi J., Lampio A., Deng X., Teeri T.H. Glucosyltransferases from Gerbera hybrida. Manuscript.
*Shared first authorship
Contribution to the articles
I I did the cloning of PKS genes, protein production in E.coli, protein purification and in vitro enzymatic assays. I did all the HPLC runs from in vitro assays and tobacco infiltration results, and with the help of MS-laboratory, analyzed the MS-results from all experiments. I did the protein models, mutagenesis, and all the work relating to the mutated proteins and in vitro results. Finally, I analyzed the transgenic gerbera plants for HMC. I participated in planning the experiments, and wrote the article with Milla Pietiäinen under the supervision of Teemu Teeri.
II I cloned the PKS-genes, made protein production in E.coli, protein purification and in vitro enzymatic assays. I screened the results with HPLC and with the help of MS-laboratory, I analyzed the MS-results. I conducted all the expression studies, qRT-PCR and in situ localizations. Additionally, I made the sequence analysis and participated in making the phylogeny. I participated in planning the experiments, and wrote the article independently under the supervision of Teemu Teeri.
III I was responsible for cloning the VIGS-constructs, agrobacterium transformations and two separate VIGS-experiments. I analyzed the samples by RT-PCR and HPLC. Finally I did the phylogeny analysis, and participated in planning the experiments and writing the article under the supervision of Teemu Teeri.
6 ABSTRACT
The type III polyketide synthases (PKSs) are a group of enzymes involved in the production of structurally diverse secondary metabolites, polyketides in plants. Compounds, such as chalcones, chromones, stilbenes and coumarins possess a variety of important biological roles for the plants, such as protection against oxidative stress, pests and diseases, herbivory and UV irradiation. From the human use perspective, polyketides or their derivatives serve as antifungal compounds, immunosuppressants, anticancer drugs, antibiotics and insecticides. Type III PKS are small (40-45 kDa) proteins functioning as homodimers. They catalyze the sequential decarboxylative condensation reaction between malonyl-CoA and a variety of CoA-linked starter molecules in a biosynthesis, which closely resembles fatty acid biosynthesis. Gerbera (Gerbera hybrida) expresses three genes encoding chalcone synthases (CHS), which are enzymes involved in the biosynthesis of flavonoids in plants. In addition, it has three genes encoding 2-pyrone synthases (G2PS1-3), which differ from the CHSs with altered substrate specificity and the amount of decarboxylative malonyl-CoA condensation reactions. It was previously shown that G2PS1 is a triketide synthase, responsible for the biosynthesis of 4-hydroxy-6- methyl-2-pyrone (triacetolactone, TAL), a putative precursor for two antimicrobial compounds, gerberin and parasorboside in gerbera. In this study, the G2PS2 and G2PS3 were functionally characterized as pentaketide synthases and their role in the biosynthesis of another antimicrobial compound, 4-hydroxy-5methylcoumarin (HMC) was demonstrated. Using protein modelling and mutagenesis studies, the structural differences in the active site cavity of gerbera 2-pyrone synthases were demonstrated. The gerbera type III PKS family was extended by characterizing two additional 2-pyrone synthases and determining their expression patterns. The newly discovered GASCL1 and GASCL2 are anther specific chalcone synthase like enzymes (ASCLs), which in many other plant species have been shown to take part to the biosynthesis of sporopollenin, the main component of a exine layer of the pollen grain. The last part of the thesis focus on glycosyltransferses (GTs), a group of enzymes that catalyze the addition of a sugar moiety to the plant secondary metabolites, including the products of the gerbera type III PKSs.
7 1. INTRODUCTION
1.1 Type III Polyketide synthases (PKS)
Plant secondary metabolites are small molecular compounds, which are not directly involved in the growth or development, but are essential for the survival of the plant. Based on the biosynthetic origins, plant secondary metabolites can be divided to terpenoids, alkaloids and phenylpropanoids. Polyketides belong to the subset of phenylpropanoids. Some polyketides acts as intermediates in phenylpropanoid metabolism, but others are end products of the biosynthetic pathways (Croteau et al., 2000; Yu and Jez, 2008). Plant polyketides such as stilbenes, chalcones, chromones, phloroglucinols, coumarins and curcuminoids are a large class of natural secondary metabolites, produced by the plant type III polyketide synthases. In plants, the compounds serve as a protection against UV irradiation, oxidative stress and as antimicrobial defense. Additionally, they act as flower pigments and in some species possess a role in pollen fertility. They are also important compounds for human health, serving as immunosuppressants, antifungal agents, anticancer drugs, antibiotics and insecticides (Dixon R.A. 2001; Stauton and Weissman, 2001; Austin and Noel, 2003; Kim et al., 2010; Ferreyra et al., 2012; Jiang et al., 2016).
Type III PKS enzymes are typical to vascular plants but they have also been identified from the moss Physcomitrella patens (Kim et al., 2013). Type III PKSs differ from the microbial origin type I (modular type) and type II (subunit) both mechanistically and structurally and they utilize free CoA thioesters as a substrate without the help of acyl carrier proteins (ACPs). The type III PKSs share 30-95 % similarity in their amino acid sequence to each other, but only 21-31 % to the type I and type II PKSs (Abe and Morita, 2010). At least 20 functionally different plant type III PKSs have been characterized. Originally, the type III PKSs were regarded as plant-specific enzymes and called CHS-superfamily enzymes. Later, CHS-related enzymes were discovered from bacteria and fungi (Funa et al., 1999; Moore and Hopke, 2001; Abe and Morita, 2010).
Type III PKS are relatively small (40-45 kDa) homodimeric proteins that catalyze the assembly of polyketides in sequential decarboxylative condensation reactions of acetate units derived from malonyl-CoA onto CoA- linked starter substrates, in a biosynthesis sequence which closely resembles fatty acid biosynthesis. Type III PKS share a similar overall fold and catalytic mechanism as fatty acid synthases (FAS), and most likely they have evolved from FAS (Austin and Noel 2003). Still, differences exists between the enzymes. FAS enzymes complete each biosynthesis cycle by catalyzing one condensation followed by three reduction steps, with the help of other enzymes. While the substrates and intermediates are maintained as thioester conjugates to the carrier molecule called acyl carrier protein (ACP) in fatty acid biosynthesis, the type III PKSs catalytic steps do not require ACPs. The FAS substrates are first activated through binding to the ACP by an acyl transferase (AT). Then, the substrate is transferred to a ketoacyl synthase (KAS). Similarly as in type III PKS reaction, substrate is loaded to the catalytic cysteine of KAS, and the condensing enzyme catalyzes the addition of a two carbon acetate unit to the enzyme bound thioester end of the fatty acid, through a decarboxylative condensation with malonyl-ACP (Figure 1). The product is then further processed by a ketoacyl reductase (KR) and other FAS enzymes (Austin and Noel 2003; Yu et al., 2012).
8 Figure 1. The enzyme activity of keto acyl synthase (KAS), which is enzyme involved in the fatty acid biosynthesis (a) and the initial reaction catalyzed by a type III PKS (b), which uses acetyl-CoA as a starter.
Type III PKSs are functionally divergent, which arises from the ability to use a wide range of various starter substrates, variating in number of acetate unit condensations, and the type of cyclization and aromatization reactions. The enzymes are capable of using a CoA-linked starter substrates varying from small (acetyl-CoA) to bulky (p-coumaroyl-CoA), aliphatic-CoA (hexanoyl-CoA) to aromatic-CoA (benzoyl-CoA), and from polar (malonyl-CoA) to non-polar CoA (isovaleryl-CoA), which gives an unusual functional diversification for the plants. The number of condensation reactions of malonyl-CoA may vary from one up to eight, in the PKS enzymes occurring in the nature (Austin and Noel 2003; Abe and Morita 2010). In many cases, PKSs use malonyl-CoA as an extender molecule, but enzymes that can utilize methylmalonyl- or ethylmalonyl-CoA as an extender have also been characterized (Schröder et al., 1998; Jez et al., 2000a).
Cyclization reactions can be divided into three different kind of reactions: Claisen- and aldol types of condensations and lactonization (Bringmann et al., 2006). In a Claisen type cyclization, enzymes form a C-C bond from C6 to C1 of the catalytic cysteine-bound intermediate. Enzymes catalyzing this kind of reaction include chalcone synthase (CHS, Figure 2a, Ferrer et al., 1999) and pentaketide chromone synthase (PCS, Figure 2d, Abe et al., 2005b). The aldol-type cyclization is divided into two types. For example stilbene synthase (STS, Figure 2b, Schröder et al., 1998) and biphenyl synthase (BIS, Liu et al., 2007) catalyze the reaction forming bonds from C2 position to C7 position with one additional decarboxylative reaction of C1 as a loss of CO2. The cyclization reaction catalyzed by stilbenecarboxylate synthase (STCS) takes place between the same carbons but without decarboxylation at C1 (Eckermann et al., 2003). On the other hand, for instance aloesone synthase (ALS, Abe et al., 2004) and octaketide synthase (OKS, Figure 2e, Abe et al., 2005a) of the aldol type form a new C-C bond between the carbonyl carbon of the starter substrate and methylene carbon of the third extender on the polyketide intermediate, without decarboxylation. 2-pyrone synthase (2PS, Figure 2c) is a typical enzyme performing a lactonization reaction. It has remained unclear whether the lactonization in the pyrone ring formation is enzymatic or not (Abe and Morita 2010). In addition to the three types of cyclization reactions, there are PKSs such as benzalacetone synthase (BAS, Abe et al., 2001) and curcuminoid synthase (CUS, Katsuyama et al., 2007), which only catalyze the condensation reactions without cyclization. Gagne et al., (2012) also reported that a type III PKS (tetraketide synthase) from Cannabis sativa required the presence of a separate polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which performs the C2–C7 intramolecular aldol condensation to synthetize olivetolic acid.
9 1.1.1 Chalcone synthase (CHS)
The first characterized type III PKS was CHS, the key enzyme of the biosynthesis of flavonoids in plants. CHS is a tetraketide synthase, which catalyzes the sequential condensation of the C6-C3 unit of p-coumaroyl-CoA with three acetate units of malonyl-CoA, producing an enzyme-bound tetraketide intermediate, which folds into naringenin chalcone in a Claisen type condensation reaction (Figure 2a). The product chalcone is stereospecifically isomerized by chalcone isomerase (CHI), which forms the central heterocyclic ring of the flavanone naringenin, which can be used for flavone synthesis by flavone synthase (FNS). Flavanones can also be hydroxylated by flavanone 3-hydroxylase (F3H) to produce dihydroflavonols, which serve as a starter molecules for flavonols catalyzed by flavonol synthase (FLS). Dihydroflavanol can also be reduced by dihydroflavonol reductase (DFR) to produce flavan 3,4-diols. These compounds can be either dehydrated by anthocyanin synthase (ANS) for anthocyanin biosynthesis pathway, or reduced by leucoanthocyanidin reductase (LAR) into flavan-3-ols, which are condensed with flavan 3,4-diols to form condensed tannins, also known as proanthocyanidins. The different kind of flavonoids act as a sunscreen for the plants since the compounds absorb the harmful UV-B radiation. Additionally, plants use these compounds to control the herbivory, and to attract pollinators of flowers or animals that spread seeds in colored fruits (Holton and Cornish, 1995; Harborne and Williams, 2000; Austin and Noel, 2003).
CHS has a broad substrate specificity, accepting also nonphysiological and aliphatic CoAs. Still, small aliphatic CoA thioesters (e.g. acetyl-CoA) are poor substrates for CHS and majority of the reactions with aliphatic CoA thioesters terminate after two condensation reactions of malonyl-CoA (Eckermann et al., 1998; Zuurbier et al., 1998; Ferrer et al., 1999). All gymnosperms and angiosperms examined so far possess a CHS, but the distribution and specificity of downstream enzymes varies greatly within different tissues and the different stages of plant development. As a consequence of this biochemical diversity, many plants maintain multiple copies of CHS genes (Austin and Noel 2003). Gerbera for example, has three CHS-genes, which differ in their expression patterns and timing of the plant development (Helariutta et al., 1995; Deng et al., 2014). More diversity is achieved within the plant kingdom by the huge number of tailoring enzymes which modify the flavonoid scaffold, which can be later modified at various combinations of glycosylation, hydroxylation, acylation, O-methylation, prenylation and conjugation at multiple positions, resulting the identification more than 6000 naturally occurring flavonoids (Ferrer et al., 2008; Ferreyra et al., 2012). Flavonoids in the human diet have been shown to have effects since they can act as anti-microbial, anti-cancer, anti-asthmatic compounds, antioxidants and anti-inflammatory agents (Cushnie et al., 2005; Fu et al., 2013; Ravishankar et al., 2013; Lee et al., 2014; Parhiz et al., 2014).
10 Figure 2. Examples of reactions catalyzed by the plant type III PKSs. Both CHS and STS synthesize the same linear tetraketide intermediate, but CHS performs a Claisen type cyclization (a), while STS catalyzes an aldol type reaction (b). 2PS is an example for the intramolecular lactonization reaction (c). PCS is a pentaketidesynthase using five molecules of malonyl-CoA (e), OKS an octaketidesynthase condensing eight molecules of malonyl-CoA (d). ASCLs are enzymes, producing long chain triketide and tetraketide 2-pyrones.
11 1.1.2 Stilbene synthase (STS)
Like CHS, STS synthesize the same linear tetraketide intermediate from p-coumaroyl CoA and three molecules of malonyl-CoA. Still, they catalyze different ring-closure reactions. While the CHS cyclizes the intermediate molecule in an intramolecular Claisen type condensation between carbons C6 and C1, STS forms the bonds between C2 and C7 in an aldol type condensation reaction, with an additional decarboxylative loss of C1 as CO2 (Figure 2b). The released product resveratrol, has been shown to act as phytoalexin, an anti-microbial agent that is produced by the plant in response to pathogen attack. In medicine, resveratrol has been suggested to act as a chemopreventive agent against cancer and an agonist for the estrogen receptor (Hain et al., 1993; Jang et al., 1997; Austin et al., 2004, Khan et al., 2014). Not all STS enzymes produce resveratrol. STSs from pine produces pinosylvin in contrast to resveratrol because it prefers unsubstituted cinnamoyl- CoA instead of p-coumaroyl-CoA as a substrate in vitro. Still, STS efficiently accepts p-coumaroyl-CoA as a substrate in vitro, and most likely the substrate availability explains the in vivo preference (Schanz et al. 1992; Austin and Noel, 2003). In medicine, pinosylvin has been shown to act as an antioxidant and anti- inflammatory agent (Koskela et al., 2014; Moilanen et al., 2016).
1.1.3 2-pyrone synthase (2PS)
CHS being as an example for Claisen type of condensation, and STS for the aldol type, 2-pyrone synthase (2PS) is a typical enzyme for the lactonization reaction (Figure 2c). In gerbera, two CHSs (GCHS1, GCHS3) were characterized early as true CHS-enzymes (Helariutta et al., 1995). Later, GCHS4 was also added to the CHS- family (Deng et al., 2014). An enzyme originally named as GCHS2 shared 73-74% sequence similarity at the amino acid level with the true CHS-enzymes GCHS1 and GCHS3, but both for the expression pattern of the gene encoding the protein, and in vitro for its enzymatic activity, it behaved differently and could not use p- coumaroyl-CoA as a starter (Helariutta et al., 1995; Helariutta et al., 1996). In vitro results by Eckermann and coworkers (1998) showed that GCHS2 used a much smaller molecule, acetyl-CoA, as a starter in a reaction with two molecules of malonyl-CoA producing 6-methyl-4-hydroxy-2-pyrone (triacetolactone, TAL), a putative precursor for gerberin (5,6-dihydro-4-ɴ-D-glucopyranosyloxy-6-methyl-2-pyrone) and parasorboside (tetrahydro-4-ɴ-D-glucopyranosyloxy-6-methyl-2-pyrone), two compounds missing in transgenic gerbera lines down regulated for GCHS2 (Eckermann et al., 1998) and shown to have antimicrobial properties (Koskela et al., 2011). Interestingly, if acetyl-CoA is not available the enzyme is able to decarboxylate malonyl-CoA to make its own starter, acetyl-CoA. The reaction is typical for type III PKSs but no significant product formation is observed in many cases (Kreuzaler et al., 1978). GCHS2 was named as 2- pyrone-synthase (2PS), distinct from the CHS enzymes and the gene was as G2PS1. In addition to acetyl-CoA, G2PS1 accepts various small aliphatic starters, including propionyl-CoA, buturyl-CoA and hexanoyl-CoA in vitro. The enzyme also accepts benzoyl-CoA, which is used with two malonyl-CoAs to produce 6-phenyl-4- hydroxy-2-pyrone, possessing the backbone of substances that have been characterized as inhibitors of the HIV-1 protease (Helariutta et al., 1996; Thaisrivongs et al., 1996; Eckermann et al. 1998).
1.1.4 Pentaketide chromonesynthase (PCS) and Octaketidesynthase (OKS)
As an example for type III PKSs capable of catalyzing greater number of malonyl-CoA condensation reactions, PCS and OKS from Aloe (Aloe arborescens) have been described (Abe et al., 2005a; Abe et al., 2005b). PCS catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA followed by a Claisen type cyclization reaction to form the aromatic ring (Figure 2d). Acetyl-CoA, the product of decarboxylation of malonyl-CoA is also accepted by the enzyme and apparently decarboxylation of malonyl- CoA takes place as a first step in the PCS reaction cascade. Other aliphatic and aromatic CoA esters are also accepted but yielding only triketide and tetraketide 2-pyrones (Abe et al., 2005b). Additionally, a pentaketide
12 synthase, oxoalkylresorcinol synthase (OARS) has been characterized from the moss Physcomitrella patens (Kim et al., 2013).
Similarly as PCS, OKS is able to use malonyl-CoA as a sole substrate and catalyze the sequential decarboxylative reactions of eight malonyl-CoA to yield a 1:4 mixture of aromatic octaketide products, SEK4 and SEK4b (Figure 2e). OKS also accepts acetyl-CoA as a starter, which is the product formed through decarboxylation of malonyl-CoA. If acetyl-CoA is added to the reaction, the yield of octaketides stays at the same level, compared to the reaction where only malonyl-CoA is used. The observed incorporation rate is approximately 12.5 %, acetyl-CoA forming 1/8 of the building molecules of the end product. Interestingly, the decarboxylative reaction of the malonyl-CoA seems to be quite inefficient if the incorporation rate of acetyl-CoA is so close to the theoretical level (Abe et al., 2005a). OKS has been also characterized from StJohn’s wort (Hypericum perforatum) by Karppinen and coworkers (2008). They reported the production of the same octaketide compounds SEK and SEK4b, from one acetyl-CoA and seven malonyl-CoA molecules by the OKS enzyme (Figure 2e). These two enzymes construct the longest naturally occurring polyketides, in an eight-step decarboxylative condensations of malonyl-CoA followed by the aldol type cyclization (Abe et al., 2005a; Karppinen et al., 2008).
1.1.5 Anther specific chalcone synthases (ASCLs)
Anther-specific chalcone synthase-like enzymes (ASCLs) form a separate subfamily of plant PKSs of type III. The group represents ancient type III PKSs shared by all land plants, since an ASCL protein has been characterized also from the moss Physcomitrella patens (Colpitts et al. 2011). In plants, ASCL proteins take part in the biosynthesis of sporopollenin, the main component of exine layer of moss spores and mature pollen of seed plants (Kim et al., 2010). First ASCL-genes with sequence similarity to CHS were isolated from Brassica napus (Shen and Hsu, 1992), Oryza sativa (Hihara et al., 1996), Nicotiana sylvestris (Atanassov et al., 1998) and Silene latifolia (Barbacar et al., 1997). It was then suggested that ASCLs belong to a distinct subfamily of plant type III PKSs by Atanassov and coworkers (1998). They relate to CHS in gene and amino acid sequence and catalytic mechanism, but possess a different substrate preference and due to their expression pattern are often called anther-specific CHS-like enzymes (ASCLs). Later two ASCLs, PKSA and PKSB were characterized from arabidopsis (Arabidopsis thaliana). The enzymes were capable of accepting unusually long (up to C24) fatty acyl CoAs as starter substrates and catalyze the sequential condensation reaction with malonyl-CoA to produce 2-pyrones in vitro (Mizuuchi et al., 2008). Already earlier Nishikawa et al. (2005) had discovered arabidopsis mutants, which had reduced pollen-stigma adhesion and noticed that the patterning of the exine was changed, resulting in structurally weakened pollen grains that collapsed in the mutant lines (less adhesive pollen, lap). Dobritsa et al. (2010) mapped LAP6 and LAP5 to the CHS-like genes, PKSA and PKSB. At the same time, another research group discovered a role for LAP6/PKSA and LAP5/PKSB in the biosynthesis of sporopollenin, originally through coexpression analysis with ACOS5, which synthesizes the necessary fatty acyl-CoA esters (Kim et al., 2010).
In vitro, the two ASCLs, PKSA and PKSB, from arabidopsis accept aliphatic long-chain fatty acyl-CoAs from n- hexanoyl CoA (C6) up to tetracosanoyl-CoA (C24) as starters and produces triketide and tetraketide 2-pyrones as major products after a lactonization reaction (Figure 2f). The enzymes have a strong preference for hydroxylated CoAs over the unsubstituted analogs (e.g., 12-OH-C18-CoA over C18-CoA) and therefore the enzymes are also characterized as hydroxyalkyl 2-pyronesynthases. The requirement of PKSA and PKSB in the biosynthesis of sporopollenin, the main component of mature pollen of seed plants, has been demonstrated (Dobritsa et al., 2010; Kim et al., 2010). In addition to arabidopsis, ASCL proteins have been functionally characterized from rice (Oryza sativa), tobacco (Nicotiana tabacum) and StJohn’s wort (Hypericum perforatum) (Mizuuchi et al., 2008; Dobritsa et al., 2010; Kim et al., 2010;; Wang et al., 2013; Jepson et al., 2014). All angiosperms with the genome sequenced contain these two genes for ASCL proteins. ASCL proteins can be separated into two distinct groups based on whether they have either Ala or Val at position 220/214
13 (arabidopsis numbering). All angiosperm ASCLs can be identified as either Ala-clade or Val-clade proteins (Jepson et al., 2014). Interestingly, the few sequenced gymnosperm genomes (Nystedt et al. 2013; Neale et al., 2014) possess only a single ASCL-gene.
1.2 Type III PKS structure and function
The type III PKS enzyme reaction is initiated by the starter molecule loading to the active site cysteine, which is followed by condensations of malonyl-CoA, polyketide chain elongation and cyclization of the polyketide intermediate by different cyclization reactions. The PKS enzyme utilizes a single catalytic cysteine residue and every chain elongation contains the cleavage of the CoA and the formation of a new C-C bond by insertion of an additional acetate unit to build the CoA linked intermediate (Morita et al., 2007; Abe and Morita 2010).
Especially resolving the crystal structures of CHS from alfalfa (Medicago sativa) (Ferrer et al., 1999) and 2PS from Gerbera hybrida (Jez et al., 2000a) helped to understand the structural and functional basis on the control of polyketide length and selection of the starter substrate in type III PKSs. Type III PKSs possess a common three-dimensional overall structure, consisting of ɲɴɲɴɲ five-layered core and an independent active site in each monomer. The active site is composed of a CoA-binding tunnel, starter substrate binding pocket, and the three conserved residues found from all the known PKSs, Cys164, His303 and Asn336 (M.sativa CHS numbering), which defines the active site (Figure 3). The active site is buried deep inside of each monomer where the substrate enters through the long CoA-binding tunnel. Cys164 initiates the reaction, acting as a nucleophile and attacks on the thioester carbonyl of p-coumaroyl CoA, resulting in transfer of acyl moiety to the cysteine side chain. Asn336 positions the thioester carbonyl from malonyl-CoA near His303 with Phe215, providing a nonpolar environment for the terminal carboxylate. While a mutation in Cys164 can still perform the decarboxylation reaction, mutations in Asn336 and His303 lead to the CHSs inability to decarboxylate malonyl-CoA (Jez et al., 2000b). The elongated starter-acetyl-diketide-CoA is recaptured by Cys164, CoA is released, followed by additional rounds of elongation, resulting in the formation of a final tetraketide intermediate, which undergoes the intramolecular cyclization reaction. Phe215 and Phe265 are situated at the active site entrance serving as gatekeepers, which helps the folding and the internal orientation of the tetraketide intermediate during the cyclization reaction. Mutation in the residue Phe215 leads to the interruption of the polyketide chain elongation at an early stage (Abe et al., 2003; Austin and Noel, 2003; Flores-Sanchez and Verpoorte, 2009; Abe and Morita 2010).
Figure 3. Suggested mechanism for the type III PKS enzyme reactions by Abe and Morita (2010): (a) Substrate loading, (b) malonyl-CoA decarboxylation, (c) formation of the diketide, (d) diketide stabilization, (e) malonyl-CoA decarboxylation, and (f) creation of the triketide intermediate.
14 While the residues are conserved in the catalytic triad, residues lining the active-site cavity, critical for the selection of a starter substrate and the number of condensation reactions of malonyl-CoA, are specifically substituted in other type III PKSs. A lot of insight into the role of the active site cavity residues was achieved when the crystal structure of 2PS from Gerbera hybrida was published, a year after the published structure of CHS (Jez et al., 2000a). 2PS shares 74 % amino acid sequence similarity to CHS, but does not accept p- coumaroyl-CoA as a starter, and performs only two condensation reactions with the starter acetyl-CoA, yielding TAL. The crystal structure of 2PS revealed that the enzymes possess the same three-dimensional overall fold, identical catalytic triad, and conserved CoA binding sites. 2PS differs in the residues that influence the selection of the starter substrate and the volume of the active site cavity. The total cavity volume is only one-third of that of CHS from M. sativa. The combination of three amino acid substitutions (Thr197, Gly256 and Ser338 in CHS) (Leu202, Leu261 and Ile343 in 2PS) leads to reduction in the cavity volume and to selection of the small acetyl-CoA instead of the bulky p-coumaroyl-CoA as starter. CHS can be converted to 2PS by generating a triple mutant T197L/G256L/S338I protein, which is functionally identical to 2PS, using acetyl-CoA as a substrate and performing only two rounds of malonyl-CoA condensations, releasing TAL. These results showed for the first time that the active site cavity residues influences the choice of starter molecule, and controls the length of the polyketide (Jez et al., 2000a). Later the role of these residues were confirmed in other studies (Abe et al., 2006; Morita et al., 2007; Morita et al., 2011). The pentaketide synthase (PCS) from Aloe arborescens was converted to an octaketide synthase (OKS) by a single bulky to small amino acid mutation, M207G (Thr197 in CHS). Similarly, OKS G207M mutant abolished its octaketidesynthase activity, producing only a pentaketide 2,7-dihydroxy-5-methylchromone. Furthermore, also the residue 351 (338 in CHS), near the catalytic Cys at the top of the active site cavity, plays an important role determining the polyketide length (Morita et al., 2007). These results demonstrated that two residues (Thr197 and Ser338 in CHS) in the active site cavity has the control on the number of malonyl-CoA condensations. In addition, the residue Gly256 in CHS is at crucial position for loading the starter substrate p-coumaroyl-CoA, and in the enzymes such as in PCS, OKS, 2PS and ALS, which do not accept p-coumaroyl- CoA, the corresponding residues has been replaced with bulky Leu (Ferrer et al., 1999; Jez et al., 2000a; Abe et al., 2006).
1.2.1 Engineering plant polyketides
The functional diversity of plant type III PKSs is derived from small modifications in the active site, which influence the selection of the starter substrate, number of malonyl-CoA condensations and the specific cyclization reaction. Therefore, the type III PKSs possess a great catalytic potential for engineered biosynthesis of plant polyketides using a site directed mutagenesis approach (Lim et al., 2016). Based on the information from the crystal structures of PCS and the M207G mutant, Abe and coworkers (2007) generated a triple mutant F80A/Y82A/M207G of PCS where the two aromatic residues Phe80 and Tyr82, located at the at the bottom of the newly discovered buried pocket, were replaced with the small amino acid Ala. Based on a homology model of the mutant protein, the active site was predicted to extend to four times bigger than in the wild type PCS. Indeed, the mutant enzyme condensed nine molecules of malonyl-CoA to produce an unnatural, novel nonaketide naphtopyrone that was the first demonstration of the production of a nonaketide by a structurally simple type III PKS.
HsPKS1 from Huperzia serrata, which normally catalyzes chalcone formation by the sequential condensations of p-coumaroyl-CoA with three molecules of malonyl-CoA was able to utilize a synthetic nitrogen-containing nonphysiological starter substrate, 2-carbamoylbenzoyl-CoA. The wild type enzyme produced a novel pyridoisoindole product with a 6.5.6-fused ring system after two condensations of malonyl-CoA, while a large to small S348G mutated version of HpPKS1 not only extended the amount of malonyl-CoA condensation reactions up to three, but additionally altered the cyclization mechanism to synthesize a novel, unnatural ring-expanded 6.7.6-fused dibenzoazepine. Interestingly, the dibenzoazepine was biologically active,
15 inhibiting biofilm formation (Morita et al., 2011). Quite recently, another type III PKS was characterized from Huperzia serrata. HsPKS3 enzyme possessed a broad range of substrate tolerance, producing curcuminoids, benzalacetone and quinolones, and additionally unnatural 2-substituted quinolones as well as 1,3-diketones from synthetized starters (Wang et al., 2016). Thus, manipulation of the functionally divergent type III PKSs by nonphysiological substrates combined with structure based engineering provides an efficient method for production of pharmaceutically important compounds in the future.
1.2.2 Combinatorial biosynthesis
In addition to the manipulation of the active site, a combinatorial biosynthesis, where several biosynthetic enzymes are expressed together in heterologous systems, is a powerful method for producing novel polyketides. Katsuyama and coworkers (2007) developed an artificial, three step biosynthetic pathway system for the production of unnatural flavonoids in E.coli. First, the CoA-esters were synthetized from carboxylic acids by 4-coumarate-CoA ligase, which was followed by a PKS step for the conversion of CoA- esters into flavanones by CHS and chalcone isomerase (CHI), and into stilbenes by STS. Finally, the flavanones were further modified by flavone synthase (FNS), flavanone 3ɴ-hydroxylase (F3H) and flavonol synthase (FLS) leading to the identification of 87 different polyketides, including 26 unnatural flavonoids and stilbenes. More recently, Kim et al. (2015) produced long-chain 2-pyrones in yeast using the arabidopsis ASCL enzyme PKSA and a tetraketide 2-pyrone reductase (TKPR1), both enzymes which have been characterized from the sporopollenin pathway (Grienenberger et al., 2010; Kim et al., 2010).
1.3 Type III PKSs in the biosynthesis of plant secondary metabolites
In order to use combinatorial biosynthesis approach to produce industrially or pharmaceutically interesting compounds, all of the enzymes in the biosynthesis pathway should be characterized. Usually the in vitro results of the enzyme activity of type III PKSs are insufficient to tell about their roles in vivo. For example, Aloe arborescens is a medicinal plant shown to be rich in the anthrone glycoside barbaroin, which structurally is an octaketide. Still, the in vitro products of OKS, SEK4 and SEK4b are not found from the aloe plant, which is known to contain significant amounts of octaketide anthrones and anthraquinones. Therefore, the compounds SEK4 and SEK4b are regarded as shunt products produced by the enzymes, because the in vitro reaction is lacking some tailoring enzymes or still unknown factors (Abe et al., 2005a). Similar results have been published in the type III PKS research where the structure of the in vitro product differs from the naturally occurring ones since the in vitro reaction is lacking some unknown components (Eckermann et al., 1998, Springob et al., 2007; Jindaprasert et al., 2008; Karppinen et al., 2008; Gagne et al., 2012).
1.3.1 The biosynthesis of 4-hydroxy-5-methylcoumarin (HMC)
Coumarins, structurally 2-pyrones, are found in plants and many of them have been shown to be biologically active compounds. In plants, coumarins act as phototoxins, they can be toxic to herbivores or have allelopathic properties (Bourgaud et al., 2006; Niro et al., 2016). Some of the naturally occurring coumarins have also been reported to have a variety of biological activities such as antitumor activity. One coumarin structured drug, warfarin is available at the market as a blood anticoagulant and as a rat poison (Holbrook et al., 2005; Kawase et al., 2005).
Regardless of the importance of coumarins for the plants and human use, a lot of detailed information is lacking from their biosynthesis. In general, coumarins originate from the phenylpropanoid pathway. The core structure of coumarin (2H-1-benzopyran-2-one) is formed via the ortho-hydroxylation of cinnamic acid followed by cis-trans isomerization of the side chain, and finally by lactonization (Figure 4, Bourgaud et al.,
16 2006; Kai et al., 2008). Coumarins with a methyl group at carbon position 5 are uncommon and have a restricted occurrence in the plant kingdom. They are mainly found from the members of tribes Mutisieae (including gerbera) and the Vernonieae of the Asteraceae family, and sometimes from a few other taxa (Murray, 1997).
Figure 4. Formation of coumarins by ortho-hydroxylation of cinnamic acid, followed by cis-trans isomerization of the side chain, and by lactonization.
Inoue et al. (1989) made radioactive feeding studies with G. hybrida and noticed that labelled acetate and malonic acid were incorporated to 4-hydroxy-5-methylcoumarin (HMC). Labelling pattern suggested that the carbon at position 5 is derived from acetyl-CoA and the acetate units of the pentaketide backbone originate from malonyl-CoA, in contrast to the coumarins derived from coumaric acid. The model (Figure 5) also assumed that a plant polyketide reductase (PKR) would reduce the keto/hydroxyl group at carbon number 7. These kind of reductases have been discussed in many publications (Abe et al., 2005a; Abe et al., 2005b; Springob et al., 2007; Jindaprasert et al., 2008; Karppinen et al., 2008). Two biphenyl synthases (BIS) from Sorbus aucuparia have been reported to produce 4-hydroxycoumarin in vitro from salicoyl-CoA, and in vivo in elicitated cell cultures when fed with salicoyl-N-acetylcysteamine, an analogue of salicoyl-CoA that passes cell membranes. The link between these enzymes to coumarins have remained unclear since the substrate salicoyl-CoA is not naturally present in the elicitated cell culture of S. aucuparia (Liu et al., 2010). Still, in vitro some PKS products have been identified as coumarins, however they were presented in their tautomeric form as 2,7-dihydroxy-5-methylchromones in the publications (Abe et al., 2005a; Karppinen et al., 2008).
Figure 5. A model for the biosynthesis of HMC, modified from Inoue et al. (1989).
More supporting information on HMC being a polyketide derivative, was achieved by generating transgenic gerbera plants which express G2PS1 in an anti-sense orientation. These plants lack any detectable G2PS1 activity leading to radically lowered amounts of gerberin and parasorboside, which are obvious derivatives of TAL, the in vitro product of G2PS1 (Elomaa et al., 1996; Eckermann et al., 1998). Interestingly, also HMC was absent in these transgenic lines. These observations led to two alternative hypothesis about the biosynthesis of HMC in gerbera (Koskela et al., 2011). First, it was possible that G2PS1 would be responsible for the synthesis of both the triketide and pentaketide in vivo. However, the active site of G2PS1 is relatively small (Jez et al., 2000), which may limit the extension of the polyketide into a pentaketide and does not make G2PS1 a suitable candidate for the HMC synthesis. Therefore it was possible that gerbera had other, yet unidentified PKS for the biosynthesis of HMC.
17 1.3.2 The biosynthesis of sporopollenin
The surface of the pollen grain is separated into three distinct layers. The innermost layer, intine is composed of pectin, cellulose and hemicellulose, giving the structural integrity to the pollen grain. The outer layer is known as exine, a multilayered complex structure made up of a physically and chemically resistant biopolymer called sporopollenin. The pollen grain is also covered by a pollen coat, composed of proteins, lipids, pigments and aromatic compounds, which fill the cavities of exine. The exine serve as a barrier against dehydration, pathogen attack and UV irradiation and assists the pollination by interacting with the female stigmatic cells. To the present knowledge, the exine components are synthesized in the adjacent sporophytic tapetal cells and transferred to the surface of developing microspores within the locule (Edlund et al., 2004; Persson et al., 2007; Ariizumi and Toriyama, 2011; Quilichini et al., 2015). The high resistance to physical and chemical degradation has prevented resolving of the exact structure of sporopollenin, but fatty acids and phenolics have been thought to act as components (Ahlers et al., 1999; Guilford et al., 1988). Additionally, information about the biosynthesis of sporopollenin has been unclear. Recent studies concerning the fatty acid biosynthesis-related genes have brought new insights to the sporopollenin biosynthesis (Morant et al., 2007; de Azevedo Souza et al., 2009; Chen et al., 2011). The involvement of two ASCLs in the biosynthesis of sporopollenin in arabidopsis was shown by two independent research groups through mutation studies. As discussed in section 1.1.5, the enzymes were named as PKSA and PKSB. Single mutations in the genes encoding these proteins resulted abnormal exine pattering, and the double mutation to male sterility (Mizuuchi et al., 2008; Dobritsa et al., 2010; Kim et al., 2010).
Genes encoding enzymes with sequence similarity to CHS and shown to be expressed in the tapetum cells of the anther have been isolated from Brassica napus (Shen and Hsu, 1992), Oryza sativa (Hihara et al., 1996), Nicotiana sylvestris (Atanassov et al., 1998) and Silene latifolia (Barbacar et al., 1997). Atanassov et al. (1998) suggested that ASCLs represent a distinct subfamily of plant type III PKSs. They have similar gene and amino acid sequence and catalytic mechanism to CHS, but differ in substrate preference and therefore are often referred to as anther-specific CHS-like enzymes (ASCLs). Later, also from arabidopsis, two ASCLs, PKSA and PKSB were functionally characterized (see section 1.1.5). Already three years earlier Nishikawa et al. (2005) had found arabidopsis mutants (less adhesive pollen, lap), which had reduced pollen-stigma adhesion and noticed that the patterning of the exine was changed, resulting in structurally weakened pollen grains that easily collapsed in these lines. Later LAP6 and LAP5 were identified as CHS-like genes PKSA and PKSB and at the same time their role in the biosynthesis of sporollenin was confirmed (Dobritsa et al.,2010; Kim et al., 2010). Based on these results and research done with fatty-acyl metabolism enzymes, a model for sporopollenin biosynthesis in arabidopsis was formulated (Figure 6) (Grienenberger et al., 2010; Quilichini et al., 2015). The model suggests that in a reaction sequence localized to the ER, ASCLs work together with cytochrome P450-enzymes (Morant et al., 2007), fatty acyl-CoA synthetase (ACOS) (de Azevedo Souza et al., 2009), and tetraketide 2-pyrone reductases (TKPR1) (Grienenberger et al., 2010) to produce polyhydroxylated tetraketide products, which integrates directly or after modifications into the sporopollenin polymer. The polyhydroxylation of the polyketide has been suggested to provide multiple places for cross-linking the polyketide product and other potential sporopollenin components such as fatty acids and fatty alcohols by ester and ether bonds. This has been used to explain the cross-linked feature of sporopollenin and its resistance to chemical and physical degradation (Scott et al., 2004; Kim et al., 2010). Lallemand and coworkers (2014) proposed the existence of a metabolon consisting of sporopollenin biosynthetic enzymes, where the substrates are efficiently channeled from one enzyme to the other in the pathway.
18 Figure 6. Model for sporopollenin biosynthesis by Quilichini et al. (2015). Fatty acids in the cytosol are combined with CoAs by ACOS5, which is followed by condensation of fatty-acyl CoAs with malonyl-CoAs by PKSA and PKSB to produce tetraketide 2-pyrones in the ER. PKS condensation reactions could happen directly (path c) or after the hydroxylations by CYP450s (path a or b, and d). CYP450 enzymes could possibly hydroxylate free fatty acids. It is also possible that the hydroxylations of the CoA esters by CYP450s could take place directly (path b). Hydroxylation reaction could also happen to the tetraketide product (path e). Polyhydroxylated tetraketides are then reduced by TKPR1 to make tetraketide 2- pyrones possessing multiple hydroxyl groups. Finally these compounds could be transported to the anther locule and to the surface of pollen grain by ABCG26 transporters and/or lipidtransfer proteins (LTPs).
19 1.4 Glycosylation of plant metabolites
Glycosyltransferases (GTs) catalyze the stereospecific and regiospecific transfer of nucleotide-diphosphate- activated sugars to many different kind of molecules such as lipids, proteins, nucleic acids, antibiotics and other low molecular substrates, including secondary metabolites. Usually the activated sugar donor form is UDP-glucose (uridine-diphosphate glucose) but the use of UDP-galactose and UDP-rhamnose have also been reported. Glycosylation is an important, final modification step in the biosynthesis of plant secondary metabolites such as flavonoids, phenylpropanoids, coumarins, steroids and terpenoids. Together with methylation, acetylation and hydroxylation reactions, GTs are increasing the diversity and chemical complexity of plant secondary metabolites (Bowles et al., 2006; Yonekura-Sakakibara and Hanada, 2011; Tiwari et al., 2016). Also in gerbera, the secondary metabolites gerberin, parasorboside and HMC accumulate as glucosidic forms (Koskela et al., 2011).
1.4.1 The function of GTs
Usually GTs are classified based on the amino acid sequence similarity, reaction mechanisms, substrate specifity, three dimensional structure and the type of reaction. The enzymes are classified into 100 families where the family 1 glycosyltransferases (GT-1), usually referred as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom (www.cazy.org/glycosyltransferases). For example, the fully sequenced genome of arabidopsis contains 96 GT sequences, all members of GT-1 (Paquette et al., 2003; Tiwari et al., 2016). GTs categorized to family 1 add sugar residues to diverse nucleophile acceptors, mostly oxygen, carbon, nitrogen and sulfur, and with the exception of C-glycosylating enzymes, all GTs perform heteroatom glycosylation (N- glycosylation, S-glycosylation and O-glycosylation, Figure 7) (Chang et al., 2011). For example, arabidopsis UGT72B1 is a bi-functional enzyme and forms an N-glucosidic bond with 3,4-dichloroaniline and an O- glucosidic bond with 3,4-dihydroxybenzoic acid (Loutre et al., 2003; Brazier-Hicks et al., 2007). Recently, Ferreyra and coworkers (2013) characterized a bi-functional C and O specific glucosyltransferase from Z. mays that was able for the first time form also flavonoid C and O glucosides. In addition, some GTs have the ability to glycosylate both glycoside and aglycone molecules, while some plant GTs catalyze the glycosylation only of the aglycone moiety of secondary metabolites (Lim et al., 2004, Lim E.-K, 2005).
Figure 7. The chemical structures of some glucosylated plant natural products. (a) quercetin 3’-O-glucoside, (b) apigenin 6-C-glucoside, (c) 3,4 dichloroaniline N-glucoside, (d) desulfobenzylglucosinolate.
20 1.4.2 The physiological role of GTs
Similarly as with the type III PKS research, in vitro substrate specificity may provide only some indications of the role of the GTs in planta. First GTs were thought to have limited influence on the physiology of plants but identification and functional characterization many plant GTs have revealed their function in various physiological processes. GTs takes part in many different cellular processes, which include modification of plant hormones, detoxification of biotic toxins and xenobiotics (including herbicides, pesticides and pollutants), and in plant-pathogen interactions. The sugar addition onto a lipophilic acceptor compound changes the acceptor in terms of stability, solubility, bioactivity, subcellular localization and the binding property to other compounds. The glycosylation makes the acceptor more polar and may inhibit the possibility of its free diffusion through lipid bilayers and between intracellular compartments. The sugar addition has also been used to explain the detoxification of exogenous and endogenous toxic compounds. Therefore, GTs play an important role in plant stress tolerance, maintaining cell homeostasis and regulation plant growth (Vogt and Jones, 2001; Loutre et al., 2003; Grubb et al., 2004; Lim and Bowles, 2004; Bowles et al., 2005; Ferreyra et al., 2013). GTs also possess a role in the defense against pathogens. For example, Chong and coworkers (2002) used antisense approach to silence tobacco (Nicotiana tabacum) GTs that had been shown to be upregulated by salicylic acid (SA) and during the hypersensitive response (HR), and in vitro accept hydroxycinnamates and hydroxycoumarins, particularly scopoletin as substrates (Horvath et al., 1996; Fraissinet-Tachet et al., 1998). As a result, the GT activity in plants decreased and the amounts of the glucoside form of scopoletin (scopolin) and, unexpectedly, also the free form, scopoletin were lowered, resulting in transgenic plants more susceptible to Tobacco Mosaic Virus (TMV) infection. More recent studies have shown the involvement of GTs in defense against Pseudomonas syringae in arabidopsis (Langlois- Meurinne et al., 2005; Simon et al., 2014), and the role in detoxification of fungal toxins in stiff brome (Brachypodium distachyon, Pasquet et al., 2016). Interestingly, also the two antifungal compounds gerberin and parasorboside, from the G2PS1 pathway, are not antifungal as glycones but are thought to be precursors or storage forms of the aglycone bioactive molecules in gerbera. In vitro bioassays with these two aglycone forms and HMC, clearly showed the effect of these compounds against Botyritis cinerea and other pathogenic fungi (Koskela et al., 2011). In addition, in G2PS1 antisense gerbera lines, where the amounts of these compounds were drastically lowered, the plants became more susceptible to Botyritis cinerea (Eckermann et al., 1998; Koskela et al., 2011).
1.4.3 Structure of GTs
The first cDNA sequence of a GT, which was involved in plant secondary metabolism was the maize bronze-1 gene, which encoded a flavonol 3-O-glucosyltransferase (Fedoroff et al., 1984). In the following years, many other putative GTs were cloned based on this sequence. With the help of these and the sequence data from animal UDP-glucuronosyl-transferases, a common consensus sequence for all GTs in plant in secondary metabolism was identified. This sequence was called PSPG, and was regarded as a conserved domain found in GTs that are involved in the glycosylation of natural products. (Bairoch, 1992; Hughes and Hughes, 1994; Altschul et al., 1997; Kapitonov and Yu, 1999). The PSPG box is highly characteristic, and with overall sequence identity of 60-80 %, it represents well the GT-1 family enzymes involved in natural product glycosylation (Vogt and Jones, 2000).
GTs are globular proteins, which have been divided into two structural superfamilies, GT-A and GT-B, each having a different C-terminal and N-terminal domain. In 2008, the new GT-C family was established (Igura et al., 2008). GT-A enzymes have two different kind of domains. The C-terminal domain, made largely of mixed ɴ sheets holds the acceptor binding site. The N-terminal domain consists of several ɴ sheets, each flanked by ɲ helices (Rossman folds), recognizes the sugar donor. Enzymes in the GT-B category hold two similar Rossmann-like folds. The C-terminal domain is involved in binding the sugar donor, while the N-terminal holds the acceptor binding site. Most of the plant GTs that take part in natural product glycosylation belong
21 to the GT-B-family (Krauth et al., 2009; Tiwari et al., 2016). While numerous GTs have been isolated in the past years, the problems in their heterologous production, purification and crystallization have made structural studies difficult. Still, some plant GT crystal structures are available. The structure of a flavonoid/triterpene glycosyltransferase was reported from Medicago truncatula, a multifunctional enzyme possessing a structure similarity to GT-B family of proteins, and capable of glycosylating flavonoids, isoflavonoids, and triterpenes (Shao et al., 2005; He et al., 2006). Later the structures of two additional GTs from M. truncula were determined (Li et al., 2007; Modolo et al., 2009b), from Vitis vinifera (Offen et al., 2006), and recently from Clitoria ternatea (Hiromoto et al., 2015), helping to understand the structural basis of GTs.
1.4.4 Protein engineering of GTs and biotechnological applications
Similarly as with the type III PKSs, the structural studies have helped to characterize the amino acids essential for the catalytic mechanism of GTs. With help of public crystal structures and mutation studies, it has been possible to modify donor recognition and to engineer sugar specificity by modifying the catalytic key amino acid residues in the PSPG motif, making GT able to use other UDP-sugars in addition to glucose (Osamani et al., 2008; Noguchi et al., 2009). On the other hand, the acceptor recognition can be modified by manipulating the N-terminal part of the protein for glycosylation selectivity and efficiency. By mutating the key amino acids determining the regioselectivity, it has been possible to change the selectivity of the target molecule, producing differentially glycosylated compounds (He et al., 2006; Modolo et al., 2009a). It is further possible to do domain swapping between two enzymes to generate GT chimeras, with altered preference and specificity of glycosylation (Cartwright et al., 2008; Hansen et al., 2009). Finally, as some GTs possess the ability to catalyze glycosylation and deglycosylation reactions, the feature can also be re-engineered since the key amino acids for the deglycosylation activity have also been determined (Modolo et al., 2007; Modolo et al., 2009a; Modolo et al., 2009b). Using the capability for the reverse reaction for some specific GTs, it would be possible to synthetize activated sugars (Wang, 2009). Interestingly, in contrast, some glucosyltransferases themselves seem to have originated from hydrolytic enzymes (Miyahara et al., 2013; Ishihara et al., 2016)
GTs usually catalyze the reaction in the last biosynthetic steps, making them perfect candidate candidates for protein engineering. From the plant breeding perspective, modification of a flower color and pattern has been an interesting biotechnical aim for years. The genetically engineered pigmentation would be possible since the GTs involved in the blue and red color pigments have been characterized (Fukuchi-Mizutani et al., 2003; Sawada et al., 2005). Also the flavor of the plant can be target for biotechnological modification, since GTs are involved in the stabilization of volatile phenolics and terpenoids, and the glycosylation pattern for example determines the sweetness and bitterness in Citrus species (Kita et al., 2000; Frydman et al., 2004). Finally, GTs take part in the hormone balance of plants. GTs involved in homeostasis of plant hormones would offer new possibilities to manipulate hormone levels, and therefore affect the plant phenotype (Tiwari et al., 2016).
Engineering of the function of a glycosyltransferases is interesting also from the human health perspective. For example, positive health effects of C-glycosylated flavones that have cytotoxic and antioxidant properties have been demonstrated (Nichemenetla et al., 2006; El-Alfy et al., 2011; Xiao et al., 2014). Additionally, the glycosylation of a bioactive compound is an important feature in modifying the pharmaceutical properties of the compound, increasing its solubility and, hence bioavailability (Kren and Martinkova, 2001; La Ferla et al, 2011). Since the organic synthesis of specific glycoconjugates is challenging, the genetic engineering of GTs offers a better alternative for targeted glycosylation. Finally, using a combinatorial biosynthesis approach, glycosylated compounds such as flavones can be produced using heterologous organisms (De Bruyn et al., 2015; Wang et al., 2016; Yang et al., 2016).
22 2. AIMS OF THE STUDY
The aims of this PhD project were to study the biosynthesis of polyketide compounds in gerbera and the involvement of type III PKSs in the biosynthetic pathways. The first aim was to find possible type III PKS that would be responsible for the biosynthesis of HMC. Secondly, I wanted to expand the well characterized type III PKS family from gerbera using transcriptomic data, and study the anther expressed type III PKSs, which by their nucleotide sequence looked like ASCLs. Thirdly, I was interested in finding GTs, which could be involved in the glucosylation of secondary metabolites in gerbera.
3. MATERIALS AND METHODS
The materials and methods used in the publications (I-III) are summarized in Table 1.
Table 1. Materials and methods used in the publications. Method Publication Gerbera callus generation and transgenic plants I Gerbera plant growth I, II, III Genomewalking II GT enzyme assays III HPLC-MS I, II,III In situ hybridization II Mutagenesis PCR I PCR and RT-PCR I, II, III Phylogenetic analysis II, III PKS enzyme assays I, II Protein production and purification I, II, III Protein active site modelling I Quantitative real time PCR I, II Thin layer chromatography (TLC) III Total RNA isolation and cDNA synthesis I, II, III Transient protein expression in tobacco I Virus induced gene silencing III Western blot I
23 4. RESULTS AND DISCUSSION
4.1 Characterization of new 2PS enzymes, G2PS2 and G2PS3 from Gerbera hybrida
Previously Helariutta et al. (1996) showed that based on the screen of genomic libraries, gerbera encodes at least two genes similar to G2PS1, at that time named as GCHS17 and GCHS26. The clones GCHS17 and GCSHS26 shared 84-85 % sequence similarity to G2PS1 at the nucleotide level, so it was hypothesized that they also might be 2-pyrone synthases (Eckermann et al., 1998). Previously, the full length cDNA for GCHS17 was amplified from cDNA made from gerbera leaf. GCHS26 was found to be expressed in roots of gerbera, and the cDNA was PCR amplified from roots. Based on sequence similarity and enzymatic properties described in this study, GCHS17 and GCHS26 were renamed to G2PS2 and G2PS3. The link between these enzymes and HMC biosynthesis came from the observation that transgenic gerbera plants expressing G2PS1 in an anti-sense orientation, showed also downregulation for G2PS2 and G2PS3 genes, and drastically lowered HMC amounts (Elomaa et al., 1996; Eckermann et al., 1998; Koskela et al., 2011). Since the active site of G2PS1 had been described to be relatively small (Jez et al., 2000a), possibly limiting the extension of the polyketide to a pentaketide level, G2PS2 and G2PS3 enzymes seemed better candidates for the HMC biosynthesis.
4.1.1 In vitro enzymatic activity of G2PS2 and G2PS3
G2PS2 and G2PS3 were produced as His-tagged recombinant proteins in E.coli and purified using Ni-affinity chromatography. The enzymatic activity of recombinant proteins were tested in enzyme assays using different CoA-starters with malonyl-CoA. Both enzymes accepted acetyl-CoA, acetoacetyl-CoA, benzoyl-CoA and hexanoyl-CoA as starters, but not p-coumaroyl-CoA. Most of the focus was on acetyl-CoA since it has been suggested by Inoue and coworkers (1989) that HMC is an acetyl-CoA derivative. Both G2PS2 and G2PS3 produced the triketide TAL similarly as G2PS1 (Eckermann et al., 1998), based on comparison of UV-spectrum, retention time, and mass with the reference compound. G2PS2 and G2PS3 also produced an additional compound with an m/z of 209 in MS-ESI-. Based on the mass, fragmentation pattern, and previous publications (Springob et al., 2007; Jindaprasert et al., 2008), the product with m/z of 209 was proposed to be the pentaketide 4-hydroxy-6-(2´,4´-dioxo-pentyl)-2-pyrone (Figure 8). The product was observed in the MS-analysis when malonyl-CoA was used in the assays alone. No HMC was detected in the in vitro assays and compound was not the two ring structured coumarin but a pentaketide 2-pyrone also reported as a PKS product in few other publications (Springob et al., 2007; Jindaprasert et al., 2008).
Figure 8. Model for HMC biosynthesis in Gerbera hybrida and the characterized in vitro and in planta products.
24 4.1.2 G2PS2 and G2PS3 expression in planta
To further study the function of G2PS2 and G2PS3, two in planta methods were used. First, G2PS2 and G2PS3 genes were transiently expressed in Nicotiana benthamiana and the metabolites were analyzed from methanol extracts after ɴ-glucosidase treatments. The UPLC-ESIMS results showed that both G2PS2 and G2PS3 produced a compound with m/z of 191 [M-H]-, which was identified as a 4,7-dihydroxy-5- methylcoumarin (Figure 8) based on the mass, fragmentation pattern and previous publications (Abe et al., 2005a, Karppinen et al., 2008). Function of G2PS2 and G2PS3 was further studied in transgenic gerbera callus overexpressing G2PS2 or G2PS3 under the CaMV 35S promoter. Expression of the transgenes were verified first with RT-PCR and the metabolites were analyzed with UPLC-ESIMS. Based on the MS-ESI- and MS/MS results, the compound found from the transgenic callus (Figure 8) had a m/z of 175, and was identical to the reference compound HMC. Furthermore, the calli overexpressing G2PS2, were regenerated to mature plants and accumulation of HMC was observed in organs, where in wild type plants, the HMC is missing. HPLC analysis of these organs confirmed the accumulation of HMC in scape, bracts and receptacle in transgenic plants only. This indicates that, in contrast to tobacco, gerbera expresses the still unknown reductase which removes the oxygen function at position 7 and takes part of the HMC biosynthesis as the model by Inoue et al. (1989) predicted (Figure 5). The role of such reductases, which are part of different polyketide pathways has been has been discussed in many publications (Abe et al., 2005a; Abe et al., 2005b; Springob et al., 2007; Jindaprasert et al., 2008; Karppinen et al., 2008).
4.1.3 Active site of G2PS2 and G2PS3
The resolved crystal structure of G2PS1 showed that the restricted size of the active site cavity is the reason why the enzymes accepts the small starter molecule acetyl-CoA and performs only two rounds of condensation of malonyl CoA. It has demonstrated that it is possible to convert a CHS to 2PS by changing the three active site amino acid residues to correspond the ones in G2PS1 (Jez et al., 2000a). Therefore, protein modelling and enzyme assays with mutated proteins were used to study why G2PS2 and G2PS3 are able to make a pentaketide product while G2PS1 produces only TAL. Using homology based modelling, the active site of G2PS2 and G2PS3 was modelled against the solved structure of G2PS1. G2PS2 and G2PS3 share with G2PS1 the amino acid residues responsible for CoA binding, starter substrate specificity and the structural residues of the active site cavity (Jez et al., 2000a). The G2PS2 and G2PS3 differ from G2PS1 in the three residues lining the initiation/elongation cavity that controls the size of the polyketide. The roles of these three amino acids have been described in several articles (Jez et al., 2000a; Abe et al, 2005a Abe et al., 2006; Morita et al., 2007; Abe and Morita 2010; Morita et al., 2011). The homology based protein models of G2PS2 and G2PS3 showed that all of the residues forming the active site are identical in G2PS2 and G2PS3 compared to G2PS1, except in two amino acid residues. The L202 and I343 in G2PS1 are substituted by I203 and T344 in G2PS2 and G2PS3. The visualized protein models suggest that the cavity is more open in G2PS2 and G2PS3 and the distance between amino acids I203 and T344 is increased to 7Å, where in G2PS1 the corresponding distance is only 4 Å (Figure 9).
25 Figure 9. Comparison of the active site cavities between G2PS1 (a), G2PS2 (b) and G2PS3 (c) modelled with the reaction intermediate acetoacetyl-CoA. The residues lining the cavity with the catalytic center (Cys-His-Asn) are shown. In G2PS2 and G2PS3, L202 and I343 in G2PS1 are substituted by I203 and T344, which makes the active site cavity more open.
4.1.4 Enzyme activity of mutated G2PS1 and G2PS2
To test the role of these amino acid residues in relation to the enzymes’ activity, in vitro enzymatic activity of mutated G2PS1 and G2PS2 was tested. The amino acids I203 and T344 in G2PS2 were converted to the corresponding ones in G2PS1. I203L substitution did not change the enzymatic activity of G2PS2 when asetyl- CoA was used as a starter. T344I mutation in G2PS2 made the protein unable to produce the pentaketide product but only the triketide TAL. The same result was observed also for the double mutant (I203L, T344I). Similarly, L202 and I343 in G2PS1 were converted to the corresponding ones in G2PS2. As a result, the double mutant of the protein produced the same open pentaketide product as G2PS2, where G2PS1 with single mutations (L202I or I343T) produced only TAL. These results indicates that T344 in G2PS2 and G2PS3 is a crucial amino acid for increasing the active site volume and making the proteins able to perform four condensation reactions with malonyl-CoA. In G2PS1 it was necessary to mutate both amino acids (L202I and I343T) for the pentaketide product formation, proving that also I203 has a role in the pentaketide formation in G2PS2. In fact, a reduced amount of pentaketide product was observed when I203L mutated version of G2PS2 was used in the assays.
4.1.5 Expression patterns of G2PS2 and G2PS3
In addition to the enzymatic properties of G2PS2 and G2PS3, their gene expression patterns were determined. The qRT-PCR results showed that the expression patterns of G2PS2 and G2PS3 genes were different. G2PS2 was expressed mostly in aerial parts of the plant where the accumulation of HMC was observed. No expression of G2PS2 was observed in the roots. Conversely, the expression of G2PS3 was strictly root specific. A clear correlation between the G2PS2 and G2PS3 expression with the HMC accumulation suggested that G2PS3 is responsible for the HMC production in roots, and G2PS2 in aerial parts of the plant, and that HMC is not transported within the plant.
26 4.2 Functional characterization of GASCL1 and GASCL2, two ASCL enzymes from Gerbera hybrida
In addition to G2PS2 and G2PS3, two additional 2-pyrone synthases were characterized that extended the type III polyketide synthase family to eight members in Gerbera hybrida. RNA-sequencing of gerbera anther libraries generated in our laboratory showed expression of two CHS-like genes, which were not previously identified. GASCL1 was full length, but GASCL2 lacked ca. 60 nucleotides from the 5’ of the gene. After genome walking techniques the full length cDNA was amplified from gerbera anthers using gene specific primers. GASCL1 and GASCL2 proteins were orthologous to PKSA and PKSB from arabidopsis, where they have been identified as anther specific chalcone synthase like proteins (ASCLs), involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants (Dobritsa et al., 2010; Kim et al., 2010). The ASCL proteins are believed to be functionally and evolutionally conserved in land plants (Wang et al., 2013; Quilichini et al., 2015), so the hypothesis was that gerbera GASCL1 and GASCL2 belong to this ancient group of type III PKSs.
All angiosperms with a sequenced genome possess these two genes for ASCL proteins, but the first sequenced gymnosperm genomes (Nystedt et al. 2013; Neale et al., 2014) contain only one ASCL gene. All ASCLs can be identified as either Ala-clade or Val-clade proteins on the basis whether they have either Ala or Val at position 220/214 (arabidopsis numbering) (Jepson et al., 2014). Phylogenetic tree constructed with gerbera ASCL gene sequences and other putative or characterized ASCLs showed that the gerbera genes are part of the ASCLs and distinct from non-ASCL type III PKSs. GASCL1 belongs to the Ala-, and GASCL2 to the Val-subclade of ASCLs
4.2.1 In vitro enzyme activity of GASCL1 and GASCL2
GASCL1 and GASCL2 were produced as His-tagged recombinant proteins in E.coli and purified using Ni-affinity chromatography. Enzyme activity of purified GASCL1 and GASCL2 were tested in in vitro assays. Both enzymes accepted acetyl-CoA, hexanoyl-CoA, octanoyl-CoA and stearoyl CoA but not p-coumaroyl-CoA or benzoyl- CoA. When acetyl CoA was used as a starter, GASCL1 produced TAL, where GASCL2 did not show any activity. The result is similar with the arabidopsis PKSA and PKSB enzymes, where only PKSA was able to use short- chain CoA starters (Mizuuchi et al., 2008). With hexanoyl-CoA and octanoyl-CoA, both enzymes performed two rounds of condensation reactions of malonyl-CoA, producing 2-pyrone triketides (Table 2), based on the mass, elemental composition, fragmentation pattern and publications for other ASCLs (Mizuuchi et al., 2008; Jepson et al., 2014).
When stearoyl-CoA was used in the assays, GASCL1 and GASCL2 performed two to three rounds of condensation reactions with malonyl-CoA. Based on the observed m/z values, elemental composition, fragmentation pattern typical to 2-pyrones (loss of CO2) and on previously published (Kim et al., 2010) the products were identified as triketide- and tetraketide 2-pyrones (Table 2). The result is similar as with Jepson and coworkers (2014) who observed that HpPKS1 from Hypericum perforatum was able to produce a tetraketide product when the carbon chain was extended to C14-CoA or C18-CoA. Based on the studies on the biosynthetic enzymes from the sporopollenin pathway, a tetraketide reductase has been characterized, which is reducing the carbonyl function of the tetraketide 2-pyrone but does not act on the triketide. Therefore, it is thought that the tetraketide is the physiological product of the ASCL-enzymes in the sporopollenin pathway (Grienenberger et al. 2010, Quilichini et al., 2015).
27 Table 2. In vitro products in the GASCL1 and GASCL2 enzyme assays. Both enzymes possessed identical activity except that GASCL2 did not produce compound TAL from acetyl-CoA.
Starter Proposed structure of the compound Elemental Retention m/z m/z - - CoA composition time [M-H] [M-CO2-H] (HPLC) (rel. int. to major ion) 15 eV
Acetyl C6H5O3 0.38 125.0234 81.0365 CoA (-)
Hexanoyl- C10H13O3 2.09 181.0878 137.0977 CoA (100)
Octanoyl- C12H17O3 3.65 209.1185 165.1289 CoA (100)
Stearoyl- C22H37O3 8.94 349.2751 305.2848 CoA (22)
Stearoyl- C24H39O4 8.39 391.2855 347.2982 CoA (12)
28 4.2.2 Expression of GASCL1 and GASCL2
The expression of GASCL1 and GASCL2 genes were tested in gerbrera organs. The highest expression was observed in the floral organs, the strongest expression in anthers. No expression was detected outside the flowers. In situ hybridization confirmed the expression of the genes to the tapetal cells of the anthers (Figure 10). Tapetum is the innermost sporophytic layer of the anther and provides both phenylpropanoid and lipidic precursors for the developing microspores. Based on the current understanding, the exine components are synthesized in these surrounding tapetal cells and transferred to the surface of developing microspores within the locule in arabidopsis (Ariizumi and Toriyama, 2011; Wang et al., 2013; Quilichini et al., 2015).
Figure 10. GASCL1 and GASCL2 mRNA were localized to the tapetum of the anthers using gene specific antisense and sense probes. The control (sense) probe did not give any hybridization signal.
29 4.3 Characterization of gerbera GTs
The aim of the third part of thesis was to characterize GTs involved in the anthocyanin pathway or in the glucosylation of the anti-fungal compound, gerberin. First, suitable candidates were selected from the gerbera EST collection (Laitinen et al., 2005), and they were functionally characterized in in vitro assays. Finally, a virus induced gene silencing method (VIGS) was used to downregulate the selected GT genes.
4.3.1 In vitro activity of gerbera GTs
Several glucosyltransferase candidates were produced in a soluble from in E.coli, purified as His-tagged forms using Ni-affinity chromatography. Using UDP-[14C] glucose as a sugar donor, the enzyme activity of purified proteins were tested in enzyme assays with various plant metabolites and results were analyzed using thin layer chromatography (TLC). One GT, named Gh14h had highest activity against luteolin, eriodictyol, esculetin and quercetin, which all have a dihydroxylated B-ring structure. For Gh21c, the preferred acceptor seemed to be pelargonidin, and it also showed some activity towards apigenin, eriodictyol, kaempferol and umbelliferone. The most interesting candidate was Gh9e, which was able to transfer glucose to several gerbera metabolites including naringenin and pelargonidin. Most interestingly, TAL and gerberin aglycones were among the acceptor molecules.
4.3.2 Virus induced gene silencing of glucosyltransferases
Using Virus induced gene silencing (VIGS), the aim was to silence the functionally characterized GTs and to study how down regulation affects the balance between glycone and aglycone forms of the metabolites, mainly in the G2PS1 pathway. A gene fragment with the size of 300 to 400 bp was cloned into a TRV2 vector, transformed to agrobacterium and the plants were vacuum treated for gene silencing. Gh9e was selected for experiments, since in vitro it was able to glucosylate TAL and gerberin aglycone. In addition another candidate, Gh16e, which could not be produced in a soluble form in E.coli, was chosen due to high nucleotide sequence similarity (83 %) to Gh9e. For Gh9e and Gh16e, two separate VIGS-constructs and a common construct were designed. For some reason the cloning of the Gh16e specific construct was not successful.
After growing the plants for five weeks, plant leaves were ground, divided in two parts and the expression of selected GTs was analyzed. Sample collection was always done based on the assumption that the GT-VIGS construct spreads similarly as the PDS-VIGS construct, which causes the bleaching of leaves (Deng et al., 2012). The co-infiltration of the PDS and the GT-VIGS-construct was not possible, since based on the observations made in this laboratory, the silencing of the PDS gene causes downregulation of G2PS1 for unknown reason. When the specific Gh9e construct was used, no down regulation was seen in RT-PCR compared to the untreated controls. When the construct 9e_16e was used, the expression of the Gh16e remained unchanged but obvious downregulation of Gh9e was observed in the leaves. This may be caused by the fact that the VIGS-construct was 100 % identical to Gh9e but shared only 90 % similarity to Gh16e. Still, only 23 nucleotides should be enough to achieve silencing in the VIGS-method but longer homology results better silencing (Brigneti et al., 2004).
The other half of the ground leaf was extracted with methanol and analyzed by HPLC. Based on the assumption, downregulation of a GT (Gh9e or Gh16e) would lead to detection of TAL or gerberin aglycones at the expense of the glycosides due to the in vitro results. In samples, where the downregulation of the desired gene was observed none of the compounds could be detected, and only the glucosylated gerberin peak was observed in the chromatograms. As previously described, GTs are regiospecific enzymes, not compound specific (Bowles et al., 2006; Tiwari et al., 2016). Therefore, it is possible that the Gh9e, which in
30 vitro was able to act on TAL and gerberin, has a different physiological role in gerbera. It is also possible that there are several GTs expressed in the gerbera possessing a similar function to Gh9e, including Gh16e. Blast search of a recently completed gerbera transcript assembly for GTs using the 96 arabidopsis GT sequences yielded 461 putative gerbera GTs, which have potential activity in gerbera secondary metabolism. Among these, there were five additional putative GTs, which shared over 80 % amino acid sequence similarity with Gh9e. Therefore it is quite likely that there is redundancy among these enzymes
31 5. CONCLUSION AND FUTURE PROSPECTS
In this study, the gerbera type III PKS family was extended first by functionally characterizing the G2PS2 and G2PS3. Although the presence of these genes (GCHS17 and GCHS26) were discovered already in the 1990s, it took a while to find where they are expressed and to functionally characterize them as type III PKSs. Especially the work done with G2PS1, characterized as the first type III PKS utilizing acetyl-CoA as a starter molecule (Eckermann et al., 1998) made the ground work for identifying G2PS2 and G2PS3 as 2-pyrone synthases. Also the work done with gerbera feeding studies (Inoue et al., 1998) and the gerbera G2PS1 antisense lines (Elomaa et al., 2003; Koskela et al., 2011), which were lacking HMC, helped to build the hypothesis that HMC would be the product of these two PKSs. Functional characterization of an enzyme in heterologous systems is a powerful tool to examine its function in vitro. Still, it gives only a limited information of the in vivo role. Therefore with the help of two in planta methods, the involvement of G2PS2 and G2PS3 in the biosynthesis of HMC was shown, and the unusual route to coumarins in plants was demonstrated in enzymatic terms. Further studies are needed to study why in vitro the enzymes produce an open pentaketide. Second, an enzyme for catalyzing the reduction step in the route to HMC should be characterized. In the second part, two ASCL proteins were characterized, which furthermore extended the gerbera type III PKS family. Although the involvement of GASCL1 and GASCL2 in the biosynthesis of sporopollenin could not be shown, the enzymes were characterized as long-chain 2PS enzymes and a strict tapetum specific expression was observed. This study also extended the number of vascular plant species to five, where the ASCLs have been enzymatically characterized. Further studies would be needed to examine the enzymes’ preference for hydroxylated long chain CoAs as a starters, as it has been described in other plant species (Kim et al., 2010; Wang et al., 2013; Jepson et al., 2014). In the study of the gerbera GTs, no specific GT for the G2PS1 pathway could be uncovered, although several active GTs were produced in E.coli and their function in transferring UDP-glucose to several gerbera secondary metabolites was demonstrated. The VIGS method is a powerful method for silencing genes in plants as it has been described with gerbera in earlier studies (Deng et al., 2012; Deng et al., 2014). In this study it was not possible to observe any visible phenotype in plants where the GTs were silenced, which made the experiment challenging. As GTs act regiospecifically more than substrate dependently, it is difficult to say anything conclusively about their function in vivo, based on the in vitro results. Therefore, some more suitable method for characterizing GTs from the G2PS1 pathway is needed. Especially, if the missing reductase from the G2PS1 pathway could be found, it would be possible to make an in vitro assay with the synthase, reductase and a GT.
32 6. ACKNOWLEDGEMENTS
This work would have not been possible without great people working aside me. Mostly I am grateful to my supervisor Teemu Teeri for your guidance and inspiration towards scientific work. Quite rarely, I have seen people with so much passion towards scientific work. Being strict and accurate towards science and work, but having a relaxed attitude for other things, makes you an ideal supervisor. Secondly, I want to acknowledge my deepest gratitude to PhD Milla Pietiäinen, who helped me especially in the beginning of my work, and participated in writing a great scientific paper. Without your help, I guess I would be still in the lab. I also want to thank my follow-up group member Jussi Joensuu, who gave me great advices to the PhD work. This thesis would have not been possible without the great technical staff in our lab. Therefore I am deeply grateful to our laboratory technicians Marja Huovila, Anu Rokkanen and Eija Takala for the great technical knowledge and skills. In addition, I want to thank all the other staff in the department and in the greenhouse; especially Marjo Kilpinen and Sanna Peltola. Aarno Pelkonen, Arto Nieminen and Kaj-Roger Hurme from the University Instrument Centre are also thanked for great technical advices. Nina Sipari from the metabolomics unit is thanked for the MS-analysis. I also want to thank the DPPS coordinator Karen Sims-Huopaniemi, who helped with many practical issues during the PhD. I want to thank Anna Kärkönen for organizing the phenolics meeting, which helped to give new ideas to the work. From the VTT, Hannu Hotti, Suvi Häkkinen and Heiko Rischer are thanked for organizing the PKRED meetings. Finally I want to thank my girlfriend Emma for being there for me when there has been problems and challenges at work. In addition, I want to thank my son Noel for cheering up me with your funny stories, when picking up you from the kindergarten after the work days.
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