# Patent

## Title
Primer for amplifying farnesyl pyrophosphate synthase from mango

## Abstract

The present invention discloses primers for amplifying farnesyl pyrophosphate synthase gene, having sequence selected from the group consisting of Seq Id. nos. 1-3 and 5-7, from mango. Also disclosed herein is a novel nucleotide sequence of sequence ID no. 8 encoding said amplified farnesyl pyrophosphate synthase (FPPS) for enzyme production in an artificial system thus generating the desired flavor in food products.

## Background
PRIORITY CLAIM TO RELATED APPLICATIONS
This application is a U.S. national stage application filed under 35 U.S.C. §371 from International Application Serial No. PCT/IN2013/000071, which was filed Feb. 1, 2013, and published as WO 2013/114406 on Aug. 8, 2013, and which claims priority to India Application No. 0303/DEL/2012, filed Feb. 3, 2012, which applications and publication are incorporated by reference as if reproduced herein and made a part hereof in their entirety, and the benefit of priority of each of which is claimed herein.
FIELD OF THE INVENTION
The present invention relates to primers for amplifying farnesyl pyrophosphate synthase gene from mango. The invention further relates to a nucleotide sequence encoding said amplified Farnesyl Pyrophosphate Synthase (FPPS) for enzyme production in an artificial system thus generating the desired flavor in food products.
BACKGROUND OF THE INVENTION
Terpenoids form the largest class of plant secondary metabolites, comprising more than 55000 compounds. These molecules play very important ecological and physiological functions in plant life including attraction of pollinators, seed disseminators and predators of herbivores, repulsion of herbivores and pathogens, photosynthetic pigments, protein management (prenylation and ubiquitination) and growth regulation (gibberellins) (Vandermoten et al., 2009). In spite of such vast diversity in structure and function, all the terpenoids are derived from the common C5 precursors: dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). These two building blocks are condensed together by a group of enzymes known as isoprenyldiphosphate synthases or short-chain prenyltransferases. Addition of one molecule of IPP to DMAPP yields geranyl pyrophosphate (GPP, C10). Sequential addition of two IPP units to DMAPP results in the production of farnesyl pyrophosphate (FPP, C15); whereas, addition of three IPP molecules to DMAPP yields geranylgeranyl pyrophosphate (GGPP, C20) (Vandermoten et al., 2009). These prenyl pyrophosphates formed by isoprenyldiphosphate synthases are the substrates in turn for terpene synthases, which produce the parent carbon skeletons of the terpenes. Being rich in the terpene flavorants, mango forms an appropriate system to study biosynthesis and regulation of monoterpenes. Farnesyl pyrophosphate synthase (FPPS) is one of the central enzymes in the terpenoid biosynthetic pathway in mango. The genes encoding FPPS have been isolated and characterized from several plant genera such as Lupinus (Attucci et al., 1995), Parthenium (Pan et al., 1996), Oryza (Sanmiya et al., 1997), Lycopersicon (Gaffe et al., 2000; Sallaud et al., 2009) Ginkgo (Wang et al., 2004), Centella (Kim et al., 2005), Taxus (Liao et al., 2006), Picea (Schmidt and Gershenzon, 2007), Chimonanthus (Xiang et al., 2010) and Panax (Kim et al., 2010). The most intensive work on FPPS has been carried out with Arabidopsis (Cunillera et al., 1996; Cunillera et al., 1997, 2000; Delourme et al., 1994), (Masferrer et al., 2002) (Closa et al., 2010; Manzano et al., 2006) and Artemisia (Chen et al., 2000) (Han et al., 2006) (Hemmerlin et al., 2003) (Matsushita et al., 1996) (Banyai et al., 2010) (Zhao et al., 2003). These and other studies provide evidence on the importance of FPPS in providing the precursor (FPP) for sesquiterpene volatiles and also for the higher terpenoids such as dolichols, phytoalexins, sterols, ubiquinones, farnesylated proteins and prenylatedheme group of cytochrome a and a3 (Chappell, 1995; Weinstein et al., 1986). FPP also contributes to the biosynthesis of carotenoids, chlorophylls, tocopherols, gibberellins and geranylgeranylated proteins, when it is involved in the formation of GGPP (Szkopinska and Plochocka, 2005). In fruit, which produces the whole range of these compounds, FPPS could well acts as a key regulatory point in terpenoid biosynthesis as well as an important player in controlling cell cycle progression, growth, development and general metabolism (Chappell, 1995; Gaffe et al., 2000; Grunter et al., 1994). Being such an important component of fruit physiology and metabolism, it has been hypothesized that FPPS plays a key role in the variable fruit quality that mango (Mangifera indica cv. Alphonso) exhibits among′ localities (Kulkarni et al., 2012). This hypothesis is based on the fact that such variation is prominently observed in taste, flavor and texture, as well as the color of the fruit, and FPP might affect the development of each of these properties. For example, glycosylated sesquiterpenes contribute to the taste and flavor of fruits, volatile sesquiterpenes to the odor, carotenoids to the color and sterols to the texture (Chappell, 1995; Clark et al., 1987; Seigler, 1998).
Article titled “Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein” by Attucci et. al published in Arch Biochem Biophys. 1995 Aug. 20; 321(2):493-500, discloses the molecular cloning, expression, and purification of Farnesyl pyrophosphate synthase from white lupin. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The deduced amino acid sequence of lupinfarnesyl pyrophosphate synthase pFPS2 shares 90% and 79% identity with those from Lupinusalbus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human.
Article titled “A cDNA Encoding Farnesyl Pyrophosphate Synthase in White Lupin” by Attucciet. al. published in Plant Physiol. (1995) 108: 835-836 discloses a method of isolating DNA encoding farnesyl pyrophosphate synthase from White Lupin. A λZapIIcDNA library was constructed from poly(A)+ RNA extracted from 10-day-old seedling roots of white lupin (Lupinusalbus). A cDNA clone, pFPS1, which contained an insert of 1354 base pairs, was selected and sequenced. The deduced amino acid sequence of the encoded protein has 80% identity with and 90% similarity to that of FPP synthase identified from A. thaliana (GenBank accession No. X75789).
Article titled “Cloning and sequencing of a cDNA encoding farnesyl pyrophosphate synthase from Gossypium arboretum and its expression pattern in the developing seeds of Gossypium hirsutum” by Liu Chang-Jun et. al. published in Acta Botanica Sinica 1998, Volume 40 (8), 703-710, discloses the isolation of a cDNA encoding farnesyl pyrophosphate synthase from Gossypium arboretum. Nucleotide sequencing revealed that it is a full length cDNA of 1.28 kb and the putative amino acid sequence exhibited 80.7%, 78.9% and 71.6% identities with the FPP synthases of Artimisia annua, Arabidopsis thaliana and Zea mays respectively.
U.S. Pat. No. 6,600,094 titled “Recombinant plant expression vector comprising isolated cDNA nucleotide sequence encoding farnesyl pyrophosphate synthase (FPS) derived from seedlings of sunflower (Helianthus annus)” reports a process of amplifying and sequencing the sunflower FPS cDNAs by using a pair of universal FPS oligonucleotides probe and then expressing the FPS cDNAs in bacterial cells, carrying out functional complementation assay in yeast mutant to verify its function and generating a line of transgenic tobacco plants, in order to observe the influence of the overexpression of sunflower Farnesyl Pyrophosphate Synthase (SFPS) in vivo.
Article titled ‘Expression profiling of various genes during the fruit development and ripening of mango’ by Pandit et. al. published in Plant Physiology and Biochemistry, 48 (2010) explores several flavor related genes along with a few associated to the physiology of developing and ripening in ‘Alphonso’ mango. The temporal and spatial regulation of the genes during development and ripening of ‘Alphonso’ mango has been analysed. Genes implicated in terpenoid metabolism include geranyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase.
As seen from the above disclosures, isolation of farnesyl pyrophosphate synthase (FPPS) which plays an important role in the terpenoid biosynthetic pathway and the nucleotide sequence encoding the same, from mango is not known hitherto and there is a long standing need in the prior art for such sequences. Hence the Inventors have attempted in this research to provide artificial sequences which may be used to impart color, flavor and smell as in natural Alphonso mangoes.
OBJECTIVE OF THE INVENTION
It is therefore the object of the present invention to provide primer sequence for amplifying farnesyl pyrophosphate synthase from mango and to provide a novel nucleotide sequence thereof.
SUMMARY OF THE INVENTION
In accordance with the above, the present invention provides primer sequence for amplifying farnesyl pyrophosphate synthase from mango.
In an aspect, the invention provides an isolated nucleotide sequence encoding farnesyl pyrophosphate synthase (FPPS) from mango and a process of isolating the gene encoding functional farnesyl pyrophosphate synthase from mango.
In another aspect of the present invention a novel nucleotide sequence encoding farnesyl pyrophosphate synthase (FPPS) from mango is elucidated which is useful for enzyme production in an artificial system by appropriately mixing with the mango pulp to generate the desired flavor. The nucleotide sequences are also useful in the flavor industry for semi-biosynthesis of flavors via various approaches such as enzyme immobilization, single cell culture, etc., as well as to improve other varieties of mango.
In another embodiment of the present invention primers useful for amplification of farnesyl pyrophosphate synthase gene of mango wherein the said sequence is selected from the group consisting of Seq ID Nos 1-3 and 4-7.
In yet another embodiment the present invention a forward and reverse gene specific primers of FPPS for amplification of the ends of cDNA by rapid amplification of cDNA ends (RACE).
In still another embodiment of the present invention primers corresponding to the terminal regions of the mRNA which are designed for FPPS and which are used for the PCR amplification with mango cDNA as a template are provided.
In yet another embodiment of the present invention methods of biochemical characterization of the FPPS isolated from mango are provided.



BRIEF DESCRIPTION OF THE DRAWINGS & FIGURES
FIG. 1: Complete open reading frame encoding farnesyl pyrophosphate synthase isolated from mango. (Seq ID 8)
FIG. 2: (a) Alignment of MiFPPS with the most similar sequences of other characterized plant FPP synthases. Five regions which are conserved among the isoprenyldiphosphate synthases (I-V) are indicated in dark cyan colour; FARM (region II) and SARM (region V) motifs are indicated in coral colour and the chain length determining stretch in the region II is indicated in purple colour. NCBI accession numbers of the sequences were: AAP74719 (Artemisia tridentate; SEQ ID NO:9), AAV58896 (Centellaasiatica; SEQ ID NO:10), AAR27053 (Ginkgo biloba; SEQ ID NO:11), AAA86687 (Lupinusalbus; SEQ ID NO:12), AAK63847 (Mentha×piperita; SEQ ID NO:13), AAY87903 (Panax ginseng; SEQ ID NO:14), AAS19931 (Taxus×media; SEQ ID NO:15), NP_001105039 (Zea mays; SEQ ID NO:16) ACA21460 (Piceaabies; SEQ ID NO:17) and Mango (SEQ ID NO:18).
(b) Genomic organization of MiFPPS. Numbers on the top and the bottom indicate sizes (base pair) of the introns and the exons, respectively.
FIG. 3: LC-MS/MS chromatogram of the in vitro assay products formed from IPP and DMAPP with the protein expressed from the empty vector (upper panel), and the purified MiFPPS (middle panel). Chromatogram of standards for GPP, FPP and GGPP is presented in the lower panel.
FIG. 4: Optimum temperature, pH and MgCl2 concentration of recombinant MiFPPS. For each parameter, the maximum activity was set to 1. Letters over each point indicate the significance of ANOVA (p≦0.05) carried out by Fisher's LSD test independently for each of the three parameters; the values having different letters are significantly different from each other.
FIG. 5: Homology model of MiFPPS generated using Avian FPPS (IFPS) as a template.
(a) Overall structure of MiFPPS showing the five conserved regions (I-V) in dark cyan colour and FARM and SARM in the region H and V in coral colour. The helical regions have been labelled by the letters A to J; α-1, α-2 and α-3 are the short helices between helices H and I.
(b) Top-view of the model showing central reaction cavity and part of the structure harbouring the residues involved in the catalysis. Side chains of the aspartate residues in the FARM and SARM are indicated in dark cyan colour, those of the other important residues in the conserved regions are shown in coral colour and that of phenylalanine in the chain-length determining region is indicated in green colour.
FIG. 6: Abundance of MiFPPS transcripts relative to EF1α during ripening of mango fruits from three cultivation localities, Dapoli, Deogad and Vengurle, in India (DAH: days after harvest). Values presented are averages of four independent biological replicates each of which was represented by at least two technical replicates. Letters over the columns indicate the significance of ANOVA (p≦0.05) carried out by Fisher's LSD test for the comparison between localities for each ripening stages; the values having different letter are significantly different from each other. The ANOVA based comparison is independent for each ripening stage and therefore, is represented by different series of letters (0 DAH: A, B, C; 5 DAH: a, b, c; 10 DAH: m, n, o and 15 DAH: x, y, z).



The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. The embodiments as described are not limiting or restricting the scope of the invention.
DETAILED DESCRIPTION OF THE INVENTION
As used herein ‘Farnesyl pyrophosphate synthase’ means and refers to an enzyme that catalyzes formation of farnesyl pyrophosphate.
In an embodiment, the present invention relates to a primer sequence for amplifying farnesyl pyrophosphate synthase having sequence selected from the group consisting of Seq Id. nos. 1-3 and 5-7.
Accordingly, the degenerate primers for amplification of mango cDNA are:









for1:  



(Seq ID-1)



5′- TKGAYTACAAYGTVCCHGGAGG -3′



 



for2:  



(Seq ID-2)



5′- CYCTYGGYTGGYGYATTGAATGG -3′,



 



rev:  



(Seq ID-3)



5′- YTAYTTYTGCCTCTTRTADATYTT -3′






Forward and reverse gene specific primers for amplification of the ends of cDNA by rapid amplification ends (RACE) are as described below:









for



(Seq ID-4)



5′- AGTATTCATTGCCACTTCATTGCCAG -3′,



 



rev 



(Seq ID-5)



5′- ACTTTCATACTCTGCAAACGCACCC -3′






Primers corresponding to the terminal regions of the mRNA which are designed for FPPS and for the PCR amplification with mango cDNA as a template represent the following sequences:









for



(Seq ID-6)



5'- ATGAGTGATTTGAAGTCCAAGTTCG -3',



 



rev



(Seq ID-7)



5'- CTACTTCTGCCTCTTATATATCTTGG -3'






In another embodiment, the present invention discloses an isolated novel nucleotide sequence encoding FPPS derived from mango (NCBI sequence ID: EU 513268).
The complete open reading frame encoding farnesyl pyrophosphate synthase isolated from mango is as shown in FIG. 1.
A cDNA encoding farnesyl pyrophosphate synthase from mango has been isolated and sequenced, and the corresponding amino acid sequence has been deduced. Accordingly, the present invention relates to isolated DNA sequences which code for the expression of farnesyl pyrophosphate synthase, such as the sequence designated SEQ ID No:8 which encodes farnesyl pyrophosphate synthase from mango.
The nucleotide sequence encoding FPPS is useful for enzyme production in an artificial system. This artificially synthesized enzyme can be appropriately mixed with the mango pulp to generate the desired flavor. The nucleotide sequence is also useful for semi-biosynthesis of fla vors via various approaches such as enzyme immobilization, single cell culture, etc., as well as to improve other varieties of mango. Mature raw fruits of mango used in the present invention are collected from Dapoli, Deogad and Vengurle regions of Maharashtra.
According to the present invention, the complete open reading frame encoding farnesyl pyrophosphate synthase isolated from mango is as shown in FIG. 1. The degenerate primers of farnesyl pyrophosphate synthase useful for the amplification of the mango cDNA are:









for1:



(Seq ID-1)



5′- TKGAYTACAAYGTVCCHGGAGG -3′



 



for2:



(Seq ID-2)



5′- CYCTYGGYTGGYGYATTGAATGG -3′,



 



rev:



(Seq ID-3)



5′- YTAYTTYTGCCTCTTRTADATYTT -3′






Forward and reverse gene specific primers of farnesyl pyrophosphate synthase for amplification of the ends of cDNA by rapid amplification of cDNA ends (RACE). The gene specific primers useful for the amplification of the ends of mango cDNA are:









for



(Seq ID-4)



5′- AGTATTCATTGCCACTTCATTGCCAG -3′,



 



rev



(Seq ID-5)



5′- ACTTTCATACTCTGCAAACGCACCC -3′






Primers corresponding to the terminal regions of the mRNA which are designed for FPPS and which are used for the PCR amplification with mango cDNA as a template. The terminal primers are:









for



(Seq ID-6)



5′- ATGAGTGATTTGAAGTCCAAGTTCG -3′,



 



rev



(Seq ID-7)



5′- CTACTTCTGCCTCTTATATATCTTGG -3′






In yet another embodiment, the present invention disclose a process for isolating full-length nucleotide sequence encoding farnesyl pyrophosphate synthase from ripe mangoes designated as MiFPPS (JN388975) (Mangifera indica farnesyl pyrophosphate synthase). The process comprises the following steps:
i. isolating RNA by CTAB method;
ii. treating total RNA with DNase and carrying out reverse transcription to obtain cDNA;
iii. designing degenerate primers for FPPS based on conserved regions in the nucleotide sequences of farnesyl pyrophosphate synthase reported in the NCBI database;
iv. amplifying cDNA using the degenerate primers of step (ii);
v. designing gene specific primers for FPPS based on the sequence of the fragments obtained in step (iv);
vi. amplifying the ends of the cDNA using gene specific primers of step (v) by Rapid Amplification of cDNA Ends (RACE);
vii. designing primers corresponding to the terminal regions of mRNA based on the alignment of 5′ and 3′ RACE fragments with the FPPS sequences reported from other plants; and
viii. amplifying mango cDNA using primers obtained in step (vii) by PCR (polymerase chain reaction) using the Expand High Fidelity PCR System (La Roche, Switzerland) to obtain the full length sequence of mango FPPS (MiFPPS).
The process of isolating full-length sequence of farnesyl pyrophosphate synthase (MiFPPS) from ripe mango fruits comprises isolating RNA by CTAB method, treating isolated RNA with DNase and carrying out reverse transcription of the isolated RNA. Based on the conserved regions in the nucleotide sequences of orthologous farnesyl pyrophosphate synthase reported in the NCBI database degenerate primers are designed. These primers are used for the amplification of cDNA prepared from ripe fruits of mango. This is followed by designing gene specific primers based on the sequence of the fragments obtained by amplification over the cDNA. The gene specific primers are used for amplification of the ends of the cDNA by rapid amplification of cDNA ends (RACE). Based on the alignments of the 5′ and 3′ RACE fragments with the FPPS sequences reported from the other plants, primers corresponding to the terminal regions of the mRNA are designed and are used for obtaining full-length sequence of MiFPPS.
According to the process, after each amplification step, the fragments are eluted from the agarose gel, ligated in a pGEM-T Easy vector and transformed in E. coli cells. Positive colonies are identified by colony PCR and the presence of desired insert is confirmed by sequencing. Sequences are aligned and analysed for the presence of uninterrupted reading frame in the MEGA 4.1 software.
The degenerate primers designed in step (iii) of the process for isolating full-length nucleotide sequence encoding FPPS from ripe mangoes are as follows;









for1:



(Seq ID-1)



5′- TKGAYTACAAYGTVCCHGGAGG -3′,



 



for2:



(Seq ID-2)



5′- CYCTYGGYTGGYGYATTGAATGG -3′,



 



rev:



(Seq ID-3)



5′- YTAYTTYTGCCTCTTRTADATYTT -3′.






The gene specific primers designed in step (v) of the process of isolating full-length nucleotide sequence encoding FPPS from ripe mangoes are as follows;









for



(Seq ID-4)



5′- AGTATTCATTGCCACTTCATTGCCAG -3′,



 



rev



(Seq ID-5)



5′- ACTTTCATACTCTGCAAACGCACCC -3′.






The terminal primers designed in step (vii) of the process of isolating full-length nucleotide sequence encoding FPPS from ripe mangoes are as follows;









for



(Seq ID-6)



5′- ATGAGTGATTTGAAGTCCAAGTTCG -3′,



 



rev



(Seq ID-7)



5′- CTACTTCTGCCTCTTATATATCTTGG -3′






The complete open reading frame (ORF) of MiFPPS (JN 035296) thus obtained is 1029 base pair long and is flanked by 40 untranslated nucleotides at 5′ end up to the first in-frame ATG and 259 nucleotides at the 3′ end including the AATAAAA motif between the stop codon and the polyadenylation sequences. The reading frame encodes a protein of 343 amino acids with a calculated molecular weight of 39.5 kD and an isoelectric point of 5.35. When ends of the cDNAs are amplified by RACE, the obtained fragments show sequence similarity to the 5′ and 3′ ends of full-length FPP synthase genes reported from the other plants. No intervening stop codons are detected upon in silico translation of the DNA sequence constructed from these fragments. These observations suggest that the overlapping 5′ and 3′ fragments obtained represent the complete open reading frame of the M. indica FPP synthase (MiFPPS). The genomic organization of MiFPPS, shows the presence of 11 introns and 12 exons, consistent with that of an Arabidopsis FPP synthase gene, the only previous such gene whose genomic structure has been reported (Cunillera et al., 1996). The length of the gene sequence isolated is 331 base pair.
Accordingly, in an assay when isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate (DMAPP) is used as an allylic substrate, the recombinantly expressed MiFPPS produces farnesyl pyrophosphate (FPP) as its major product. It is well-known that during the biogenesis of isopentenyl pyrophosphates higher than GPP, additions of two or more units of IPP to DMAPP occur sequentially. DMAPP condenses with the first molecule of IPP to produce gerenyl pyrophosphate (GPP) which remains enzyme bound (Elitzur et al., 2010) and this is used as an allylic substrate for the sub-sequential addition of another molecule of IPP. Thus, the detection of trace amounts of GPP (1-3% of the total) during the assay with MiFPPS can be hypothesized to be a consequence of a truncated in vitro reaction, as well as attributed to residual E. coli enzyme. During the enzymatic reaction catalyzed by prenyltransferases, the participation of divalent metal ions such as Mg2+ is necessary for the dissociation of pyrophosphate moiety from the allylic substrate. Mg2+ is also required for the binding of the substrate molecules to the enzyme (King and Rilling, 1977). These facts clearly explain the absence of MiFPPS activity in the absence of MgCl2. A few of the FPP synthases reported till now are at least partially active with Mn2+ as a cofactor instead of Mg2+ (Hemmerlin et al., 2003; Ogura et al., 1985). Strikingly, MiFPPS does not show any activity when Mn2+ is used as a divalent metal ion. Since mango fruits contain a more than 600 fold higher concentration of magnesium as compared to manganese (Malik et al., 2004), the lack of activity with Mn2+ might be explained as an adaptation of MiFPPS to the higher concentration of Mg2+ in the fruits. The absence of activity with the other two divalent cations, Zn2+ and Ca2+ is consistent with most of the isopentenyldiphosphate synthases reported till now. The optimum pH of 7.5 observed for MiFPPS is similar to the near-neutral optimal pH observed for FPP synthase from pumpkin fruit (Ogura et al., 1985), Ricinus communis (Green and West, 1974) and cotton (Widmaier et al., 1980). At the unripe stage, the pH of the mango fruit pulp is highly acidic and there is a sharp increase in fruit pH during ripening (Yashoda et al., 2006) which could result in increased activity of MiFPPS because of shift of the physiological pH towards the optimal range of MiFPPS. The temperature of mango fruits rises almost up to 40° C. because of increased rate of respiration while ripening (Kumar et al., 1990). This balances the increased activity of MiFPPS due to increased pH.
The present invention further provides three-dimensional structure of MiFPPS using avian FPPS as a template. The avian FPPS shows 50% sequence identity with MiFPPS. Accordingly, homology modeling with the avian FPPS as a template is performed and the quality of the generated model is assessed by a Ramachandran plot, which shows the presence, of 98% residues in the allowed region. All isoprenyl diphosphate synthases which carry out additions of IPP units to allylic substrates show the presence of five conserved regions (I-V). MiFPPS contains all of these regions with regions II and V having the first and second aspartate-rich motifs (FARM and SARM), respectively. The aspartate residues in these regions are involved in binding with the pyrophosphate moiety of substrate through Mg2+ bridges (King and Rilling, 1977). The replacement of aspartate residues in these regions with other amino acids results in drastic reduction in enzymatic efficiency (Marrero et al., 1992) (Joly and Edwards, 1993; Song and Poulter, 1994). In the modeled structure of MiFPPS, extension of the carboxylic side chains of these aspartate residues into the substrate binding cleft supports their role in binding the pyrophosphate moieties of the substrate. A stretch of about seven amino acids before FARM forms the chain-length determining (CLD) region of the prenyltransferases, with the amino acids at the fourth and fifth position before FARM deciding the chain-length of the products. If these amino acids are aromatic, their bulky side chains protrude into the activity cavity preventing further chain elongation of the prenyl pyrophosphate product and such enzymes usually synthesize shorter products such as GPP or FPP (Wang and Ohnuma, 1999). On the other hand, in the isoprenyldiphosphate synthases synthesizing longer products, such as geranylgeranyl pyrophosphate (GGPP) synthase, these amino acids are usually smaller (Ohnuma et al., 1996). In the case of MiFPPS, these chain-length determining residues are tyrosine and phenylalanine, and in the modeled structure the side-chain of phenylalanine is observed to protrude into the central cavity supporting the functional characterization of MiFPPS as a farnesyl pyrophosphate synthase. Further, FPPSs are classified into two types based on their chain-length determining mechanisms. In Type I FPPS, two aromatic amino acids in the CLD region are solely responsible for determining the product length and the FARM is constituted by DDX2D. On the other hand, in Type II FPPS, only the 4th amino acid upstream of FARM is aromatic and FARM is formed by DDX4D (Ohnuma et al., 1997). Based on these reports and the sequence analysis, MiFPPS can be classified as a Type I FPPS.
Profiles the transcripts of MiFPPS in order to get insight into the involvement of MiFPPS in the regulation of sequisterpene biosynthesis is provided herein. The transcripts of MiFPPS are profiled through the four ripening stages of mango from Dapoli, Deogad and Vengurle regions of Maharashtra. The transcript profiling of MiFPPS reveals that the profile is consistent through the ripening process for all three localities. Such consistency suggests a key ripening-related role of the MiFPPS protein. The highest expression is observed at 10 days after harvest DAH stage. This is an important stage in the ripening of mango fruits in which the color turns yellowish and fruit softening begins. In tomato, it has been shown that during ripening, especially, during the color-turning stage (between mature green and red) there is a substantial accumulation of sterols (Chow and Jen, 1978; Whitaker, 1988), which has been hypothesized to play a role in the structural changes, in the cell membrane associated with fruit softening. A similar ripening-related accumulation of sterols has also been reported in apple (Galliard, 1968). Although no reports are available for mango, being a climacteric fruit such an accumulation of sterols during ripening is very likely and can explain the rise, in MiFPPS transcripts at the 10 DAH stage. Secondly, in tobacco the overexpression of yeast FPPS resulted in the increased accumulation not only of sterols, but also of carotenoids (Daudonnet et al., 1997). Considering that the carotenoids are the major components of ripe mango color, a role of MiFPPS in this aspect is also likely. The novel nucleotide sequence encoding FPPS may be used to generate a transgenic variety of mango.
INDUSTRIAL ADVANTAGES
Sesquiterpenes represent one of the important classes of flavor and fragrance chemicals. These chemicals are also nowadays being considered for the biofuel applications. The coding sequence of the enzyme, farnesyl diphosphate synthase, characterized in this study can be used for biotechnological production of the recombinant enzyme which can be further used for the production of sesquiterpenes. The degenerate primers described here have been designed by homology-based approach based on the putative gene sequences reported from the other plants. These primers can thus be used for isolating similar genes from the other plants also.
The novel nucleotide sequences of the present invention can be used for enzyme production in an artificial system and later this artificially synthesized enzyme can be mixed appropriately with any desired food product for generating the desired flavor. The nucleotide sequences can also be used for semi-biosynthesis of flavors via various approaches such as enzyme immobilization, single cell culture, etc., as well as to improve other varieties of mango. Also farnesyl pyrophosphate (FPP), which is the product of FPPS, is a starting material for the synthesis of anticancer drugs (for example, artemisin). The dephosphorylated derivative of FPP, farnesol, as well as the downstream biological product of FPP, sesquiterpenes can be used as fragrance materials to impart floral odor. Farnesol also has the potential to be used as a biofuel component.
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EXAMPLES
The following examples are given by way of illustration of a simple portable type charcoal kiln for wood, sticks and leaves, in actual practice and should not be constructed to limit the scope of the present invention.
Example 1
Plant Material
Mature raw fruits of mango were collected from the orchards of Konkan Krishi Vidyapeeth at Dapoli (N17°45′ E73°11′) and Deogad (N16°31′ E73°20′) and from a private orchard at Vengurle (N15°51′ E73°39′). For each of the three localities, fruits were collected from four plants. After harvesting, fruits were put in hay, carried to the laboratory and allowed to ripen at ambient temperature. At intervals of five days, fruits were cut, immediately frozen in the liquid nitrogen and stored at −80° C. until use, giving rise to four measurement points during ripening: 0, 5, 10 and 15 DAH (days after harvest) from each of the three localities.
Example 2
RNA Isolation and cDNA Synthesis
RNA was isolated by CTAB method (Pandit et al., 2007). After treating total RNA with DNase, reverse transcription was carried out over 1 μg of total RNA using Enhanced Avian RT First Strand Synthesis Kit (Sigma, St. Louis, Mo., USA). The cDNA so obtained was used to amplify the MiFPPS transcript fragments using degenerate primers and later to measure the MiFPPS transcript abundance through real time PCR.
Example 3
Isolation of Full-Length MiFPPS
Based on the conserved regions in the nucleotide sequences of farnesyl pyrophosphate synthase genes reported in the NCBI database, degenerate primers (for1: 5′-TKGAYTACAAYGTVCCHGGAGG-3′ (SEQ ID NO:1), for2: 5′-CYCTYGGYTGGYGYATTGAATGG-3′ (SEQ ID NO:2), rev: 5′-YTAYTTYTGCCTCTTRTADATYTT-3′ (SEQ ID NO:3)) were designed. These primers were used for the amplification over the cDNA prepared from ripe fruits of mango. The gene specific primers (for 5′-AGTATTCATTGCCACTTCATTGCCAG-3′ (SEQ ID NO:4), rev 5′-ACTTTCATACTCTGCAAACGCACCC-3′ (SEQ ID NO:5)) designed based on the sequence of the fragments obtained were used for amplification of the ends of the cDNA by rapid amplification of cDNA ends (RACE) using a SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, Calif., USA). Based on the alignments of the 5′ and 3′ RACE fragments with the FPPS sequences reported from other plants, primers corresponding to the terminal regions of the mRNA were designed (for 5′-ATGAGTGATTTGAAGTCCAAGTTCG-3′ (SEQ ID NO:6), rev 5′-CTACTTCTGCCTCTTATATATCTTGG-3′ (SEQ ID NO:7)) and were used for the PCR with mango cDNA as a template. The genomic fragment MiFPPS was isolated by carrying out PCR using the above-mentioned terminal primers over the genomic DNA isolated from mango leaves by the method described earlier (Pandit et al., 2007). After each step of PCR mentioned above, the fragments were eluted from the agarose gel, ligated in pGEM-T Easy vector (Promega, Madison, Wis., USA) and the ligation reactions were transformed in E. coli cells (Top 10, Invitrogen, Carlsbad, Calif., USA). Positive colonies were identified by colony PCR and the presence of desired insert was confirmed by sequencing. Sequences were aligned and analyzed for the presence of an uninterrupted reading frame in the MEGA 4.1 software (Tamura et al., 2007).
The complete open reading frame of 1029 bp was flanked by 40 untranslated nucleotides at 5′ end up to the first in-frame ATG and 259 nucleotides at the 3′ end including the AATAAAA motif between the stop codon and the polyadenylation sequences. The reading frame encoded a protein of 342 amino acids with a calculated molecular weight of 39.5 kD and an isoelectric point of 5.35. The in silico translated amino acid sequence of MiFPPS showed the highest homology with FPPS from Panax ginseng (86% identity) (Kim et al., 2010), Centellaasiatica (85% identity) (Kim et al., 2005) and Heveabrasiliensis (84% identity) (Adiwilaga and Kush, 1996). Alignment of the Mangifera indica FPPS (MiFPPS) with the FPPS sequences from other plants revealed the presence of five regions which are conserved among prenyltransferases (Chen et al., 1994). Out of these, regions II and V contained the first aspartate rich motif (FARM, between amino acids 93 and 97) and the second aspartate rich motif (SARM, between amino acids 232 and 236), respectively which are essential for substrate binding and catalysis (FIG. 2a).
To obtain information about the genomic organization of MiFPPS, PCR was carried out on the genomic DNA of mango using the terminal primers. The resulting fragment of about 3.8 kb was cloned and its sequence was determined by primer walking. The genomic sequence of MiFPPS showed the presence of 11 introns having a total size of 2717 base pairs (FIG. 2b). The shortest and the longest introns had sizes of 87 base pairs and 897 base pairs, respectively. Almost all the introns followed the “GT-AG” rule (Breathnach and Chambon, 1981): the introns began with GT at the 5′ ends and ended with AG at the 3′ end. The length of the gene sequence isolated was 331 base pair.
Example 4
Expression Cloning and Recombinant Expression in E. coli 
The full length sequence of mango FPPS (MiFPPS) was amplified using the Expand High Fidelity PCR System (La Roche, Basel, Switzerland) with the terminal primers described above. cDNA prepared from the ripe fruit was used as template and the resulting fragment was cloned in the pEXP5-CT/TOPO expression vector (Invitrogen). The ligation reaction was transformed in the E. coli strain TOP10F′ (Invitrogen) and the transformants were selected on the LB-agar media containing 100 μg/ml carbenicillin. After confirming the correct orientation of the insert and the presence of an uninterrupted reading frame, the recombinant plasmids were transformed in the BL21 (DE3) pLysS cells (Invitrogen). Five ml of starter culture grown at 18° C. for 48 hours was used as inoculum for the expression in 100 ml LB medium with the Overnight Express Autoinduction System 1 (Novagen, Madison, Wis., USA). Cultures were grown for 24 hours at 18° C. and the pellet obtained after centrifugation was resuspended in the buffer containing 25 mM MOPSO (pH 7.2), 10 mM MgCl2 and 10% (v/v) glycerol and lysed by sonication. The (his)6-tagged recombinant protein was purified by passing the cleared lysate through Ni-NTA resin (Qiagen, Hilden, Germany) following the manufacturer's instructions. Elution was carried out with a buffer containing 250 mM imidazole, 25 mM MOPSO (pH 7.2), 10 mM MgCl2 and 10% (v/v) glycerol. Both crude lysate and the purified protein were checked for the presence of the recombinant protein by SDS-PAGE.
Before actual expression, the deduced amino acid sequence of MiFPPS was analyzed on the PROSO server to predict the solubility of the recombinantly expressed protein in E. coli. The prediction that MiFPPS would be soluble upon heterologous expression with the probability of 0.507 proved correct and a substantial amount of recombinant protein was obtained in the soluble fraction upon expression. Purification of the crude soluble fraction through a Ni-NTA agarose matrix yielded a protein of the expected size.
Example 5
Assay for Enzymatic Activity
In vitro assay for determining the activity of MiFPPS was carried out in a final volume of 200 μl containing about 0.5 μg of the purified protein, 25 mM MOPSO (pH 7.0), 10% (v/v) glycerol, 2 mM DTT, 10 mM MgCl2 and 67 μM of each DMAPP and IPP (Echelon Biosciences, Salt Lake City, USA). In a qualitative test to find the allylic specificity of MiFPPS towards substrate, 67 μM GPP was used as an allylic substrate instead of DMAPP in the separate reaction having the same composition as mentioned above. For the optimum pH determination of the recombinant enzyme, assays were performed in 25 mM MOPSO (pH 6 and 6.5), 25 mM HEPES (pH 7 and 7.5) or 25 mM TRIS (pH 8, 8.5 and 9) containing the other required components as mentioned above. The assays carried out for determining the optimum Mg2+ concentration, contained varied concentrations of MgCl2 along with the other required components as mentioned above. After overnight incubation, the assay reactions were deproteinized by washing with equal volume of chloroform and used directly for LC-MS/MS analysis.
Consistent with the sequence-based characterization of the isolated cDNA as a FPPS, recombinantly expressed MiFPPS produced FPP as its major product. MiFPPS was confirmed to produce E,E-FPP as its main product along with about 1-3% of GPP. The very minute amounts of GPP and FPP detected in the assays with the protein expressed from an empty vector can be attributed to the activity of the enzymes from E. coli (FIG. 3). Neither FPP nor GPP were detected in the enzyme assays with the boiled protein, without protein and without substrates.
The optimum temperature for the activity of recombinant enzyme was found to be 25° C. and more than 75% of the optimum activity was retained at 15° C. and 20° C.; however, the activity was sharply reduced beyond 25° C. (FIG. 4). When the activity of MiFPPS was assayed at varying pH, maximum FPP production was observed at pH 7.5 with retention of more than 80% activity between pH 7 and 9; however, only about 10% of the optimum activity was detected below pH 7. The enzyme also required Mg2+ as a divalent metal ion cofactor for its activity. The optimum MgCl2 concentration was found to be 0.3 mM with 67 μM DMAPP and IPP and the enzyme possessed more than 75% of the maximum activity at 1 mM MgCl2 after which the activity declined slowly. Strikingly, MiFPPS specifically required Mg2+ for catalysis and was found to be inactive with other divalent metal ions such as Mn2+, Zn2+ and Ca2+ in the concentration range of 0.5-12 mM. To test whether the enzyme shows any specificity towards allylic substrates, GPP was added as the allylic substrate instead of DMAPP. It was observed that MiFPPS was able to condense GPP and IPP to produce FPP.
Example 6
LC-MS/MS Analysis
Analysis of isoprenoid pyrophosphates was performed on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, Calif., USA) coupled to an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, Calif., USA). The column used was an Agilent ZORBAX Extended C-18; 1.8 μm, 50×4.6 mm (Agilent Technologies). Mobile phase consisted of 5 mM ammonium bicarbonate in water as solvent A and acetonitrile as solvent B, flow rate was set at 0.8 ml/min and column temperature at 20° C. Separation was achieved using a gradient starting at 0% B increasing to 10% B in 2 min, 64% B in 12 min and 100% B in 2 min keeping it at 100% B for 1 min followed by a change to 0% B in 1 min and keeping it for another 5 min before the next injection. Injection volume for samples and standards was 10 μl. The mass spectrometer was used in the negative electrospray ionization mode. Optimal settings were determined using standards purchased from Sigma-Aldrich. Ion source gas 1 and 2 were set at 60 and 70 psi having a temperature of 700° C., curtain gas was set at 30 psi and collision gas at 7 psi. Ion spray voltage was maintained at −4200 V. Monitored MRM transitions were m/z 312.9/79 for GPP, m/z 380.9/79 for FPP and m/z 449/79 for GGPP. Data analysis was performed using Analyst Software 1.5 Build 3385 (Applied Biosystems).
Example 7
Homology Modeling
The three-dimensional structure of MiFPPS was determined on the CPH model 3.0 server. Avian FPPS (PDB ID IFPS) (Tarshis et al., 1994), which shows 50% sequence identity with. MiFPPS, was used as a template. Ramchandran plot assessment of the structure was carried out on the RAMPAGE server (Lovell et al., 2003). Further quality parameters of the generated model were assessed on a web-based program, ProSA (Sippl, 1993; Wiederstein and Sippl, 2007).
The quality of the generated model was assessed by a Ramachandran plot, which showed the presence of 98% residues in the allowed region. Further evaluation of the structure by ProSA-web yielded a Z-score of −9.46 and negative energy values for all the residues in the energy plot. Root mean square deviation for superimposition of the modeled structure of MiFPPS with the template (avian FPPS) was 0.61 Å. All of these assessments point towards the good quality of the model generated for MiFPPS.
The avian FPPS has 10 helices (A-J). While the structure of MiFPPS at the region corresponding to helix A of avian FPPS could not be determined because of low sequence identity of this region, the other nine helices (B-J) were evident and surrounded the central reaction cavity (FIG. 5a). All the five regions conserved in isoprenyl diphosphate synthases lined the substrate binding cavity. The FARM and SARM regions were present on the opposite walls of the cavity on the D and H helix, respectively. The carboxylate side chains of all six aspartate residues in FARM and SARM were extended into the substrate binding cleft. The side-chain of the phenylalanine residue present before FARM, known to be involved in the chain-length determination of the product, also protruded into the active-site cavity (FIG. 5b).
Example 8
qRT-PCR
Quantitative PCR was performed with Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, Calif., USA). Mango elongation factor 1α (MiEF1α) transcript was used for normalization (Pandit et al., 2010). Primers used for amplifying a fragment of MiFPPS transcript were: for 5′-TGGGAAAGCAGATCCAGCCTGTGT-3′ (SEQ ID NO: 19) and rev 5′-TGCACTTTCATACTCTGCAAACGCA-3′ (SEQ ID NO: 20). For MiEF1α the primers used were same as described earlier (Pandit et al., 2010). For both MiFPPS and MiEF1α at least three amplicons were sequenced to confirm the primer specificity. Transcript abundance was quantified with a Mx3000P Real Time PCR Thermocycler (Stratagene, La Jolla, Calif., USA) using a program with 45 cycles of 95° C. for 30 seconds, 63° C. for 30 seconds and 72° C. for 30 seconds, followed by a melting curve analysis of transcripts. The relative transcript abundance of MiFPPS for the raw stage (0 DAH) from Dapoli was considered 1 and the fold difference for the rest of the tissues was calculated. Each measurement was repeated with four independent biological replicates, each of which was represented by at least two technical replicates.
When the ripening stages were compared to each other, the level of expression of MiFPPS for all three localities was similar for the raw (0 days after harvest) and 5 DAH fruits. Such consistency suggested a key ripening-related role of the MiFPPS protein. The highest expression was observed at 10 DAH stage. About 6.6, 7.6 and 16 fold increase in the expression was observed at the 10 DAH stage as compared to the 0 DAH stage for Vengurle, Deogad and Dapoli, respectively (FIG. 6). This is an important stage in the ripening of mango fruits in which the color turns yellowish and fruit softening begins. Although the transcripts were present in the higher amounts in the ripe (15 DAH) stage than the raw stage, the levels were reduced by about half in comparison with 10 DAH, at all three localities. When the localities were compared to each other for the expression level of MiFPPS, at 0 and 15 DAH the highest expression was depicted by the Deogad fruits; whereas, at 5 and 10 DAH, the maximum expression was shown by the Dapoli fruits (FIG. 6). Though the MiFPPS expression pattern through ripening was similar for all three localities, only the correlation between Dapoli and Vengurle was significant at p<0.05
Example 9
Statistical Analysis
All the statistical analyses were carried out using the Statview software, version 5.0 (SAS Institute Inc., Cary, N.C., USA). ANOVA was carried out to assess the significance of the difference in the relative transcript levels of MiFPPS between the ripening stages and the difference in the relative activity of the recombinant enzyme at different reaction conditions, by Fisher's LSD at p<0.05. For every two cultivation localities, the relative transcript abundance profiles of MiFPPS through the ripening stages were correlated.

## Claims

1. A population of degenerate primers for amplifying farnesyl pyrophosphate synthase, wherein the population has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

2. The population of claim 1 where the population comprises SEQ ID NO:1 and SEQ ID NO:3.

3. The population of claim 1 where the population comprises SEQ ID NO:2 and SEQ ID NO:3.

4. The population of claim 1 where the population comprises SEQ ID NO:1.

5. The population of claim 1 where the population comprises SEQ ID NO:2.

6. The population of claim 1 where the population comprises SEQ ID NO:3.