U.S. patent application number 10/781724 was filed with the patent office on 2005-04-28 for rhamnosyl-tranferase gene and uses thereof.
Invention is credited to Eyal, Yoram, Fluhr, Robert, Gressel, Jonathan.
Application Number | 20050089882 10/781724 |
Document ID | / |
Family ID | 11072405 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089882 |
Kind Code |
A1 |
Gressel, Jonathan ; et
al. |
April 28, 2005 |
Rhamnosyl-tranferase gene and uses thereof
Abstract
An isolated polynucleotide comprising a nucleotide sequence
encoding a polypeptide having a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase catalytic
activity and its uses.
Inventors: |
Gressel, Jonathan; (Rehovot,
IL) ; Eyal, Yoram; (Shimshon, IL) ; Fluhr,
Robert; (Rehovot, IL) |
Correspondence
Address: |
SOL SHEINBEIN
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
11072405 |
Appl. No.: |
10/781724 |
Filed: |
February 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10781724 |
Feb 20, 2004 |
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09889738 |
Jul 20, 2001 |
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6734343 |
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09889738 |
Jul 20, 2001 |
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PCT/IL00/00038 |
Jan 20, 2000 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12N 9/1051 20130101;
C12N 15/8243 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 22, 1999 |
IL |
128193 |
Claims
1-47. (canceled)
48. A commercial process of producing neohesperidin comprising the
steps of: (a) obtaining hesperidin; (b) treating said hesperidin
with a hesperidinase, thereby obtaining hesperetin-7-glucoside; and
(c) treating said hesperetin-7-glucoside with a
flavanone-7-O-glucoside-2"-O-rhamnosyl- -transferase in the
presence of activated rhamnose, thereby obtaining
neohesperidin.
49. The commercial process of claim 48, wherein said activated
rhamnose is selected from the group consisting of UDP-rhamnose or
dTDP-rhamnose.
50. The commercial process of claim 48, wherein said hesperidinase
is immobilized on a solid support, whereas said hesperidin is
treated with said hesperidinase while passing over said solid
support.
51. The commercial process of claim 48, wherein said treatment of
hesperetin-7-glucoside with a
flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase in presence of
activated rhamnose of step (c) is effected in vivo by a cell
genetically modified to overexpress said
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase, which said cell
is producing activated rhamnose and is capable of intake of said
hesperetin-7-glucoside.
52. The commercial process of claim 51, further comprising the step
of extracting said neohesperidin from said cell.
53. A commercial process of producing neohesperidin dihydrochalcone
comprising the steps of: (a) obtaining hesperidin; (b) treating
said hesperidin with a hesperidinase, thereby obtaining
hesperetin-7-glucoside; (c) treating said hesperetin-7-glucoside
with a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the
presence of activated rhamnose, thereby obtaining neohesperidin;
(d) treating said neohesperidin with an alkali, thereby obtaining
neohesperidin chalcone; and (e) reducing said neohesperidin
chalcone, thereby obtaining neohesperidin dihydrochalcone
54. The commercial process of claim 53, wherein said activated
rhamnose is selected from the group consisting of UDP-rhamnose or
dTDP-rhamnose.
55. The commercial process of claim 53, wherein said hesperidinase
is immobilized on a solid support, whereas said hesperidin is
treated with said hesperidinase while passing over said solid
support.
56. The commercial process of claim 53, wherein said treatment of
hesperetin-7-glucoside with a
flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase in presence of
activated rhamnose of step (c) is effected in vivo by a cell
genetically modified to overexpress said
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase, which said cell
is producing activated rhamnose and is capable of intake of said
hesperetin-7-glucoside.
57. The commercial process of claim 56, further comprising the step
of extracting said neohesperidin from said cell prior to treating
said neohesperidin with said alkali.
58.-83. (canceled)
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention relates to a nucleic acid encoding a
1-2-rhamnosyl-transferase and uses thereof. More particularly, the
present invention relates to a multistep process of converting
hesperidin from orange peels to the sweetener neohesperidin
dihydrochalcone (NHDC), in which, in one of the steps,
1-2-rhamnosyl-transferase in the presence of activated rhamnose is
used in a rhamnosylation reaction to convert hesperidinase-treated
hesperidin (H7G) to neohesperidin. Further particularly, the
present invention relates to genetically modified plants of the
Citrus genus including an antisense or sense (for co-suppression)
construct which comprises the above nucleic acid or a gene
knock-out integrated construct to provide less bitter grapefruits,
pomelos and other citrus containing bitter flavanoid
glycosides.
[0002] The bitter flavanones naringin and neohesperidin (FIG. 1)
are produced only in young leaves and fruits of a few citrus
species, such as grapefruit and pomelo, accumulate in a brief few
week period, and remain through maturity (Castillo et al., 1992).
Isomerically structured, yet tasteless flavanones, such as
hesperidin (FIG. 1) are produced in oranges at the same time in
development (Castillo et al., 1993). The differences between the
tastelessness of orange hesperidin and the bitterness of grapefruit
and pomelo naringin and neohesperidin depend on a specific set of
glycosylation reactions (glucosylation and rhamnosylation) as
further detailed hereinunder.
[0003] Low consumption of grapefruit is due in part to the bitter
flavor of its juice and flesh (Matthews et al., 1990). This
bitterness is due to the presence of large amounts of the flavanone
glycoside naringin, as well as limonin in the juice. While limonin
is a problem limited to the juice (Guadagni et al., 1973), in the
intact fruit it appears in the tasteless A ring monolactone and
forms the bitter dilactone only after maceration in the acidic
juice environment (Matthews et al., 1990).
[0004] Thus, bitterness in the fruit can be decreased by reducing
naringin levels. The bitterness in commercially prepared grapefruit
juice is presently diminished to levels more acceptable by
consumers by using resins that adsorb some bitter compounds or by
treating the juice with the immobilized enzyme naringinase (Jimeno
et al., 1987; Nikdel et al., 1989).
[0005] Commercial naringinase preparations typically consist of two
enzymes, mainly .alpha.-rhamnosidase and some .beta.-glucosidase,
which successively hydrolyze one or both the sugar groups from
naringin, leaving the tasteless compound prunin
(naringenin-7-O-glucoside). Removal of the terminal rhamnose
removes about 95% of the bitterness. Evidently, this procedure does
not solve the problem of bitterness of whole fruit or home prepared
juice.
[0006] A better approach which also addresses the above problem
would, therefore, be to regulate the levels of naringin within the
plant itself.
[0007] However, because of high heterozygousity of the commercial
citrus varieties, classical plant breeding programs, will be hard
put to yield identical varieties, yet having significantly less
bitter fruit by reducing naringin. It will be appreciated in this
respect that the presently available low naringin varieties (such
as the Texas Red) are also low yielding.
[0008] While reducing the present invention to practice, studies of
the naringin flavonoid metabolism (see below) assisted (i) in
developing novel approaches to the problem; (ii) to further study
the critical glycosylation steps that produce naringin; and (iii)
in using this knowledge to modulate the degree of bitterness.
[0009] Citrus flavonoids ubiquity and biosynthesis: The genus
Citrus contains many flavonoid glycosides that differ either in the
structure of the aglycone or their sugar moieties. The major
flavonoid in pomelo and grapefruit peel is naringin, while sweet
orange peel contains hesperidin (Horowitz and Gentili, 1977 and
FIG. 1). Peels of sour orange, trifoliate orange and Ponderosa
lemon contain neohesperidin (FIG. 1). Neohesperidin and naringin
are flavanone glycosides that contain the same disaccharide,
.beta.-neohesperidose (2-O-.alpha.-L-rhamnosyl-.beta.-D-glucose),
which is attached at position C-7 of different aglycones,
hesperetin and naringenin, respectively. Hesperidin and narirutin
are isomers, respectively, of the above compounds that contain the
disaccharide rutinose
(6-O-.alpha.-L-rhamnosyl-.beta.-D-glucose).
[0010] These flavonoids have some remarkable taste properties.
Naringin and neohesperidin are extremely bitter, while narirutin
and hesperidin are nearly tasteless (Horowitz and Gentili, 1986;
Naim et al., 1986).
[0011] Flavonoids in Citrus: The highest naringin levels are
associated with very young developing leaves and fruit tissue.
Undifferentiated cells of Citrus paradisi (grapefruit) were able to
biotransform exogenous naringenin and hesperetin, into prunin
(naringenin-7-O-glucoside) and hesperetin-7-O-glucose (H-7-G),
respectively. Further 1-2-rhamnosylafion resulting in naringin or
neohesperidin synthesis was not detected, although
1-6-rhamnosylation resulting in narirutin was observed (Lewinsohn
et al., 1986, 1989b).
[0012] All of the above suggests that the blockage in the stepwise
production of the naringenin from prunin (naringenin-7-O-glucoside)
in the undifferentiated Citrus cells is caused by the absence of a
specific rhamnosyl transferase activity.
[0013] Lewinsohn et al. detected chalcone-synthase and UDP-glucose,
flavanone-7-O-glucosyl-transferase activities in cell-free extracts
of Citrus (Lewinsohn et al. 1989a). Partial purification of the
glucosyl transferase has been recently reported and some of its
characteristics are therefore known (McIntosh and Mansell,
1989).
[0014] The glucosylated flavanone was further rhamnosylated.
Chalcone-synthase activity was detected in cell-free extracts
derived from young leaves and fruits.
[0015] Young fruits (2 millimeter diameter) had the highest
chalcone synthase activity. In earlier studies it was shown that
the glycosylation of the aglycone, naringenin, in undifferentiated
Citrus cells occurs in two steps. First there was an initial
glucosylation resulting in prunin (Lewinsohn et al. 1986, 1989b),
and then a further rhamnosylation of prunin occurred when exogenous
UDP-glucose and NADPH were added to the extract forming the
end-product naringin (Lewinsohn et al 1989b).
[0016] Prunin was also shown to be a likely intermediate in the
biosynthesis of naringin in immature grapefruit fruits (Berhow and
Vandercook 1989).
[0017] Several glucosyl-transferases from plants catalyze the
transfer of the sugar moiety from an activated UDP-sugar to a
specific site on the flavonoids (Hahlbrock, 1981). Enzymatic
preparations from plants catalyze the conversion of UDP-glucose to
UDP-rhamnose in the presence of NADPH (Barber, 1962). The
conversion is due to at least three different enzymatic activities
requiring NADH or NADPH. It was not previously known how this
conversion is catalyzed in Citrus. A coupled assay using
UDP-glucose with NADPH was developed, which formed a rhamnosylated
flavanone glycoside, indicative of a
UDP-rhamnose:flavanone-7-O-glucoside- -2'-O-rhamnosyl-transferase
activity (.alpha.-1-2 rhamnosyl transferase). A system was
thereafter developed to directly biosynthesize radiolabeled
UDP-rhamnose from [.sup.14C]-UDP glucose and NADPH for use in
direct assay of the .alpha.-1-2 rhamnosyl transferase activity
during purification of the .alpha.-1-2 rhamnosyl transferase. The
direct assay facilitated the purification of the .alpha.-1-2
rhamnosyl transferase. Once both rhamnosyl transferase substrates
were available it was possible to purify the enzyme. Purity to
homogeneity of the .alpha.-1-2 rhamnosyl transferase in a four step
procedure, which includes S-200 gel filtration, affinity column,
ion exchange-FPLC and reverse-phase HPLC (Bar-Peled et al. 1991)
was achieved and an antibody to it was elicited. These findings
allowed to isolate the gene, which is the subject of this
invention.
[0018] Semi-artificial sweetener: Neohesperidin and naringin from
the peels of sour oranges and grapefruits are being converted into
neohesperidin dihydrochalcone (NHDC), an intense sweetener used in
the EU that is 2,000 fold sweeter than sugar (see right path on
FIG. 1). NHDC is especially useful when long-lasting sweetness is
desired, such as in chewing gum or in admixture with one of the
artificial sweeteners. Currently, world-wide production of this
NHDC is limited by the amount of neohesperidin and naringin
available from these lesser grown Citrus species (less than 10% of
total Citrus cultivated).
[0019] Unfortunately, the vast majority of citrus processed
(oranges) contain large amounts of the hesperidin, an isomer having
the terminal rhamnose attached by a 1.fwdarw.6 linkage instead of
the 1.fwdarw.2 linkage. Hesperidin cannot be converted to the
sweetener and is currently a waste by-product.
[0020] There is thus a widely recognized need for, and it would be
highly advantageous to have, a nucleic acid encoding a
1-2-rhamnosyl-transferase to effect a multistep process of
converting hesperidin from orange peels to the sweetener
neohesperidin dihydrochalcone (NHDC), and to provide genetically
modified plants of the Citrus genus including an antisense or sense
(for co-suppression) construct thereof, or knock-out integrated
construct, to provide less bitter grapefruits, pomelos and other
citrus containing bitter flavanoid glycosides.
SUMMARY OF THE INVENTION
[0021] While reducing the present invention to practice, a novel
rhamnosyl transferase gene from citrus responsible for producing
the bitter flavanoids naringin and neohesperidin was isolated,
sequenced, and characterized. This gene can be used in antisense or
overproducing sense constructs to decrease the bitterness in
grapefruit, and in sense constructs as part of the bioconversion of
hesperidin from orange waste products to neohesperidin, used for
the production of neohesperidin dihydrochalcone (NHDC), a
sweetener. A knock-out construct can be used to knock-out the endo
rhamnosyl transferase gene.
[0022] Thus, according to one aspect of the present invention there
is provided an isolated polynucleotide comprising a nucleotide
sequence encoding a polypeptide having a
flavanone-7-O-glucoside-2"-O-rhamnosyl-tr- ansferase catalytic
activity.
[0023] According to another aspect of the present invention there
is provided a cell genetically modified to include, in an
expressible sense orientation, a nucleotide sequence encoding a
flavanone-7-O-glucoside-2"-- O-rhamnosyl-transferase.
[0024] According to still another aspect of the present invention
there is provided a plant cell of a plant species naturally
expressing a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
messenger RNA, wherein the plant cell is genetically modified to
include, in an expressible antisense orientation, a nucleotide
sequence encoding an antisense RNA molecule being capable of in
vivo base pairing with the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
to thereby render the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
when expressed, amenable to degradation by nucleases present in the
plant cell.
[0025] According to yet another aspect of the present invention
there is provided a transgenic plant of a species naturally
expressing a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
messenger RNA, wherein the transgenic plant is genetically modified
to include, in an expressible antisense orientation, a nucleotide
sequence encoding an antisense RNA molecule being capable of in
vivo base pairing with the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
to thereby render the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
when expressed, amenable to degradation by nucleases present in the
transgenic plant.
[0026] According to a further aspect of the present invention there
is provided a plant cell of a plant species naturally expressing a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase, wherein the
plant cell is genetically modified to include, in an
overexpressible sense orientation, a nucleotide sequence encoding a
sense RNA molecule being capable of inducing a co-suppression
effect to thereby reduce production of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the plant
cell.
[0027] According to yet a further aspect of the present invention
there is provided a transgenic plant of a species naturally
expressing a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase,
the plant is genetically modified to include, in an overexpressible
sense orientation, a nucleotide sequence encoding a sense RNA
molecule being capable of inducing a co-suppression effect to
thereby reduce production of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the plant
cell.
[0028] According to still a further aspect of the present invention
there is provided a transgenic plant or cell of a plant species
naturally expressing a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase, the transgenic
plant or cell being genetically modified to knock-out a gene
encoding said flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase to
thereby abolish production of said
flavanone-7-O-glucoside-2"-O-rhamnosyl- -transferase in the
transgenic plant or cell.
[0029] According to an additional aspect of the present invention
there is provided a commercial process of producing neohesperidin
comprising the steps of: (a) extracting hesperidin from a
hesperidin producing species of the genus Citrus; (b) treating the
hesperidin with a hesperidinase, thereby obtaining
hesperetin-7-glucoside; and (c) treating the hesperetin-7-glucoside
with a flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase in the
presence of activated rhamnose, thereby obtaining
neohesperidin.
[0030] According to yet another additional aspect of the present
invention there is provided a method of modifying a
rhamnose-1-6-glucose linkage of a chemical compound to a
rhamnose-1-2-glucose linkage comprising the steps of: (a)
enzymatically treating the chemical compound to remove a rhamnose
group thereof, thereby obtaining a first derivative of said
chemical compound having a terminal glucose group; and (b)
enzymatically treating the first derivative with recombinant
flavanone-7-O-glucoside-2"- -O-rhamnosyl-transferase in the
presence of activated rhamnose, thereby obtaining a second
derivative chemical compound having the rhamnose-1-2-glucose
linkage.
[0031] According to one further aspect of the present invention
there is provided a commercial process of producing neohesperidin
dihydrochalcone comprising the steps of: (a) extracting hesperidin
from a hesperidin producing species of the genus Citrus; (b)
treating the hesperidin with a hesperidinase, thereby obtaining
hesperetin-7-glucoside; (c) treating the hesperetin-7-glucoside
with a flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase in the
presence of activated rhamnose, thereby obtaining neohesperidin;
(d) treating the neohesperidin with an alkali, thereby obtaining
neohesperidin chalcone; and (e) reducing the neohesperidin
chalcone, thereby obtaining neohesperidin dihydrochalcone.
[0032] The present invention successfully addresses the
shortcomings of the presently known configurations by providing a
nucleic acid encoding a 1-2-rhamnosyl-transferase to effect a
multistep process of converting hesperidin from orange peels to the
sweetener neohesperidin dihydrochalcone (NHDC), and to provide
genetically modified plants of the Citrus genus to provide less
bitter grapefruits, pomelos and other citrus containing bitter
flavanoid glycosides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The invention is herein described, by way of example only,
with reference to the accompanying drawings, wherein:
[0034] FIG. 1 depicts a prior art process (right path) and a
process according to the present invention (left path) of producing
the sweetener NHDC; and
[0035] FIG. 2 shows the sequence of the coding strand of a
1-2-rhamnosyl-transferase cDNA according to the present invention,
and its translation into a protein shown in single letter code.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] The present invention is of a 1-2-rhamnosyl-transferase gene
and the recombinant protein product hereof, which can be used in a
multistep process of converting hesperidin from orange peels to the
sweetener neohesperidin dihydrochalcone (NHDC), and in providing
genetically modified plants of the genus Citrus characterized by
lowered bitterness.
[0037] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0038] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and arrangement
of the components set forth in the following description or
illustrated in the drawings. The invention is capable of other
embodiments or of being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting. Generally, the nomenclature used herein and
the laboratory procedures in recombinant DNA technology described
below are those well known and commonly employed in the art.
[0039] While reducing the present invention to practice, a
polynucleotide was isolated, having a nucleotide sequence encoding
a polypeptide having a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase catalytic
activity. The isolation of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase gene was not at
all trivial. Repeated attempts to isolate the gene via screening of
either conventional cDNA libraries or expression cDNA libraries
using oligonucleotides as well as antibodies raised against
purified flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase failed
to yield positive clones. The successful approach of isolating the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase gene, which
included reverse transcriptase polymerase chain reaction (RT-PCR)
using degenerate primers followed by rapid amplification of cDNA
ends (RACE), is further described in the Examples section that
follows.
[0040] Having the cDNA in hand, screening of genomic libraries can
readily be performed for isolating a genomic clone including a
polynucleotide having introns and exons, the exons of which
correspond to the cDNA sequence or parts thereof, and encode for
flavanone-7-O-glucoside-2"-O-rh- amnosyl-transferase. Both the
complementary and genomic polynucleotides according to the present
invention can be used to direct the synthesis of a messenger RNA
(mRNA) encoding flavanone-7-O-glucoside-2"-O-rhamnosyl-tr-
ansferase.
[0041] According to a preferred embodiment of the present
invention, the isolated polynucleotide, either in its cDNA or
genomic DNA form, wholly or partially synthetic (the term
"isolated" may encompass all these possibilities) is ligated to a
vector, preferably, an expression vector, either in sense or
antisense orientation. The vector preferably selected is
propagatable in plant cells and/or in microorganism cells, such as
in bacteria. The vector can be of a type capable of directing the
integration of sequences thereof into the genome of the recipient
cell. Thus, within the cell, the nucleic acid may be incorporated,
or not, within a chromosome. There may be more than one nucleotide
sequence per haploid genome. This, for example, enables increased
expression. A vector comprising a polynucleotide according to the
present invention need not include a promoter, particularly if the
vector is to be used to introduce the nucleic acid into cells for
recombination into the genome.
[0042] However, as further detailed hereinunder, a promoter is
preferably included in the vector.
[0043] The present invention also encompasses the expression
product of any of the nucleic acid sequences disclosed and methods
of making the expression product by expression from encoding
nucleic acid therefor under suitable conditions in suitable host
cells. Those skilled in the art are well able to construct vectors
and design protocols for expression and recovery of products of
recombinant gene expression.
[0044] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator fragments, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. For
further details see, for example, Molecular Cloning: a Laboratory
Manual: 2nd edition, Sambrook et al. 1989, Cold Spring Harbor
Laboratory Press. Transformation procedures depend on the host
used, but are well known. Many known techniques and protocols for
manipulation of nucleic acids, for example, in preparation of
nucleic acid constructs, mutagenesis, sequencing, introduction of
DNA into cells and gene expression, and analysis of proteins are
described in detail in Short Protocols in Molecular Biology, Second
Edition, Ausubel et al. ed., John Wiley & Sons, 1992. The
disclosures of Sambrook et al. and Ausubel et al. are incorporated
herein by reference.
[0045] According to one embodiment of the present invention the
nucleotide sequence is as set forth in SEQ ID NO:20 or a functional
part thereof. By functional part it is meant a part capable of
directing the synthesis of a polypeptide having a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase catalytic
activity.
[0046] According to another embodiment of the present invention the
nucleotide sequence shares between 50 and 100%, preferably between
60 and 100%, more preferably between 70 and 100%, yet more
preferably between 80 and 100%, still more preferably between 90
and 100% identical bases with SEQ ID NO:20 or a functional part
thereof, as determined using a sequence analysis software package
developed by the Genetic Computer Group (GCG) at the University of
Wisconsin with gap creation penalty of 50 and gap extension penalty
of 3, provided that the nucleotide sequence encodes a polypeptide
having a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
catalytic activity.
[0047] According to yet another preferred embodiment of the present
invention, the nucleotide sequence is hybridizable with SEQ ID
NO:20 or a functional part thereof under stringent hybridization,
moderate hybridization or mild hybridization, wherein stringent
hybridization is effected by a hybridization solution of
6.times.SSC and 1% SDS, hybridization temperature of 65.degree. C.,
final wash solution of 0.1.times.SSC and final wash at 60.degree.
C., moderate hybridization is effected by a hybridization solution
of 6.times.SSC and 1% SDS, hybridization temperature of 58.degree.
C., final wash solution of 0.5.times.SSC and final wash at
50.degree. C., whereas mild hybridization is effected by a
hybridization solution of 6.times.SSC and 1% SDS, hybridization
temperature of 50.degree. C., final wash solution of 2.times.SSC
and final wash at 40.degree. C. Thus, a hybridization according to
the present invention is effected by a hybridization solution of
6.times.SSC and 1% SDS, hybridization temperature of 50-65.degree.
C., final wash solution of 0.1-2.0.times.SSC and final wash at
40-60.degree. C.
[0048] According to a preferred embodiment of the present
invention, the nucleotide sequence encodes a polypeptide as set
forth in SEQ ID NO:21 or a functional part thereof. However, the
polypeptide encoded by the nucleotide sequence of the present
invention can share between 20 and 100%, preferably between 60 and
100%, more preferably between 70 and 100%, yet more preferably
between 80 and 100%, still more preferably between 90 and 100%
identical or conserved amino acids with SEQ ID NO:21 or a
functional part thereof, as determined using a sequence analysis
software package developed by the Genetic Computer Group (GCG) at
the University of Wisconsin with gap creation penalty of 12 and gap
extension penalty of 4, provided that the polypeptide has a
flavanone-7-O-glucoside- -2"-O-rhamnosyl-transferase catalytic
activity.
[0049] A polynucleotide according to various aspects of the present
invention may have the sequence of a
flavanone-7-O-glucoside-2"-O-rhamnos- yl-transferase gene or be a
mutant, variant, derivative or allele of the sequence provided.
Preferred mutants, variants, derivatives and alleles are those
which encode a product (RNA or polypeptide) which retains a
functional characteristic of the product encoded by the wild-type
gene. Changes to a sequence, to produce a mutant, variant or
derivative, may be by one or more of addition, insertion, deletion
or substitution of one or more amino acids in an encoded
polypeptide product. Of course, changes to the nucleic acid which
make no difference to the encoded amino acid sequence are
included.
[0050] In a preferred embodiment of the present invention a
polynucleotide molecule comprises a nucleotide sequence which
encodes an amino acid sequence shown in SEQ ID NO:21. The
nucleotide sequence may comprise an encoding sequence shown in SEQ
ID NO:20 or may be a mutant, variant, derivative or allele thereof
encoding the same amino acid sequence, or a sequence which retains
a functional characteristic of the product encoded by the wild-type
gene.
[0051] Sequences comprising changes to or differences from the
sequences shown may also be employed in the present invention, as
discussed herein.
[0052] According to a preferred embodiment of the present invention
the nucleotide sequence originates from a species of the genus
Citrus, such as grapefruit and pomelo.
[0053] The nucleotide sequence information provided herein or any
part thereof may be used in a data-base search to find homologous
sequences, expression products of which can be tested for
flavanone-7-O-glucoside-2"- -O-rhamnosyl-transferase activity.
[0054] A further aspect of the present invention provides a method
of identifying and cloning a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transfer- ase gene homolog
from a plant species other than pomelo which method employs a
nucleotide sequence derived from SEQ ID NO:20. Nucleic acid
libraries may be screened using techniques well known to those
skilled in the art and homologous sequences thereby identified then
tested. Hybridization may involve probing nucleic acid and
identifying positive hybridization under suitable stringent
conditions (in accordance with known techniques) and/or use of
oligonucleotides as primers in a method of nucleic acid
amplification, such as PCR. For probing, preferred conditions are
those which are stringent enough for there to be a simple pattern
with a small number of hybridizations identified as positive which
can be investigated further. It is well known in the art to
increase stringency of hybridization gradually until only a few
positive clones remain. As an alternative to probing, though still
employing nucleic acid hybridization, oligonucleotides designed to
amplify DNA sequences may be used in PCR reactions or other methods
involving amplification of nucleic acid, using routine procedures.
See for instance "PCR protocols; A Guide to Methods and
Applications", Eds. Innis et al. 1990, Academic Press, New York.
Assessment of whether or not such a PCR product corresponds to a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transfera- se may be
conducted in various ways. A PCR band from such a reaction might
contain a complex mix of products. Individual products may be
cloned and each one individual product may be cloned and each one
individually screened. It may be analyzed by transformation to
assess function on introduction into a plant of interest.
[0055] Thus, included within the scope of the present invention are
polynucleotide molecules which encode amino acid sequences which
are homologs of flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
of pomelo. Homology may be at the nucleotide sequence and/or amino
acid sequence level. Preferably, the nucleic acid or amino acid
sequence of a homolog, or a mutant, allele, derivative or variant
(see above) shares homology with SEQ ID NOs:20 or 21, respectively,
preferably at least about 50%, or at least about 60%, or at least
about 70%, or at least about 75.%, or at least about 80% homology,
most preferably at least about 90% homology, and the encoded
product shares a phenotype with the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase gene. "Homology"
may be understood to refer to similarity, in functional terms, in
an amino acid sequence, as is standard in the art. Thus, for
example, a % similarity figure will include amino acid differences
that have little or no functional significance, such as leucine to
isoleucine. Otherwise, homology may be taken to refer to identity.
These definitions are further elaborated hereinabove. Thus, for
example, gene homologs from other members of the genus Citrus may
be identified.
[0056] In certain embodiments, an allele, variant, derivative,
mutant or homolog of the specific sequence may show little overall
homology, say about 20% or about 25%, or about 30%, or about 35% or
about 40% or about 45%, with the specific sequence. However, in
functionally significant domains or regions the amino acid homology
may be much higher. Deletion mutagenesis, for example, may be used
to test the function of a region of the polypeptide and its role in
or necessity for catalytic activity.
[0057] Also according to the invention there is provided a plant
cell having incorporated into its genome a sequence of nucleotides
as provided by the present invention, under operative control of a
promoter for control of expression of the encoded polypeptide. A
further aspect of the present invention provides a method of making
such a plant cell involving introduction of a vector comprising the
sequence of nucleotides into a plant cell and causing or allowing
recombination between the vector and the plant cell genome to
introduce the sequence of nucleotides into the genome.
[0058] The present invention further encompasses a plant comprising
a plant cell comprising nucleic acid according to the present
invention e.g., as a result of introduction of the nucleic acid
into the cell or an ancestor thereof, and selfed or hybrid progeny
and any descendant of such a plant, also any part or propagule of
such a plant, progeny or descendant, including sexually or
apomictically obtained seed and vegetatively propagated plant
material.
[0059] The principal characteristics which may be altered using the
present invention are controlling the bitterness of fruits of
plants of the genus Citrus as further detailed hereinunder.
Over-expression of the gene product of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase gene may lead to
either bitterer fruits or less bitter fruits via a co-suppression
mechanism; under-expression may lead to less bitter fruits.
Down-regulation may be achieved, for example, with "gene silencing"
techniques such as anti-sense or sense regulation, discussed
further below. It can also be achieved with gene knockout.
[0060] This degree of control is useful to provide fruits of
desired bitterness which can be used in the fresh fruit market or
in the juice production industry, diminishing the need for
de-bittering processes.
[0061] In preparing a vector according to the present invention
various DNA fragments may be manipulated as necessary to create the
desired vector or construct. This includes using linkers or
adaptors as necessary to form suitable restriction sites or to
eliminate unwanted restriction sites or other like manipulations
which are known to those of ordinary skill in the art.
[0062] Promoters which are known or found to cause transcription of
a selected gene in plant cells can be used in the present
invention. Such promoters may be obtained from plants, plant
pathogenic bacteria or plant viruses, and include, but are not
necessarily limited to, the 35S and 19S promoters of cauliflower
mosaic virus (CaMV35S and CaMV19S), the full-length transcript
promoter from the figwort mosaic virus (FMV35S) and promoters
isolated from plant genes such as EPSP synthase and ssRUBISCO
genes, and promoters obtained from T-DNA genes of Agrobacterium
tumefaciens, such as nopaline and mannopine synthases. The
particular promoter selected should be capable of causing
sufficient expression of sense or antisense RNA.
[0063] Particularly useful promoters for use in the present
invention are fruit specific promoters which are expressed, for
example, during ethylene production in the fruit and the
full-length transcript promoter from the figwort mosaic virus
(FMV35S). The FMV35S promoter is particularly useful because of its
ability to cause uniform and high levels of expression.
[0064] The DNA sequence of a FMV35S promoter is presented in U.S.
Pat. No. 5,512,466 and is identified as SEQ ID NO:17 therein.
Examples of fruit specific promoters include the E8, E4, E17 and
J49 promoters from tomato (Lincoln, J. and Fischer, R. (1988).
Diverse mechanisms for the regulation of ethylene-inducible gene
expression. Mol Gen Genet 212, 71-75), as well as the 2A11 promoter
as described in U.S. Pat. No. 4,943,674.
[0065] As used herein, the term "CaMV35S" or "FMV35S" promoter
include variations of these promoters, e.g., promoters derived by
means of ligation with operator regions, random or controlled
mutagenesis, addition or duplication of enhancer sequences,
etc.
[0066] A 3' non-translated region is preferably included in a sense
vector according to the present invention and includes a
polyadenylation signal which functions in plants to cause the
addition of polyadenylated nucleotides to the 3' end of a sense RNA
sequence. Examples of suitable 3' regions are the 3' transcribed,
non-translated regions containing the polyadenylation signal of the
tumor-inducing (Ti) plasmid genes of Agrobacterium, such as the
nopaline synthase (NOS) gene, and plant genes like the 7S soybean
storage protein genes and the pea E9 small subunit of the RuBP
carboxylase gene (ssRUBISCO).
[0067] A sense RNA produced by a vector of the present invention
also preferably contains a 5' non-translated leader sequence. This
sequence can be derived from the promoter selected to express the
gene, and can be specifically modified so as to increase
translation of the sense RNA. The 5' non-translated regions can
also be obtained from viral RNA's, from suitable eukaryotic genes,
or from a synthetic gene sequence. The present invention is not
limited to vector constructs wherein the non-translated region is
derived from the 5' non-translated sequence that accompanies the
promoter sequence. Rather, the non-translated leader sequences can
be part of the 5' end of the non-translated region of the native
coding sequence, or part of the promoter sequence, or can be
derived from an unrelated promoter or coding sequence.
[0068] In a preferred embodiment according to the present
invention, the vector that is used to introduce the sense or
antisense RNA into the host cells or plants will comprise an
appropriate selectable marker. In a more preferred embodiment
according to the present invention the vector is a plant expression
vector comprising both a selectable marker and an origin of
replication. In another most preferred embodiment according to the
present invention the vector will be a shuttle vector, which can
propagate both in E. coli (wherein the construct comprises an
appropriate selectable marker and origin of replication) and be
compatible for propagation or integration in the genome of the
organism of choice. In yet another embodiment, the construct
comprising the promoter of choice and the gene of interest is
placed in a viral vector which is used to infect the cells. This
virus may be integrated in the genome of the organism of choice or
may remain non-integrated.
[0069] The promoter of choice that is used in conjunction with this
invention will comprise any suitable promoter as further detailed
herein. It will be appreciated by one skilled in the art, however,
that it is necessary to make sure that the transcription start
site(s) will be located upstream of the open reading frame. In a
preferred embodiment of the present invention, the promoter that is
selected will comprise an element that is active in the particular
host plant cells of interest.
[0070] These elements may be selected from transcriptional
regulators that activate the transcription of genes essential for
the survival of these cells in conditions of stress or starvation,
including the heat shock proteins. Promoters containing this type
of sequence may advantageously be used according to the present
invention.
[0071] As further detailed hereinabove, DNA sequences encoding the
translational start site (ATG) of the gene to be expressed, will be
placed downstream of the transcription start site(s). Any
equivalent functional element selected from similar elements in
this or other organisms may be used as appropriate in the organism
of choice. Equivalent functional elements will include elements
with synthetic bases, or elements found in other genes of plants as
well as elements found in genes of other unicellular or
multicellular organisms.
[0072] According to one embodiment of the present invention
secretion of the protein out of the cell is preferred. In this
embodiment the construct will comprise a signal sequence to effect
secretion as is known in the art. A signal sequence that is
recognized in the active growth phase will be most preferred. As
will be recognized by the skilled artisan, the appropriate signal
sequence should be placed immediately downstream of the
translational start site (ATG), and in frame with the coding
sequence of the gene to be expressed.
[0073] Introduction of the construct or vector into the cells is
accomplished by any conventional method for transfection, infection
or the like, as is known in the art. In constructs comprising a
selectable marker the cells may be selected from those bearing
functional copies of the construct. If the plasmid comprising the
gene of interest is episomal the appropriate selective conditions
will be used during growth. Stable transfectants and stable cell
lines may be derived from the transfected cells in appropriate
cases, in order to conveniently maintain the genotype of interest.
Cell growth is accomplished in accordance with the cell type, using
any standard growth conditions as may be suitable to support the
growth of the specific cell line.
[0074] A DNA construct of the present invention can be inserted
into the genome of a plant by any suitable method. Suitable plant
transformation vectors include those derived from a Ti plasmid of
Agrobacterium tumefaciens, such as those disclosed by Bevan and
Chilton, Ann. Rev. Genetics 16, 357-384, 1982; U.S. Pat. No.
4,940,838 and others. In addition to plant transformation vectors
derived from the Ti or root-inducing (R1) plasmids of
Agrobacterium, alternative methods can be used to insert the DNA
constructs of this invention into plant cells. Such methods may
involve, for example, the use of liposomes, electroporation,
chemicals that increase free DNA uptake, particle gun technology,
and transformation using viruses.
[0075] The construction of vectors capable of being inserted into a
plant genome via Agrobacterium tumefaciens mediated delivery is
known to those of ordinary skill in the art. Typical plant cloning
vectors comprise selectable and scoreable marker genes, T-DNA
borders, cloning sites, appropriate bacterial genes to facilitate
identification of transconjugates, broad host-range replication and
mobilization functions and other elements as desired.
[0076] If Agrobacterium mediated delivery is chosen, once the
vector has been introduced into the disarmed Agrobacterium strain,
the desired plant can then be transformed. Any known method of
transformation that will work with the desired plant can be
utilized.
[0077] Thus, further according to the present invention there are
provided a plant cell and a transgenic plant of a species naturally
expressing a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
messenger RNA, such as, but not limited to, grapefruit or pomelo,
wherein the plant cell or transgenic plant are genetically modified
to include, in an expressible antisense orientation, a nucleotide
sequence encoding an antisense RNA molecule being capable of in
vivo base pairing with the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
to thereby render the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA,
when expressed, amenable to degradation by nucleases present in the
plant cell or transgenic plant. The plant cell or transgenic plant
according to this aspect of the present invention exhibit either
very low or no flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase
activity.
[0078] It will be appreciated that the nucleotide sequence encoding
an antisense RNA molecule being capable of in vivo base pairing
with the flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
messenger RNA according to the present invention can be either
extrachromosomal or intrachromosomal. In any case, as further
detailed herein, the nucleotide sequence is preferably ligated to
an expression vector in the antisense orientation downstream to a
promoter element and optionally other gene expression control
elements.
[0079] The nucleotide sequence encoding an antisense RNA molecule
being capable of in vivo base pairing with the
flavanone-7-O-glucoside-2"-O-rha- mnosyl-transferase messenger RNA
according to the present invention can be as set forth in SEQ ID
NO:20 or a portion thereof longer than 10 nucleotides, preferably
longer than 15 nucleotides, more preferably longer than 20
nucleotides, most preferably longer than 30 or 40 nucleotides, so
as to ensure specific base pairing with the endogenous
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger RNA
produced by the plant cell under physiological conditions.
[0080] Alternatively, the nucleotide sequence encoding an
antisense. RNA molecule being capable of in vivo base pairing with
the flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger
RNA according to the present invention can share between 80 and
100% identical bases with SEQ ID NO:20 or a portion thereof longer
than 10 nucleotides, preferably longer than 15 nucleotides, more
preferably longer than 20 nucleotides, most preferably longer than
30 or 40 nucleotides, as determined using a sequence analysis
software package developed by the Genetic Computer Group (GCG) at
the University of Wisconsin with gap creation penalty of 50 and gap
extension penalty of 3, so as to ensure specific base pairing with
the endogenous flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase
messenger RNA produced by the plant cell.
[0081] Still alternatively, the nucleotide sequence encoding an
antisense RNA molecule being capable of in vivo base pairing with
the flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase messenger
RNA according to the present invention can be hybridizable in vitro
with SEQ ID NO:20 or a portion thereof longer than 10 nucleotides,
preferably longer than 15 nucleotides, more preferably longer than
20 nucleotides, most preferably longer than 30 or 40 nucleotides,
under hybridization selected from the group consisting of stringent
hybridization, moderate hybridization and mild hybridization,
wherein stringent hybridization is effected by a hybridization
solution of 6.times.SSC and 1% SDS, hybridization temperature of
65.degree. C., final wash solution of 0.1.times.SSC and final wash
at 60.degree. C., moderate hybridization is effected by a
hybridization solution of 6.times.SSC and 1% SDS, hybridization
temperature of 58.degree. C., final wash solution of 0.5.times.SSC
and final wash at 50.degree. C., whereas mild hybridization is
effected by a hybridization solution of 6.times.SSC and 1% SDS,
hybridization temperature of 50.degree. C., final wash solution of
2.times.SSC and final wash at 40.degree. C., so as to ensure
specific base pairing with the endogenous
flavanone-7-O-glucoside-2"-O-rhamnosyl-t- ransferase messenger RNA
produced by the plant cell. These alternative hybridization
conditions are useful in evaluating hybridization capabilities of
sequences according to all of the aspects of the present invention.
Other hybridization conditions can also be employed. Step by step
protocols for the above specified hybridizations and the mentioned
other hybridizations are found in Molecular Cloning: a Laboratory
Manual: 2nd edition, Sambrook et al. 1989, Cold Spring Harbor
Laboratory Press, which is incorporated by reference as if fully
set forth herein.
[0082] Further according to the present invention there is provided
a cell genetically modified to include, in an expressible sense
orientation, a nucleotide sequence encoding a
flavanone-7-O-glucoside-2"-O-rhamnosyl-tra- nsferase. The
nucleotide sequence according to this aspect of the present
invention can be complementary DNA or genomic DNA, and it can be
either extrachromosomal or intrachromosomal. It is preferably
ligated to an expression vector in the sense orientation and has
any of the features described herein above with respect to the
isolated polynucleotide according to the present invention. The
cell according to this aspect of the present invention can be
either a microorganism (either a prokaryote or a eukaryote) or a
cell of a multicellular organism, such as a cell of a multicellular
plant. In any case, for reasons further detailed hereinunder, the
selected cell is preferably capable of producing an activated
rhamnose, either in the form of UDP-rhamnose or of
dTDP-rhamnose.
[0083] Suitable microorganism which are known to produce
dTDP-rhamnose include, but are not limited to, Lactobacillus Spp.,
such as Lactobacillus caseii and Lactobacillus delbrueckeii and
yeast such as Saccharomyces cerevisiae.
[0084] Cells of suitable multicellular plants include, for example,
cells of plants of the genus Citrus, such as, but not limited to,
grapefruit and pomelo, both being capable of producing
UDP-rhamnose. Additional examples include cells of tobacco
(Nicotiana tabacum), grapes (Vitis viniferis) and carrot (Daucus
carota).
[0085] Further according to the present invention there is provided
a plant cell or a transgenic plant of a plant species naturally
expressing a flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase,
such as, but not limited to, grapefruit and pomelo, the plant cell
or transgenic plant being genetically modified to include, in an
overexpressible sense orientation, a nucleotide sequence encoding a
sense RNA molecule being capable of inducing a co-suppression
effect to thereby reduce production of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the plant
cell or the transgenic plant.
[0086] The transgenic plants according to the present invention,
either expressing the nucleotide sequence according to the present
invention as herein described in an antisense form or in an
overexpressible sense form, both are characterized by reduced
production of flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase.
Plants can therefore be selected via taste tests to produce fruits
of desired degree of bitterness, to provide, for example, less
bitter fruits. Varieties with optimal perceived bitterness levels
can be propagated vegetatively by land grafting or other procedures
known to the ordinary artisan, and can be used to provide fruits to
the fresh fruit market and to produce juices, such as grapefruit
juice, that do not require de-bittering.
[0087] Furthermore, according to still another aspect of the
present invention there is provided a transgenic plant or cell of a
plant species naturally expressing a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase- . The
transgenic plant or cell is genetically modified to knock-out a
gene encoding the endogenous
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferas- e thereof to
thereby abolish production of flavanone-7-O-glucoside-2"-O-rh-
amnosyl-transferase in the transgenic plant or cell. One ordinarily
skilled in the art can readily design a knock-out construct
including both positive and negative selection genes for
efficiently selecting transfected cells that underwent a homologous
recombination event with the construct. Such cells can be cultured
and propagated and can further be induced to differentiate into
seedlings and grown plants, as well known in the art. Further
details concerning uses and procedures of plant gene knock-out are
found in Miao et al., 1995; Strepp et al., 1998; and Cooley et al.,
1998, which are incorporated by reference as if fully set forth
herein.
[0088] The flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase gene
according to the present invention can be used in a process for
enzymatically modifying a 1-6 linkage of hesperidin to a 1-2
linkage of neohesperidin by removing and re-coupling the terminal
rhamnose. This is done in a two step process as shown in the left
path of FIG. 1. First, hesperidin from orange peels is treated with
immobilized commercial hesperidinase to remove the 1-6 linkage.
This process is already used commercially as a de-bittering step
for citrus juice. The second step involves using a transgenic
organism heavily expressing the gene to re-rhammosylate H7G in the
correct 1-2 position. The organism must be capable of naturally
producing copious quantities of UDP- or dTDP-rhamnose.
[0089] Thus, further according to the present invention, there is
provided a commercial process of producing neohesperidin. The
process is effected by implementing the following steps, in which,
in a first step, hesperidin is obtained, for example, by extraction
from a hesperidin producing species of the genus Citrus such as
orange. Then, the hesperidin is treated with a hesperidinase,
thereby hesperetin-7-glucoside is obtained. Thereafter, the
hesperetin-7-glucoside is treated with a
flavanone-7-O-glucoside-2"-O-rha- mnosyl-transferase in the
presence of activated rhamnose, such as UDP-rhamnose or
dTDP-rhamnose, and thereby neohesperidin is obtained.
[0090] Further according to the present invention there is provided
a commercial process of producing neohesperidin dihydrochalcone.
The process is effected by implementing the following steps, in
which, in a first step, hesperidin is obtained, for example, by
extraction from a hesperidin producing species of the genus Citrus
such as orange. Then, the hesperidin is treated with a
hesperidinase, thereby hesperetin-7-glucoside is obtained.
Thereafter, the hesperetin-7-glucoside is treated with a
flavanone-7-O-glucoside-2"-O-rha- mnosyl-transferase in the
presence of activated rhamnose, thereby neohesperidin is obtained.
Subsequently, the neohesperidin is treated with an alkali, thereby
neohesperidin chalcone is obtained. Finally, the neohesperidin
chalcone is reduced, e.g., by a Pd catalyzed H.sub.2 surface
reduction process, thereby neohesperidin dihydrochalcone is
obtained.
[0091] Preferably, the hesperidinase is immobilized on a solid
support, whereas the hesperidin is treated with the hesperidinase
while passing over the solid support.
[0092] Treating the hesperetin-7-glucoside with a
flavanone-7-O-glucoside-- 2"-O-rhamnosyl-transferase in the
presence of activated rhamnose is preferably effected in vivo by a
cell genetically modified to overexpress the
flavanone-7-glucoside-2"-O-rhamnosyl-transferase, wherein the cell
selected produces activated rhamnose and is capable of intake of
the hesperetin-7-glucoside. The neohesperidin in then extracted
from the cell. Preferably, the neohesperidin is extracted from the
cell prior to the treatment thereof with the alkali. The cell can
be of a bacterium, a fungus, a yeast or of a higher plant, as long
as it is capable of generating activated rhamnose and is capable of
intake of the hesperetin-7-glucoside. The cell can be of a
microorganism or of a higher plant. Suitable microorganisms include
bacteria and yeast cells. Lactobacillus casei, Lactobacillus
delbrueckeii and Saccharomyces cerevisiae provide good examples for
microorganisms. Tobacco (Nicotiana tabacum), grapes (Vitis
viniferis) and carrot (Daucus carota) provide good examples for
higher plant cells which are readily propagated in culture and
qualify with the required criteria.
[0093] This aspect of the present invention is of high
significance. It is well known that in vitro formation of activated
sugar moieties is highly inefficient. Therefore, only when the
supply of activated rhamnose is effected in vivo by an organism
capable also of intake of hesperetin-7-glucoside, either naturally
or rendered capable thereof by physical, chemical, biological or
genetic means, and of overexpressing
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in
stoichiometric amounts, would the process described herein be
amenable to industrialization.
[0094] Further according to the present invention there is provided
a method of modifying a rhamnose-1-6-glucose linkage of a chemical
compound to a rhamnose-1-2-glucose linkage. The method is effected
by implementing the following method steps, in which, in a first
step, for removal of the rhamnose group, the chemical compound is
enzymatically treated, thereby a first derivative of the chemical
compound having a terminal glucose group is obtained. Then, the
first derivative is enzymatically treated with recombinant
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the presence
of activated rhamnose, thereby a second derivative chemical
compound having the rhamnose-1-2-glucose linkage is obtained.
Enzymatically treating the first derivative with a
flavanone-7-O-glucoside-2"-O-rhamnosyl-transferase in the presence
of activated rhamnose is preferably effected in vivo by a cell
genetically modified to overexpress the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transf- erase, wherein the
cell produces activated rhamnose and is capable of intake of the
first derivative. The second derivative can be extracted from the
cell.
[0095] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0096] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Isolation of a Gene Encoding a
flavanone-7-O-glucoside-2"-O-rhamnosyl-tran- sferase.
[0097] Previous attempts to isolate the gene based on purified
protein were unsuccessful. These included cDNA library screens
using oligonucleotides as well as antibodies. The successful
approach, RT-PCR using degenerate primers followed by RACE is
described herein.
[0098] A cDNA expression library of pomelo young leaves was
constructed in Lambda-gt111. Antibodies prepared against pomelo 1-2
rhamnosyl-transferase were used to screen this library.
Positive-looking clones were later found to be artifacts in that
they did not contain a relevant sequence.
[0099] Several oligonucleotides were designed according to the
partial peptide sequence and were used to screen the above library
by standard techniques. Any resulting clones were irrelevant.
[0100] Thus, isolation of the gene encoding
flavanone-7-O-glucoside-2"-O-r- hamnosyl-transferase (1-2
rhamnosyl-transferase) involved a lengthy process initiated in the
purification to homogeneity of the protein, followed by partial
peptide sequencing and finalized by cDNA isolation based on RT-PCR
and RACE.
[0101] Details of the process are as follows:
[0102] Purification of the enzyme and activity assay: Purification
and functional analysis of the
flavanone-7-O-glucoside-2"-O-rhamnosyl-transfe- rase protein from
young pomelo leaves was as described (Bar-Peled et al. 1991).
[0103] Partial peptide sequence of the protein: The purified
protein was subject to partial proteolysis, using several different
proteases (Lys-C; Asp-N; Trypsin), followed by separation of
peptides by SDS-PAGE and blotting on PVDF membranes. Partial
peptide sequences generated by Edman degradation were as follows
(only conclusive sequence data is given; amino acids in question
are not shown): Lys-C cleavage yielded two partial sequences: 1 N Y
F L H L T A (SEQ ID NO:1) and Y P F (SEQ ID NO:2); Asp-N cleavage
yielded four partial sequences: I A A I L F L (SEQ ID NO:3), Y F P
S L M G N (SEQ ID NO:4), E K M T I E E A (SEQ ID NO:5) and L F Q P
(SEQ ID NO:6); whereas, trypsin cleavage yielded a single partial
sequence: V V D N G M G M V V P R D K (SEQ ID NO:7).
[0104] Isolation of partial cDNA fragments: Partial cDNA fragments
were obtained by an RT-PCR approach on target tissue mRNA (young
pomelo leaves) using degenerate primers based on the partial
peptide sequences. Pomelo young leaf single strand cDNA was
prepared by reverse transcription of poly-A RNA using a PCR-adapted
poly-T primer (primer contains sequences identical to the -40M13
forward primer followed by 15 T's; see below). PCR was used to
amplify gene specific fragments using degenerate primers (designed
according to partial peptide sequences; see below) in conjunction
with the -40M13 forward primer. Primer sequences were as
follows:
[0105] Gene specific degenerate primers were: 5'-GAT(C)AAT(C)GGIAT
GGGIATGGT-3' (SEQ ID NO:8) for D N G M G M V (SEQ ID NO:9);
5'-GAA(G)AAG(A)ATGACIATT(CA)GAA(G)GAA(G)GC-3' (SEQ ID NO:10) for E
K M T I E E A (SEQ ID NO:5); and 5'-AAC(T)TAC(T)TTC(T)
CTICAC(T)CTIACIGC-3' (SEQ ID NO:11) for N Y F L H L T A (SEQ ID
NO:12).
[0106] General primers were: PCR-adapted poly-T primer 5'-GTTTTCCCA
GTCACGACGTTTTTTTTTTTTTTT (SEQ ID NO:13); and -40M13 forward primer
5'-GTTTTCCCAGTCACGACG-3' (SEQ ID NO: 14).
[0107] Amplified DNA fragments were separated and isolated from
agarose gels and then cloned into pGEM-T vector (Promega, Madison
Wis.). Sequence was obtained by automated sequencing using
universal primers (T7 and SP6).
[0108] Completion of cDNA sequence: The full length cDNA sequence
was obtained after isolating the missing 5' segment by 5'-RACE
(Schaefer B. C., 1995) using the following gene specific primers
which were derived from the partial cDNA sequence data: RACE1:
5'-CATGCCCATACCATTGTC-3' (SEQ ID NO:15); RACE2:
5'-GACAATGGTATGGGCATG-3' (SEQ ID NO: 16); and RACE3:
5'-CCTCAACCACCGAGCCCCAACCAC-3' (SEQ ID NO: 17).
[0109] Based on the total assembled sequence data, the complete
coding region was isolated by RT-PCR using a proof-reading
thermostable polymerase (Pfu; Stratagene, La-Jolla, Calif.) and the
following primers (each primer contains a cloning adapter sequence;
gene specific sequences are underlined):
5'-CATCTAGAATGGATACCAAGCATCAAG-3' (SEQ ID NO: 18); and
5'-CAGGATCCTTATTCAGATTTCTTGACAAG-3' (SEQ ID NO:19).
[0110] The resulting translated coding region (1359 base-pairs;
FIG. 2, SEQ ID NO: 20) contains all partial peptide sequences
previously determined by protein sequencing. The sequence of the
protein encoded by SEQ ID NO: 20 is given in SEQ ID NO:21.
[0111] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
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Sequence CWU 1
1
21 1 8 PRT Citrus X paradisi 1 Asn Tyr Phe Leu His Leu Thr Ala 1 5
2 3 PRT Citrus X paradisi 2 Tyr Pro Phe 1 3 7 PRT Citrus X paradisi
3 Ile Ala Ala Ile Leu Phe Leu 1 5 4 8 PRT Citrus X paradisi 4 Tyr
Phe Pro Ser Leu Met Gly Asn 1 5 5 8 PRT Citrus X paradisi 5 Glu Lys
Met Thr Ile Glu Glu Ala 1 5 6 4 PRT Citrus X paradisi 6 Leu Phe Gln
Pro 1 7 14 PRT Citrus X paradisi 7 Val Val Asp Asn Gly Met Gly Met
Val Val Pro Arg Asp Lys 1 5 10 8 20 DNA Artificial sequence
Synthetic oligonucleotide 8 gayaayggna tgggnatggt 20 9 7 PRT Citrus
X paradisi 9 Asp Asn Gly Met Gly Met Val 1 5 10 23 DNA Artificial
sequence Synthetic oligonucleotide 10 garaaratga cnathgarga rgc 23
11 23 DNA Artificial sequence Synthetic oligonucleotide 11
aaytayttyc tncayctnac ngc 23 12 8 PRT Citrus X paradisi 12 Asn Tyr
Phe Leu His Leu Thr Ala 1 5 13 33 DNA Artificial sequence Synthetic
oligonucleotide 13 gttttcccag tcacgacgtt tttttttttt ttt 33 14 18
DNA Artificial sequence Synthetic oligonucleotide 14 gttttcccag
tcacgacg 18 15 18 DNA Artificial sequence Synthetic oligonucleotide
15 catgcccata ccattgtc 18 16 18 DNA Artificial sequence Synthetic
oligonucleotide 16 gacaatggta tgggcatg 18 17 24 DNA Artificial
sequence Synthetic oligonucleotide 17 cctcaaccac cgagccccaa ccac 24
18 27 DNA Artificial sequence Synthetic oligonucleotide 18
catctagaat ggataccaag catcaag 27 19 29 DNA Artificial sequence
Synthetic oligonucleotide 19 caggatcctt attcagattt cttgacaag 29 20
1359 DNA Citrus X paradisi 20 atggatacca agcatcaaga taagccaagc
attctcatgt taccatggct agctcatggg 60 cacatagctc cacaccttga
acttgccaag aagctttcac agaaaaactt ccacatatat 120 ttctgctcta
ctcccaacaa tctacaatcc ttcggcagaa atgttgaaaa aaacttctca 180
tcttcaatac aactcataga actgcaactt cccaatacat tccctgaact tccttcacaa
240 aatcagacca caaaaaacct tcctccccat cttatttata ctctcgtggg
agcatttgaa 300 gacgcaaaac ctgctttttg caacatcttg gagacgctta
aaccaaccct tgttatgtat 360 gatttgttcc aaccgatggc ggcggaggca
gcttaccagt atgacatagc tgctattttg 420 ttcttaccct tatctgcagt
agcctgctct ttcttgctgc acaatatcgt aaatcccagc 480 ctgaaatacc
ctttctttga atctgattac caagatagag aaagcaagaa catcaattac 540
ttcctgcatc ttactgccaa tggcacctta aacaaagaca ggttcttaaa agctttcgaa
600 ctatcttgca aatttgtgtt catcaaaaca tcaagagaga ttgaatccaa
gtacttggat 660 tattttcctt ctttaatggg aaatgaaata attccagtag
ggcctctaat ccaagaacct 720 accttcaagg tagatgatac aaagatcatg
gactggctga gccaaaagga gcctcgttca 780 gtcgtgtatg catcctttgg
cagtgagtac tttccttcca cggatgaaat acatgacata 840 gctattgggt
tattgctcac cgaggttaat tttatatggg ctttcagatt acatcctgat 900
gagaaaatga cgatagagga agcactgcct cagggctttg ctgaggagat tgaaaggaat
960 aataagggaa tgatagtaca aggttgggtt ccgcaggcta aaattttaag
gcatggaagc 1020 atcggcggat ttttgagtca ttgtggttgg ggctcggtgg
ttgaggggat ggttttcggg 1080 gtaccaatca taggtgtgcc aatggcatat
gagcagccaa gcaatgccaa ggtggtggtt 1140 gacaatggta tgggcatggt
cgttccaaga gataagatca atcaaagact tggaggagag 1200 gaggtggcga
gggtcattaa acatgttgtg ctgcaagaag aagcgaagca aataagaaga 1260
aaagctaatg aaattagtga gagtatgaag aagatagggg acgcacagat gagtgtggtg
1320 gtggagaaac tgctgcagct tgtcaagaaa tctgaataa 1359 21 452 PRT
Citrus X paradisi 21 Met Asp Thr Lys His Gln Asp Lys Pro Ser Ile
Leu Met Leu Pro Trp 1 5 10 15 Leu Ala His Gly His Ile Ala Pro His
Leu Glu Leu Ala Lys Lys Leu 20 25 30 Ser Gln Lys Asn Phe His Ile
Tyr Phe Cys Ser Thr Pro Asn Asn Leu 35 40 45 Gln Ser Phe Gly Arg
Asn Val Glu Lys Asn Phe Ser Ser Ser Ile Gln 50 55 60 Leu Ile Glu
Leu Gln Leu Pro Asn Thr Phe Pro Glu Leu Pro Ser Gln 65 70 75 80 Asn
Gln Thr Thr Lys Asn Leu Pro Pro His Leu Ile Tyr Thr Leu Val 85 90
95 Gly Ala Phe Glu Asp Ala Lys Pro Ala Phe Cys Asn Ile Leu Glu Thr
100 105 110 Leu Lys Pro Thr Leu Val Met Tyr Asp Leu Phe Gln Pro Met
Ala Ala 115 120 125 Glu Ala Ala Tyr Gln Tyr Asp Ile Ala Ala Ile Leu
Phe Leu Pro Leu 130 135 140 Ser Ala Val Ala Cys Ser Phe Leu Leu His
Asn Ile Val Asn Pro Ser 145 150 155 160 Leu Lys Tyr Pro Phe Phe Glu
Ser Asp Tyr Gln Asp Arg Glu Ser Lys 165 170 175 Asn Ile Asn Tyr Phe
Leu His Leu Thr Ala Asn Gly Thr Leu Asn Lys 180 185 190 Asp Arg Phe
Leu Lys Ala Phe Glu Leu Ser Cys Lys Phe Val Phe Ile 195 200 205 Lys
Thr Ser Arg Glu Ile Glu Ser Lys Tyr Leu Asp Tyr Phe Pro Ser 210 215
220 Leu Met Gly Asn Glu Ile Ile Pro Val Gly Pro Leu Ile Gln Glu Pro
225 230 235 240 Thr Phe Lys Val Asp Asp Thr Lys Ile Met Asp Trp Leu
Ser Gln Lys 245 250 255 Glu Pro Arg Ser Val Val Tyr Ala Ser Phe Gly
Ser Glu Tyr Phe Pro 260 265 270 Ser Thr Asp Glu Ile His Asp Ile Ala
Ile Gly Leu Leu Leu Thr Glu 275 280 285 Val Asn Phe Ile Trp Ala Phe
Arg Leu His Pro Asp Glu Lys Met Thr 290 295 300 Ile Glu Glu Ala Leu
Pro Gln Gly Phe Ala Glu Glu Ile Glu Arg Asn 305 310 315 320 Asn Lys
Gly Met Ile Val Gln Gly Trp Val Pro Gln Ala Lys Ile Leu 325 330 335
Arg His Gly Ser Ile Gly Gly Phe Leu Ser His Cys Gly Trp Gly Ser 340
345 350 Val Val Glu Gly Met Val Phe Gly Val Pro Ile Ile Gly Val Pro
Met 355 360 365 Ala Tyr Glu Gln Pro Ser Asn Ala Lys Val Val Val Asp
Asn Gly Met 370 375 380 Gly Met Val Val Pro Arg Asp Lys Ile Asn Gln
Arg Leu Gly Gly Glu 385 390 395 400 Glu Val Ala Arg Val Ile Lys His
Val Val Leu Gln Glu Glu Ala Lys 405 410 415 Gln Ile Arg Arg Lys Ala
Asn Glu Ile Ser Glu Ser Met Lys Lys Ile 420 425 430 Gly Asp Ala Gln
Met Ser Val Val Val Glu Lys Leu Leu Gln Leu Val 435 440 445 Lys Lys
Ser Glu 450
* * * * *