U.S. patent application number 17/029348 was filed with the patent office on 2021-04-08 for fermentation methods for producing steviol glycoside using high ph and compositions obtained therefrom.
The applicant listed for this patent is CARGILL, INCORPORATED, EVOLVA, S.A.. Invention is credited to James C. Anderson, Manuel Quiros Asensio, Simon Carlsen, Ting Liu Carlson, Veronique Douchin, Ariene M. Fosmer, Hans Peter Smits.
Application Number | 20210102229 17/029348 |
Document ID | / |
Family ID | 1000005277927 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210102229 |
Kind Code |
A1 |
Anderson; James C. ; et
al. |
April 8, 2021 |
FERMENTATION METHODS FOR PRODUCING STEVIOL GLYCOSIDE USING HIGH PH
AND COMPOSITIONS OBTAINED THEREFROM
Abstract
Disclosed are methods for producing steviol glycosides, such as
rebaudioside D and rebaudioside M, using engineered yeast. In some
embodiments, the methods include fermenting with a yeast at a high
pH such as of about 5.8 or greater. In some embodiments, the
methods can be carried out by first growing the yeast at a lower
first pH, and then adjusting the pH to a higher pH.
Inventors: |
Anderson; James C.; (Eden
Prairie, MN) ; Asensio; Manuel Quiros; (Soborg,
DK) ; Carlsen; Simon; (Copenhagen, DK) ;
Carlson; Ting Liu; (Marietta, SC) ; Douchin;
Veronique; (Birkerod, DK) ; Fosmer; Ariene M.;
(Eden Prairie, MN) ; Smits; Hans Peter; (Holte,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARGILL, INCORPORATED
EVOLVA, S.A. |
Wayzata
Reinach |
MN |
US
CH |
|
|
Family ID: |
1000005277927 |
Appl. No.: |
17/029348 |
Filed: |
September 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15578125 |
Nov 29, 2017 |
10815513 |
|
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PCT/US2016/034728 |
May 27, 2016 |
|
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17029348 |
|
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62168345 |
May 29, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 27/36 20160801;
A23V 2002/00 20130101; C07H 15/256 20130101; C12P 19/56
20130101 |
International
Class: |
C12P 19/56 20060101
C12P019/56; C07H 15/256 20060101 C07H015/256; A23L 27/30 20060101
A23L027/30 |
Claims
1. A method for increasing the production of a first, lower
molecular weight steviol glycoside, relative to a second, higher
molecular weight steviol glycoside, in an engineered yeast,
comprising fermenting the engineered yeast capable of producing the
first steviol glycoside and the second steviol glycoside at a pH of
5.8 or greater in a fermentation medium.
2. The method of claim 1, wherein the pH is in the range of 5.8 to
8.
3. The method of claim 1, wherein the pH is in the range of 6.0 to
7.5.
4. The method of claim 1, wherein the pH is in the range of 6.5 to
7.5.
5. The method of claim 1, wherein the pH is in the range of 5.8 to
6.5.
6. The method of claim 1, wherein the pH is in the range of 5.8 to
6.2.
7. The method of claim 1, wherein the fermentation medium comprises
a nitrogen source selected from the group consisting of ammonium
hydroxide, yeast extract, urea, and ammonium sulfate.
8. The method of claim 7, wherein the nitrogen source comprises 90%
(wt) or greater ammonium hydroxide or urea.
9. The method of claim 1, further comprising continuous or batch
addition of additional fermentation material to the medium
comprising the engineered yeast.
10. The method of claim 1 performed for a period of time of up to
150 hours.
11. The method of claim 10 performed for a period of time in the
range of 8 to 88 hours.
12. The method of claim 11 performed for a period of time in the
range of 22 to 48 hours.
13. The method of claim 1, wherein the fermentation medium has a
biomass in the range of 20-120 g dcw/L.
14. The method of claim 1, wherein the engineered yeast produces a
ratio of the first steviol glycoside to the second steviol
glycoside at the pH of 5.8 or greater that is increased by 10% or
greater than a ratio of the first steviol glycoside to the second
steviol glycoside when the engineered yeast are fermented at a pH
of 5.0.
15. The method of claim 1, wherein the first steviol glycoside is
rebaudioside D, and the second steviol glycoside is rebaudioside
M.
16. The method of claim 15, wherein the ratio of rebaudioside D to
rebaudioside M is in the range of 1:20 to 1:1.
17. The method of claim 1, further comprising growing the
engineered yeast in a first medium at a first pH, wherein the first
pH is the same as the pH of the fermentation medium.
18. The method of claim 1, further comprising growing the
engineered yeast in a first medium at a first pH, wherein the first
pH is lower than the pH of the fermentation medium.
19. The method of claim 18, wherein the first pH is a pH less than
5.8.
20. The method of claim 18, wherein the first pH is a pH less than
5.5.
21. A method for increasing the production of a first, lower
molecular weight steviol glycoside relative to a second, higher
molecular weight steviol glycoside in an engineered yeast, the
method comprising a step of fermenting engineered yeast capable of
producing one or more steviol glycoside(s) at a pH of 5.8 or
greater in a fermentation medium, wherein the engineered yeast
produces a ratio of the first and second steviol glycosides at pH
of 5.8 or greater that is greater than a ratio of the first and
second steviol glycosides produced at a pH that is less than
5.8.
22. The method of claim 21 wherein the ratio of the first and
second steviol glycosides at pH of 5.8 or greater is 10% or greater
than the ratio of the first and second steviol glycosides produced
at a pH that is less than 5.8.
23. The method of claim 22 wherein the ratio of the first and
second steviol glycosides at pH of 5.8 or greater is 25% or greater
than the ratio of the first and second steviol glycosides produced
at a pH that is less than 5.8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/168,345 filed May 29, 2015, which is herein
incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] This application contains references to amino acid sequences
and/or nucleic acid sequences which have been submitted
concurrently herewith as an ASCII text file entitled
"CAR0210WO_Sequence_Listing.txt," created on May 27, 2016, and
having a size of 92 KB. The sequence listing is hereby incorporated
by reference in its entirety pursuant to 37 C.F.R. .sctn.
1.52(e)(5).
FIELD
[0003] The present invention relates fermentation methods for
producing steviol glycosides, fermentation compositions, and
steviol glycoside compositions produced by fermentation.
BACKGROUND
[0004] Sugars, such as sucrose, fructose and glucose, are utilized
to provide a pleasant taste to beverages, foods, pharmaceuticals,
and oral hygienic/cosmetic products. Sucrose, in particular,
imparts a taste preferred by consumers. Although sucrose provides
superior sweetness characteristics, it is caloric. Non-caloric or
lower caloric sweeteners have been introduced to satisfy consumer
demand, and there is desire for these types of sweeteners that have
favorable taste characteristics.
[0005] Stevia is a genus of about 240 species of herbs and shrubs
in the sunflower family (Asteraceae), native to subtropical and
tropical regions from western North America to South America. The
species Stevia rebaudiana, commonly known as sweetleaf, sweet leaf,
sugarleaf, or simply stevia, is widely grown for its sweet leaves.
Stevia-based sweeteners may be obtained by extracting one or more
sweet compounds from the leaves. Many of these compounds are
steviol glycosides, which are glycosides of steviol, a diterpene
compound. These diterpene glycosides are about 150 to 450 times
sweeter than sugar. Steviol glycosides differ from each other by
sweetness power as well as other sensory features contributing to
taste quality such as bitterness, lingering aftertaste and the
like. See Kinghorn, A. D., Stevia: The genus Stevia, Taylor &
Francis, London (2002).
[0006] Examples of steviol glycosides are described in WO
2013/096420 (see, e.g., listing in FIG. 1); and in Ohta et. al.,
"Characterization of Novel Steviol Glycosides from Leaves of Stevia
rebaudiana Morita," J. Appl. Glycosi., 57, 199-209 (2010) (See,
e.g., Table 4 at p. 204). Structurally, the diterpene glycosides
are characterized by a single base, steviol, and differ by the
presence of carbohydrate residues at positions C13 and C19, as
presented in FIGS. 2a-2k. See also PCT Patent Publication WO
2013/096420.
[0007] Typically, on a dry weight basis, the four major steviol
glycosides found in the leaves of Stevia are dulcoside A (0.3%),
rebaudioside C (0.6-1.0%), rebaudioside A (3.8%) and stevioside
(9.1%). Other glycosides identified in Stevia extract include one
or more of rebaudioside B, D, E, F, G, H, I, J, K, L, M, N, O,
steviolbioside and rubusoside.
[0008] While the major steviol glycoside Reb A is commonly used as
sweetener in beverage applications it has off-taste issues. More
recently, there has been focus on certain minor steviol glycosides
which have better taste properties. For example, rebaudioside M has
higher sweetness intensity and is more potent than other steviol
glycosides (e.g., see Prakash, I., et al. (2013) Nat. Prod.
Commun., 8: 1523-1526, and WO 2013/096420). Rebaudioside D tastes
about 200-220 times sweeter than sucrose and in a sensory
evaluation it had a slow onset of sweetness and was very clean,
namely sweeter overall than sucrose, less sweet lingering
aftertaste compared to sucrose (e.g., see Prakash, I., et al.
(2012) Int. J. Mol. Sci., 13:15126-15136).
[0009] Molecular techniques have been used to prepare recombinant
organisms capable of synthesizing steviol glycosides via
fermentation. For example, recombinant strains of S. cerevisiae
having multiple transgenes encoding enzymes involved in steviol
glycoside synthesis have been used for the production of
rebaudioside M and rebaudioside D (see, for example,
WO2014/122227).
SUMMARY
[0010] The present invention generally relates to methods for
producing steviol glycosides using engineered yeast, as well as
fermentation compositions, and fermentation products that include
one or more steviol glycosides. Fermentation conditions of the
disclosure can promote increased production of steviol glycosides
from the engineered yeast, and can also provide desirable steviol
glycoside ratios, such as fermentation compositions that have high
rebaudioside D to rebaudioside M ratios. For example, in some
embodiments of the invention, a fermentation composition may
include a ratio of rebaudioside D to rebaudioside M of 1:20 or
greater. In still other embodiments, the ratio of rebaudioside D to
rebaudioside M is in the range of 1:20 to 1:1. In other
embodiments, the ratio of rebaudioside D to rebaudioside M at a
second pH is greater than a ratio of rebaudioside D to rebaudioside
M produced when an engineered yeast is maintained at the first pH
throughout fermentation.
[0011] In one embodiment, the invention provides a method for
producing steviol glycoside which involves changing the medium to a
higher pH condition during fermentation for production of the
steviol glycoside(s).
[0012] The method includes a step of growing engineered yeast in a
first medium at a first pH, wherein the engineered yeast are
capable of producing one or more steviol glycoside(s). An
"engineered yeast" refers to yeast cells having at least one
exogenous DNA sequence that is introduced into the cell, either
integrated into the cell's genome or present on an extrachromosomal
construct, such as a plasmid or episome. Next, a composition is
added to the first medium to provide a second medium having a
second pH that is greater than the first pH. In the second medium
the engineered yeast is fermented to produce the one or more
steviol glycoside(s). The composition added to the medium can
include a nitrogen-containing compound, such as one selected from
ammonium hydroxide, urea, ammonium sulfate. The composition added
to the medium can be used to control the pH. The pH can also be
controlled by a non-nitrogen containing base, such as potassium
hydroxide or sodium hydroxide or calcium hydroxide and
supplementing the nitrogen in the medium with a yeast nitrogenous
base, ammonium sulfate, urea, yeast extract or other nitrogen
containing nutrients.
[0013] In the second medium, the pH can be adjusted to greater than
about 5, greater than about 5.5, or greater than about 5.8, such as
in the range of about 5.8 to 7.5 or 5.8 to 6.2. The
nitrogen-containing compound added to the medium can be a base,
such as ammonium hydroxide, and be used to form the second, higher,
pH condition. Alternatively, a nitrogen-containing compound can be
used with a non-nitrogen base to provide the higher pH. The
nitrogen-containing compound, such as yeast extract, ammonium
hydroxide, urea, ammonium sulfate, or combinations thereof, can be
the predominant nitrogen component in the second medium during
fermentation conditions. The non-nitrogen bases can include
potassium hydroxide, sodium hydroxide, and calcium hydroxide.
[0014] An exemplary method involves (a) growing the engineered
yeast in a medium having a carbohydrate (e.g., glucose) at a lower
pH, such as below 5.8, and then (b) adding a composition with a
nitrogen-containing compound, such as ammonium hydroxide, urea,
ammonium sulfate, or combinations thereof, and optionally a
non-nitrogen containing base, to the medium, along with additional
carbohydrate, and optionally other fermentation compounds, to
provide a medium having a pH of 5.8 or greater, and then fermenting
the medium with the engineered yeast to produce steviol
glycoside(s).
[0015] In another embodiment, the invention provides a method for
producing steviol glycosides wherein a pH shift is not required,
but rather optional. Accordingly, another embodiment is a method
for producing steviol glycoside, where the method comprises a step
of growing and fermenting a medium with an engineered yeast at a pH
of 5.8 or greater in medium comprising a nitrogen source. The
nitrogen source is selected from ammonium hydroxide, urea, and
ammonium sulfate, yeast extract and one or a combination of these
compounds is the primary nitrogen source during fermenting. During
fermenting the engineered yeast produces one or more steviol
glycoside(s). For example, the steviol glycosides include
rebaudioside D, rebaudioside M or rebaudioside D and rebaudioside
M. In some embodiments, the molar ratio of rebaudioside D to
rebaudioside M is 1:20 or greater.
[0016] In another embodiment, the invention provides a method for
increasing the production of a first, lower molecular weight
steviol glycoside relative to a second, higher molecular weight
steviol glycoside in an engineered yeast. The method includes a
step of fermenting engineered yeast capable of producing one or
more steviol glycoside(s) at a pH of 5.8 or greater in a
fermentation medium, wherein the engineered yeast produces a ratio
of the first and second steviol glycosides at pH of 5.8 or greater
that is greater than a ratio of the first and second steviol
glycosides produced at a pH that is less than 5.8. For example, the
method can increase the ratio of the first and second steviol
glycosides at pH of 5.8 or greater by about 10% or greater over the
ratio when then engineered yeast are grown at the lower pH.
[0017] In another embodiment, the invention also provides
compositions comprising steviol glycosides produced by a
fermentation process. Accordingly, in another embodiment, the
invention provides a composition obtained from a fermentation
process comprising rebaudioside D and rebaudioside M, wherein the
molar ratio rebaudioside D to rebaudioside M is 1:20 or
greater.
DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows a representative mevalonate pathway.
[0019] FIG. 2 shows a representative non-mevalonate pathway.
[0020] FIG. 3 shows a representative pathway for steviol
production.
[0021] FIG. 4 shows representative pathways for the biosynthesis of
steviol glycosides from steviol.
DETAILED DESCRIPTION
[0022] Embodiments of the disclosure described herein are not
intended to be exhaustive or to limit the invention to the precise
forms disclosed in the following detailed description. Rather a
purpose of the embodiments chosen and described is so that the
appreciation and understanding by others skilled in the art of the
principles and practices of the present invention can be
facilitated.
[0023] Fermentation methods of the disclosure use engineered yeast
capable of producing steviol glycosides. The engineered yeast
capable of producing steviol glycosides can include one or more
exogenous nucleic acids that encode enzyme(s) that promote
formation of one or more steviol glycosides in the cell. For
example, the engineered yeast can have a set of enzymes that
provide a pathway for the synthesis of the steviol glycosides RebM
and RebD.
[0024] As used herein, the term "steviol glycoside(s)" refers to
glycosides of steviol. Exemplary steviol glycoside, include, but
not are not limited to, rebaudioside A, rebaudioside B,
rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,
rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J,
rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N,
rebaudioside O, stevioside, steviolbioside, dulcoside A,
rubusoside. Engineered yeast can produce steviol glycosides that
are the same as steviol glycosides found in nature ("naturally
occurring") as well as steviol glycosides that are not found in
nature. Steviol glycosides can be formed in engineered yeast by
enzymatic processes.
[0025] Structurally, steviol glycosides have a central molecular
moiety, which is a single steviol base, and glucopyranosyl residues
attached to the C13 and/or C19 atoms of the steviol base, according
to the atom numbering on the base shown below. That is,
glucopyranosyl residues represent groups R.sub.1 and R.sub.2 in the
following formula:
##STR00001##
[0026] Table A below shows the various steviol glycosides and the
corresponding R.sub.1 and R.sub.2 groups:
TABLE-US-00001 TABLE A Compound name R.sub.1 (C-19) R.sub.2 (C-13)
Steviol H H Stevioside .beta.-Glu .beta.-Glu-.beta.-Glu (2->1)
Rebaudioside A .beta.-Glu ##STR00002## Rebaudioside B H
##STR00003## Rebaudioside C .beta.-Glu ##STR00004## Rebaudioside D
.beta.-Glu-.beta.-Glu (2->1) ##STR00005## Rebaudioside E
.beta.-Glu-.beta.-Glu (2->1) .beta.-Glu-.beta.-Glu (2->1)
Rebaudioside G .beta.-Glu .beta.-Glu-.beta.-Glu (3->1)
Rebaudioside M ##STR00006## ##STR00007## Rebaudioside N
##STR00008## ##STR00009## Rebaudioside O ##STR00010## ##STR00011##
Glu: glucose Rha: rhamnose
[0027] According to the current disclosure, steviol glycosides are
produced in a process that includes fermenting the engineered yeast
at a pH that is higher than typical yeast fermentation conditions.
By comparison, the yeast Saccharomyces cerevisiae is typically
fermented at a pH in the range of 4 to 5.
[0028] The method of the disclosure can use various yeast host
cells engineered to provide a pathway to one or more steviol
glycosides. Such cells used in the methods of the disclosure can be
transformed with one or more DNA construct(s) encoding enzymes for
steviol glycoside synthesis. Exemplary yeast that can be used for
hosts for exogenous DNA constructs encoding steviol glycoside
pathway enzymes, include, but are not limited to species of
Candida, Kloeckera (Hanseniaspora), Kluyveromyces, Lipomyces,
Pichia (Hansenula), Rhodotorula, Saccharomycete, Saccharomyces,
Schizosaccharomyces, Torulopsis, Torulaspora, Yarrowia, and
Zygosaccharomyces. Exemplary species are Candida albicans, Pichia
pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe,
and Yarrowia lipolytica. Further, host cells can also include
genetic modifications other than those of the steviol glycoside
pathway that may provide improved performance during
fermentation.
[0029] The term "exogenous" refers to a molecule, such as a nucleic
acid, or an activity, such as an enzyme activity, that is
introduced into the host yeast. An exogenous nucleic acid can be
introduced into the yeast host by well-known techniques and can be
maintained external to the hosts chromosomal material (e.g.,
maintained on a non-integrating vector), or can be integrated into
the yeast's chromosome, such as by a recombination event.
Generally, the genome of an engineered yeast is augmented through
the stable introduction of one or more recombinant genes. An
exogenous nucleic acid can encode an enzyme, or portion thereof,
that is either homologous or heterologous to the yeast. An
exogenous nucleic acid can be in the form of a "recombinant gene or
DNA construct" referring to a nucleic acid that is in one or more
ways manipulated through molecular techniques to be in a form that
does not naturally exist.
[0030] The term "heterologous" (e.g., "non-native") refers to a
molecule or activity that is from a source that is different than
the referenced molecule or organism. Accordingly, a gene or protein
that is heterologous to a referenced organism is a gene or protein
not found in that organism. In the context of the disclosure, a
"heterologous glycosyltransferase" refers to a glycosyltransferase
polypeptide that is different from any glycosyltransferase
polypeptide that may be native to the host organism. For example, a
specific glycosyltransferase gene found in a first species and
exogenously introduced into a host yeast organism that is different
than the first species is "heterologous" to the host yeast.
[0031] The engineered yeast can use an auxotrophic marker suitable
for selecting for a transformant having a nucleic acid encoding a
steviol glycoside pathway enzyme. The host yeast can include
modifications (deletions, etc.) in one or more genes that control
auxotrophies, such as LYS2, LEU2, HIS3, URA3, URA5, and TRP1. Using
a host cell having a desired genetic background for introduction of
one or more exogenous genes, one or more gene construct(s) is
introduced into a cell to integrate into the genome, or to be
stably maintained and allow for expression. Methods for introducing
a gene construct into a host cell include transformation,
transduction, transfection, co-transfection, and electroporation.
In particular, yeast transformation can be carried out using the
lithium acetate method, the protoplast method, and the like. The
gene construct to be introduced may be incorporated into a
chromosome in the form of a plasmid, or by insertion into the gene
of a host, or through homologous recombination with the gene of a
host. The transformed yeast into which the gene construct has been
introduced can be selected with a selectable marker (for example,
an auxotrophic marker as mentioned above). Further confirmation can
be made by measuring the activity of the expressed protein, or the
production of a bioproduct associated with the introduced gene(s)
such as a steviol glycoside.
[0032] The transformation of exogenous nucleic acid sequences
including the steviol pathway genes can be confirmed using methods
well known in the art. Such methods include, for example, nucleic
acid analysis such as Northern blots or polymerase chain reaction
(PCR) amplification of mRNA, or immunoblotting for expression of
gene products, or other suitable analytical methods to test the
expression of the introduced nucleic acid sequences or their
corresponding gene product. It is understood by those skilled in
the art that the exogenous nucleic acid is expressed in a
sufficient amount to produce the desired product, and it is further
understood that expression levels can be optimized to obtain
sufficient expression using methods well known in the art and as
disclosed herein.
[0033] The terpenoid compounds isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP) can serve as chemical precursors
to steviol glycosides in an engineered yeast. Some organisms,
including plants, insect, and some microbial species, have a
mevalonate (MVA) pathway that converts acetyl-CoA through a series
of chemical intermediates to IPP and DMAPP. Some organisms produce
IPP and DMAPP through the non-mevalonate pathway (also known as the
methyl D-erythritol 4-phosphate or MEP pathway) starting with
glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR).
[0034] The yeast Saccharomyces cerevisiae naturally expresses genes
of the mevalonate pathway. Mevalonate pathway genes that encode
enzymes that include: (a1) acetoacetyl CoA thiolase (EC 2.3.1.9),
(b1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC
4.1.3.5); (c1) HMG-CoA reductase (EC 1.1.1.34); (d1) mevalonate
kinase (EC 2.7.1.36); (e1) phosphomevalonate kinase (EC 2.7.4.2);
and (f1) mevalonate diphosphate decarboxylase (EC 4.1.1.33).
Enzymes of the mevalonate pathway converts acetyl-CoA to IPP as
follows:
acetyl-CoA.fwdarw.acetoacetyl-CoA.fwdarw.3-hydroxy-3-methylglutaryl-CoA.f-
wdarw.mevalonate.fwdarw.mevalonate-5-phosphate.fwdarw.mevalonate-5-pyropho-
sphate.fwdarw.IPP. See also FIG. 1
[0035] In some embodiments, the engineered yeast can include one or
more modifications to increase the flux from acetyl-CoA to IPP
and/or DMAPP, thereby providing an increased pool of IPP and/or
DMAPP for use in a pathway to steviol. The modifications can
include, for example, increasing expression or activity of one or
more mevalonate pathway enzymes (a1)-(f1), such as by placing a
nucleic acid encoding an enzyme that is homologous or heterologous
to the yeast cell under the control of a promoter that provides
increased expression, using multiple copies of the nucleic acid,
and/or using a heterologous enzyme, a variant enzyme (e.g., one
including one or more amino acid substitutions), or a variant
heterologous enzyme that provides a higher level of enzymatic
activity as compared to the native enzyme.
[0036] Alternatively, the non-mevalonate (MEP) pathway can be used
to provide IPP and DMAPP as precursors to steviol glycoside
production. The yeast Saccharomyces cerevisiae do not naturally
express genes of the MEP pathway, but can optionally be engineered
to provide MEP pathway genes. Theoretically, the MEP pathway is
more energetically efficient generally because it loses less carbon
as CO2 as compared to the MVA pathway (MEP pathway: 1 CO2/IPP; MVA
pathway: 4 CO2/IPP; sugar as carbon source).
[0037] In particular, in the non-mevalonate (MEP) pathway compounds
isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP)
are generated through a series of intermediates leading from
glyceraldehydes-3-phosphate (G3P) and pyruvate (PYR), and a number
of enzymes are responsible for this conversion. Enzymes involved in
a biosynthetic pathway from G3P and PYR to IPP and DMAPP include
(a2) 1-deoxy-D-xylulose-5-phosphate synthase (DXS), (b2)
1-Deoxy-D-xylulose-5-phosphate reductoisomerase (ispC)-, (c2)
4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD), (d2)
4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), (e2)
2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase (IspF), (f2)
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG),
(g2) 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase
(IspH), and (h2) isopentenyl-diphosphate isomerase (IDI), see FIG.
2.
[0038] The methods of the disclosure for producing steviol
glycoside(s) by fermentation can use engineered yeast that have one
or more genetic modifications to increase the flux from G3P and PYR
to IPP and/or DMAPP, thereby providing an increased pool of IPP
and/or DMAPP for use in a pathway to steviol. The modifications can
include, for example, increasing expression or activity of one or
more enzymes (a2)-(h2), such as by placing a nucleic acid encoding
an enzyme that is heterologous to the yeast cell under the control
of a promoter that provides increased expression, using multiple
copies of the nucleic acid, and/or using a heterologous enzyme, a
variant enzyme (e.g., one including one or more amino acid
substitutions), or a variant heterologous enzyme that provides a
high levels of enzymatic activity.
[0039] The methods of the disclosure for producing steviol
glycoside(s) by fermentation can use engineered yeast can also
include a pathway to convert IPP and/or DMAPP to steviol. For
example, in some aspects the engineered yeast can include exogenous
nucleic acids expressing the following enzymes: (a3) geranyl
geranyldiphosphate synthase (GGPPS), (b3) copalyl diphosphate
synthase (CDPS), (c3) kaurene synthase (KS), (d3) kaurene oxidase
(KO), and (e3) kaurenoic acid 13-hydroxylase (KAH). Enzymes of the
mevalonate pathway convert IPP and/or DMAPP to steviol as follows:
IPP/ DMAPP.fwdarw.geranyl geranyldiphosphate.fwdarw.copalyl
diphosphate.fwdarw.kaurene.fwdarw.kaurenoic acid.fwdarw.steviol.
(See FIG. 3) Exogenous nucleic acids encoding enzymes (a3)-(e3)
that are heterologous to the yeast cell can be placed under the
control of a promoter that provides increased expression, using
multiple copies of the nucleic acid, and/or using a variant enzyme
(e.g., one including one or more amino acid substitutions), or a
variant heterologous enzyme that provides a high levels of
enzymatic activity.
[0040] The methods of the disclosure for producing steviol
glycoside(s) by fermentation can use engineered yeast having any
pathway to convert steviol to a steviol glycoside. If more than one
steviol glycoside pathway enzyme is present in the engineered
yeast, the yeast may be able to produce different steviol
glycosides. For example, the yeast may be able to produce two,
three, four, five, six, seven, eight, nine, ten, or more than ten
different steviol glycoside species.
[0041] The steviol glycoside pathway can include one or more
uridine diphosphate (UDP) glycosyltransferases (UGTs) that mediate
the transfer of glycosyl residues from activated nucleotide sugars
to acceptor molecules. In the case of a steviol glycoside pathway,
a monosaccharide unit can be transferred to a hydroxyl or carboxyl
moiety on a steviol or steviol glycoside molecule, or to a hydroxyl
group on a glucose group that is attached to the steviol base. See
FIG. 4 UGTs have been classified into families and subfamilies
based on sequence homology. See Li, et al., 2001, J. Biol. Chem.
276:4338-4343. A superfamily of over 100 genes encoding UGTs, each
containing a 42 amino acid consensus sequence, has been identified
in the model plant Arabidopsis thaliana, and genes encoding UGTs
have also been identified in several other higher plant
species.
[0042] Exemplary UDP-glucosyltransferase can be any
UDP-glucosyltransferase capable of adding at least one glucose unit
to the steviol and or steviol glycoside substrate to provide the
target steviol glycoside. In one embodiment, the engineered yeast
can include one or more UDP-glucosyltransferase selected from group
UGT74G1 (SEQ ID NO: 1), UGT85C2 (SEQ ID NO: 2), UGT76G1 (SEQ ID NO:
3), UGT91D2 (SEQ ID NO: 4), and also UGTs having substantial
identity (e.g. >85%, >75%, >65%, >55%, >45% and
>35%) to these polypeptides. An engineered yeast can include one
or more exogenous nucleic acid molecule(s) that code for these
UGTs.
[0043] The engineered yeast can also include one or more UGT and
UDP-glucose recycling enzyme(s). An exemplary
UDP-glucosyltransferase capable of adding at least one glucose unit
to rubusoside to form stevioside is UGT91D2 (SEQ ID NO: 4). An
exemplary UDP-glucosyltransferase capable of adding at least one
glucose unit to stevioside to form rebaudioside A is UGT76G1 (SEQ
ID NO: 3). An exemplary UDP-glucosyltransferase capable of adding
at least one glucose unit to rebaudioside A to form rebaudioside D
is UGT91D2 (SEQ ID NO: 4). An exemplary UDP-glucosyltransferase
capable of adding at least one glucose unit to rebaudioside D to
form rebaudioside M is UGT76G1 (SEQ ID NO: 3).
[0044] Exemplary publications that describe engineered
microorganisms for steviol glycoside production and steviol
glycoside pathway enzymes include, for example, US2014/0357588,
WO2014/193934, WO2014/193888, and WO2014/122227, each of which is
incorporated herein by reference in their entirety.
[0045] In one embodiment, an engineered yeast useful for the
production of steviol glycosides expresses the following enzymes:
geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl
diphosphate synthase (CDPS), kaurene oxidase (KO), kaurene synthase
(KS); steviol synthase (KAH), cytochrome P450 reductase (CPR),
UGT74G1, UGT76G1, UGT91D2, UGT85C2 and a EUGT11. WO2014/122227
describes an engineered yeast strain that express these enzymes.
The UDP-glucosyltransferases can be a gene encoding a polypeptide
for example, UGT74G1 (SEQ ID NO: 1), UGT85C2 (SEQ ID NO: 2),
UGT76G1 (SEQ ID NO: 3), UGT91D2 (SEQ ID NO: 4), and a EUGT11 (SEQ
ID NO: 13); these genes encode polypeptides capable of carrying out
a number of reactions such as a) a gene encoding a polypeptide
capable of beta 1,2 glucosylation of the C2' of the 19-O glucose of
a steviol glycoside; (b) a gene encoding a polypeptide capable of
beta 1,2 glucosylation of the C2' of the 13-O-glucose of a steviol
glycoside; (c) a gene encoding a polypeptide capable of beta 1,3
glucosylation of the C3' of the 19-O-glucose of a steviol
glycoside; (d) a gene encoding a polypeptide capable of beta 1,3
glucosylation of the C3' of the 13-O-glucose of a steviol
glycoside; (i) a gene encoding a polypeptide capable of
glucosylation of the 13-OH of steviol or a steviol glycoside; (j) a
gene encoding a polypeptide capable of glucosylation of the C-19
carboxyl of steviol or a steviol glycoside. For example, UGT85C2
carries out reaction (i); UGT74G1 carries out reaction (j); UGT91D2
carries out reactions (a; weakly), (b); UGT76G1 carries out
reactions (c) and (d) EUGT11 carries out reactions (a), (b; less
well).
[0046] Aspects of the disclosure can be described with reference to
stages of cell culture. For example, the process may include one or
more "stages" or "phases" of culturing the engineered yeast. For
example, the process can include a "seed/growth phase". As used
herein "seed phase" refers to a period during which the cells are
grown in a medium to become acclimated to the medium components
(carbohydrates, nitrogen source, salts, vitamins, trace metals)
that will be used in the subsequent growth phase and to increase
cell numbers. "Growth phase" as used herein refers to the period
during which the cells multiply (e.g. exponentially). During the
seed/growth phase, the engineered yeast can begin to multiply by
budding, referred to as yeast division.
[0047] The seed/growth phase can be characterized by rapid
multiplication of the engineered yeast. The seed/growth phase can
be described in terms of the doubling times of the engineered
yeast. In some embodiments of the disclosure, growth of the
engineered yeast can be performed at a lower (first) pH (e.g.,
about 5.8 or below or about 5.0 or below), and then at a time later
in the growth phase, or at time in the subsequent fermentation
phase, the pH can be increased to a higher (second) pH (e.g., about
5.8 or greater or about 6.0 or greater). In other embodiments of
the disclosure, growth of the engineered yeast can be performed at
the higher (second) pH, and therefore an adjustment to a higher pH
during the growth and fermentation phases is not required.
[0048] After the growth phase, the engineered yeast can enter a
"fermentation phase" in which growth has at least slowed and the
engineered yeast are actively assimilating carbohydrate and
producing the desired product, e.g. steviol glycoside(s). As used
herein "fermentation," "fermenting," or variants therefor is used
to describe the phase of significant production of steviol
glycoside(s) by converting a substrate with a yeast, which can
occur in partially aerobic, aerobic or anaerobic conditions. In
partially aerobic conditions, both fermentative and respiratory
pathways can be active, and some cell growth may occur. In
partially aerobic conditions the amount of oxygen consumed can be
less than during the seed/growth phase. As used herein, the phrase
"throughout the process" or "throughout" when used in reference to
the various phases or treatments means from growth phase through
formation of product.
[0049] In some modes of practice, the pH of the medium during the
fermentation phase can be at the higher pH if the pH of the medium
was increased during, or at the beginning of the growth phase. In
other modes of practice, the pH can be adjusted to the higher pH at
a predetermined point during the fermentation phase. If the pH is
increased during fermentation, it is preferably increased closer to
the beginning of the fermentation phase than the end of the
fermentation phase, and more preferably at the beginning or very
close to the beginning of the fermentation phase.
[0050] In some modes of practice, the pH of the medium during the
growth phase is carried out at the same pH as the fermentation
phase. For example, the growth phase is carried out at the higher
(second) pH. For example, the higher (second) pH is from about 5.8
or greater.
[0051] In some embodiments, the method of the disclosure for
producing steviol glycoside(s) involves altering the medium in
which the engineered cells are in to a higher pH for fermentation
and production of steviol glycoside(s). Therefore, a step in the
method can involve growing the engineered yeast in a first medium
at a first, lower pH (e.g., pH 5.8 or less or pH 5.0 or less) and
then after a period of time at the first, lower, pH the engineered
yeast are fermented in a medium at a second pH that is higher than
the first pH. The engineered yeast are fermented at the second,
higher, pH (e.g., pH 5.8 or greater, or pH 6.0 or greater) to
produce the one or more steviol glycoside(s), and the higher pH
conditions can result in increased amounts of steviol glycosides,
as well as a shift to more desirable ratios of steviol glycosides
among those produced.
[0052] The term "medium" refers to a liquid composition in which
the engineered yeast can be maintained, can grow, can be fermented,
or combinations thereof. A "medium" may also be referred to as a
"broth" or "cell culture," and terms such as "starting" or
"fermentation" may be used to more specifically define the medium
and the cellular activity that is occurring therein. A medium can
be defined with regards to the components present in the medium,
and amounts thereof, such as carbon sources, including (a)
carbohydrates such as glucose and starch products such as
maltodextrin; (b) nitrogen sources, such as yeast nitrogen base,
ammonium hydroxide, urea, ammonium sulfate, yeast extract or any
combination thereof; (c) salts, such as potassium phosphate
(monobasic, dibasic), magnesium sulfate, sodium chloride, and
calcium chloride; (d) vitamins, such as biotin, calcium
pantothenate, folic acid, (myo)-inositol, nicotinic acid,
p-aminobenzoic acid, pyridoxine HCl, riboflavin, thiamine HCl, and
citric acid; (e) trace metals such as boric acid, copper sulfate,
cobalt chloride, calcium chloride, potassium iodide, ferric
chloride, magnesium sulfate, manganese chloride, sodium molybdate,
and zinc sulfate. Components in the medium can be defined on a dry
weight basis. Further, the medium is water-based, or an "aqueous"
composition. The medium can also be defined with regards to its pH,
and biocompatible acids, bases, and buffers that are used to
control the pH in the medium.
[0053] Fermentation of the engineered yeast can be performed using
a medium that has a starch and/or sugar containing plant material
derivable from any plant and plant part, such as tubers, roots,
stems, leaves and seeds. Starch and/or sugar-containing plant
materials can be obtained from cereal, such as barley, wheat,
maize, rye, sorghum, millet, barley, potatoes, cassava, or rice,
and any combination thereof. The starch-and/or sugar-containing
plant material can be processed, such as by methods such as
milling, malting, or partially malting. In some embodiments, medium
(with higher pH or lower pH) includes a treated starch. For
example, the medium for growth and/or fermentation can include a
partially hydrolyzed starch. The partially hydrolyzed starch can
include high molecular weight dextrins and high molecular weight
maltodextrins. A partially hydrolyzed starch product can be used
that has amounts of starch and starch degradation products within
desired ranges beneficial for steviol glycoside production.
[0054] Optionally, a starch degrading enzyme can be added to the
medium that includes a starch material in order to increase the
concentration of monomeric sugars such as glucose that can be
utilized by the engineered yeast. Exemplary starch-degrading
enzymes include amylolytic enzymes such as glycoamylase and
amylase.
[0055] In some modes of practice, fermentation can be carried out
in medium that includes steviol-containing compounds. Such
compounds can be directly used by the glucosyltransferases in the
engineered yeast. For example, optionally, fermentation can be
carried out in medium containing steviol, steviol-13-O-glucoside or
steviol-19-O-glucoside. Using this medium, the microorganism may
contain and express genes encoding a functional EUGT11 (SEQ ID NO:
13), a functional UGT74G1 (SEQ ID NO: 1), a functional UGT85C2 (SEQ
ID NO: 2), a functional UGT76G1 (SEQ ID NO: 3), and a functional
UGT91D2 (SEQ ID NO: 4). Compounds such as rebaudioside A,
rebaudioside D, and rebaudioside M may be obtained from the
fermentation medium. As another option, fermentation can be carried
out in medium containing rubusoside. Using this medium, the
microorganism may contain and express genes encoding a functional
EUGT11 (SEQ ID NO: 13), a functional UGT76G1 (SEQ ID NO: 3), and a
functional UGT91D2 (SEQ ID NO: 4). Compounds such as rebaudioside
A, D, and M may be obtained from the medium following fermentation.
The terms "rebaudioside DM", "Reb DM", and variations thereof, as
used herein, refer to glycosides that are primarily rebaudioside D
and rebaudioside M (hence, the "DM"), their related isomers (e.g.
natural or synthetic), and/or salts thereof. This terminology
format may be used for glycosides having any other combination of
glycosides, for example, but not limited to Reb DA, Reb MA, Reb DMA
and the like.
[0056] In some cases fermentation is carried out in industrial
capacity fermenters in order to achieve commercial scale economic
benefits and control. In an embodiment, the fermentation is carried
out in a fermenter that has a capacity of about 10,000 liters or
more.
[0057] The terms "first medium" and "second medium" (and
optionally, "third," "fourth," fifth," etc., if necessary) may be
used to describe aspects of the method of producing steviol
glycosides. In one mode of practice, a first medium at a lower pH
(e.g., less than 5.8 or less than 5.0) and containing the
engineered cells is provided and engineered yeast therein are
cultured for a period of time. Subsequently, a liquid composition
free of cells (e.g., a "feed composition") is added to the first
medium to provide a second medium having the same or a higher pH,
which can be used for fermentation of the engineered yeast. The
feed composition can be added to the first medium in a continuous
or batch process. In a preferred mode of practice, the feed
composition is added in a continuous process to more precisely
control fermentation conditions in the medium. In some embodiments,
the feed composition is the same composition of feed added to the
first medium and added to the second medium.
[0058] As another example, a first medium at the low pH (e.g., less
than 5.8 or less than 5.0) and including the engineered yeast can
be cultured for a period of time. The first medium can then be
added to a predetermined volume of a liquid composition free of
cells in a batch or bulk step to create a second medium which has a
higher pH (e.g., 5.8 or greater or 6.0 or greater), which can be
used for fermentation of the engineered yeast and steviol glycoside
production. It is understood that there are a variety of ways a
second medium having a higher pH can be prepared starting with a
first medium with a lower pH. Therefore, formation of the second
medium can be by a process of "adding to," "adding into," or
"mixing," using bulk or continuous addition of one or more feed
components. The feed components can be in liquid or in solid form.
In some cases formation of the second medium can be by a multi-step
process. In other cases the formation of the first medium and the
second medium has the higher pH of the second medium.
[0059] In some modes of practice, the first medium is present is a
vessel, and then the pH of the medium is adjusted to provide a
second medium at a higher pH, which is formed in the same vessel.
In other modes of practice, the first medium is formed in a first
vessel and then it is transferred to a second vessel wherein the
second medium having a higher pH is formed by combining the first
medium with other components or materials.
[0060] The first medium having a lower pH can be formed by adding a
seed culture to a liquid composition that includes a
carbohydrate(s), a nitrogen source, such as yeast nitrogen base,
ammonium hydroxide, urea, ammonium sulfate, yeast extract or any
combination thereof; salts, vitamins, and trace metals. In some
modes of practice the first medium includes ammonium hydroxide,
urea, ammonium sulfate, or combinations thereof, as the sole
nitrogen source in the medium. An "initial" concentration of
components in the first medium may be described, with an
understanding that the concentration of components may decrease in
the first medium over time as the engineered cells consume the
components. When the second medium with the higher pH (e.g., 5.8 or
greater, or 6.0 or greater) is formed, the ammonium hydroxide,
urea, or ammonium sulfate can be the sole nitrogen source in the
medium.
[0061] In some modes of practice, the first medium, such as the
medium where yeast growth takes place, can have a pH less than
about 6.0, less than about 5.9, less than about 5.8, less than
about 5.7, less than about 5.6, less than about 5.5, less than
about 5.4, less than about 5.3, less than about 5.2, less than
about 5.1, less than about 5.0, such as in the range of about 3.0
to about 5.5, about 3.5 to about 5.3, or about 4.0 to about 5.0. An
exemplary pH in the first medium is about 5.0. During the period of
growth in the first medium, the pH may fluctuate. For example,
growth of the yeast cells may cause the first medium to become more
acidic after a period of time. Optionally, the pH in the first
medium can be controlled by monitoring the pH over time, and, if
necessary, adjusting the pH such as with a base or a buffer so it
remains within a desired range during growth in the first medium.
For example, the pH of the first medium can be controlled using a
nitrogen-containing base, such as ammonium hydroxide so the pH is
maintained in the range of about 4.8 to about 5.2. A
nitrogen-containing base used in the first medium can be the same
as a nitrogen-containing base used in the second medium (e.g.,
ammonium hydroxide), with a difference being that the base used in
the second medium is at a higher concentration to provide a higher
pH.
[0062] In some modes of practice, the first medium can have an
initial concentration of glucose that is less than about 50 g/L,
less than 25 g/L, such as in the range of about 5 g/L to about 50
g/L, or about 10 g/L to about 35 g/L. The glucose concentration in
the first medium may also be defined relative to the glucose
concentration in the second medium.
[0063] In exemplary modes of practice, growth in the first medium
is performed at a temperature in the range of about 25-35.degree.
C., or 28-32.degree. C., and most preferably at about 30.degree.
C.
[0064] Also, growth of the engineered yeast can be performed with
aeration, and with agitation.
[0065] For example, in the first medium and during the growth
phase, aeration can be performed. Aeration may be described in
terms of dissolved oxygen transfer rate to the medium in units of
mg min.sup.-1 liter.sup.-1. (For example, see Anderlei, T., and
Buchs, J. (2000) Biochem. Engin. J. 3478:1-6). A sparging technique
that promotes the formation of fine gas bubbles can be performed to
provide desired aeration. In some modes of practice, during the
growth phase in the first medium, agitation and aeration is
increased, such as in a stepwise manner. Aeration conditions can
have an effect on the amount of oxygen dissolved in the medium, and
therefore the oxygen available to the engineered yeast. The amount
of oxygen uptake by the engineered yeast can be controlled by the
rate at which oxygen is supplied and the formation of small oxygen
bubbles in the medium, which can be achieved through agitation
and/or sparging. Limited aeration can also be performed during the
fermentation phase.
[0066] Growth of the engineered yeast in the first medium can be
carried out for a desired period of time before adjusting to the
second medium at the higher pH. In some modes of practice, growth
of the engineered yeast in the first medium can be carried out for
a desired period of time before adjusting to the second medium,
where the first medium and second medium have the higher pH. For
example, growth in the first medium can be carried out for a time
of about two hours or greater, or about 10 hours or greater, such
as a period of time in the range of about two hours to about 30
hours, or about 10 hours to about 24 hours. The time in the first
medium may encompass all or part of the lag phase of growth, and
all or part of the log (exponential) phase of growth of the
engineered yeast. Further, during the time in the first medium at
the lower pH, the engineered yeast can have a predetermined growth
rate. For example, in the first medium the engineered yeast can
have a doubling time in the range of about 2.31 hours to about
13.86 hours, or about 2.77 hours to about 7.3 hours. Alternatively,
growth rate can be expressed as the dilution rate, which can be in
the range of about 0.05-0.31/h, or about 0.095-0.251/h.
[0067] Growth of the engineered yeast can be performed to provide a
desired amount of biomass. As used herein "biomass" refers to the
weight of the engineered yeast, which can be measured in grams of
dried cell weight per liter of medium (DCW/L). In some mode of
practice, the engineered yeast are grown to a biomass amount of
about 20 g dcw/L or greater, about 30 g dcw/L or greater, such as
in the range of about 20 g dcw/L to about 120 g dcw/L, or about 40
g dcw/L to about 80 g dcw/L.
[0068] In forming the second medium, a base can be added to the
first medium, which causes an increase from the lower pH to the
higher pH. The time at which the base is added can be chosen based
on aspects such as the time the engineered yeast have spent in the
first medium, the concentration of components in the first medium
at a particular time point, or the growth characteristics of the
engineered yeast at a particular time point, or combinations of
these aspects. In some modes of practice, the pH of the first
medium is increased at a time at least halfway through the
exponential (growth) phase of the engineered yeast in the first
medium. For example, the pH of the first medium can be increased to
about 5.8 or greater, or to about 6.0 or greater as the engineered
yeast are coming out of the exponential phase, and their growth is
slowing. Therefore, addition of base to raise the pH can be
performed prior to the engineered yeast entering the fermentation
phase with lower growth rates. Therefore, the pH of the medium can
be increased following any significant production of steviol
glycoside(s) from the engineered yeast in a fermentation period.
However, the high pH conditions preferably encompass a period of
fermentation that provides production of steviol glycoside(s) from
the engineered yeast. Alternatively, addition of base to raise the
pH can be performed after the engineered yeast enter the
fermentation phase.
[0069] The base can be a nitrogen-containing base, such as ammonium
hydroxide, or a non-nitrogen base suitable for use in a
fermentation medium. A composition that includes a mixture of a
nitrogen-containing base and a non-nitrogen-containing base can
optionally be used. Other optional nitrogen-containing bases that
can be used in the second medium can be anhydrous ammonia or an
ammonium hydroxide/potassium hydroxide blend. Other optional
non-nitrogen-containing bases that can be used in the second medium
can be potassium hydroxide, sodium hydroxide, and calcium
hydroxide. A composition that includes a nitrogen containing base
in concentrated form (e.g., ammonium hydroxide up to about 15%
(w/v) or greater) can be used to change the pH.
[0070] The base can be added to the first composition as a liquid
composition, or can be added as a solid, for formation of the
second medium. The base can be added to the first composition in a
bulk method or a continuous process. In some modes of practice, the
base is added in a continuous process to achieve a desired pH over
a desired period of time. For example the change from the first pH
to the second pH can be carried out over a short period of time
(minutes), or longer times (hours), or any time in between. For
example, the change can occur for a period of time in the range of
about two minutes to about four hours, about five minutes to about
four hours, or about 30 minutes to about three hours. In an
exemplary mode of practice, a change from about pH 5.0 to about pH
7.0 can occur in a period of time in the range of about 30 minutes
to about 180 minutes.
[0071] Formation of the second medium can also include providing a
feed medium to the first medium. In some modes of practice, a base
is added to the first medium to increase the pH from a lower to a
higher pH, and then a feed composition is added to the medium at
the higher pH. In other modes of practice, the feed medium includes
a base and has a higher pH, and then when the feed medium is added
to the first medium it increases the pH to provide the second
medium. The feed medium, with or without base, can be the same or
different than the first medium in which the engineered yeast are
grown. In some embodiments, the feed medium added to the first
medium is the same as the feed medium added to the second
medium.
[0072] The feed medium can include a carbohydrate(s), a nitrogen
source, such as yeast extract, ammonium hydroxide, urea, ammonium
sulfate, or any combination thereof; salts, vitamins, and trace
metals such. The concentration of the components in the feed medium
can be greater than the concentration of components in the first
medium so that when the feed medium is added it provides desired
amounts of components in the second medium suitable for
fermentation of the engineered yeast. In exemplary embodiments, the
concentration of glucose in the feed medium or during
fermentation(e.g. second medium) is kept in the range of about 0
g/L to about 5 g/L, or 0 g/L to about 2 g/L. In exemplary
embodiments, the concentration of a nitrogen source (total amount)
in the feed medium such as yeast extract, nitrogen base, ammonium
hydroxide, urea, ammonium sulfate, is kept in the range of about 5
g/L to about 40 g/L. In exemplary embodiments, the concentration of
salts (total amount) in the feed medium such as salts including
magnesium sulfate in the range of about 0 g/L to about 12 g/L, and
potassium phosphate in the range of about 0 g/L to about 22 g/L. In
exemplary embodiments, the concentration of trace metals (total
amount) in the feed medium is kept in the range of about 0 g/L to
about 0.4 g/L, or 0 g/L to about 0.2 g/L.
[0073] During the period the engineered yeast are in the second
medium, such as during a period of fermentation, the pH may
fluctuate. However, the pH is preferably kept at about pH 5.8 or
greater, or about pH 6.0 or greater, such as in the range of about
pH 5.8 to about pH 8.0, pH 5.8 to about pH 7.5 or greater, about pH
6.0 to about pH 7.0, about 5.8 to about 6.5 or about 5.8 to about
6.2. During the period in the second medium, the pH can be
monitored (e.g., periodically or continuously) and adjustments to
the second medium can be made if the pH falls outside a desired
range. For example, additional ammonium hydroxide can be added to
the second medium if the pH drops below 5.8, so as to adjust the pH
to about 5.8 or greater. In exemplary embodiments, approximately
0.17 kg to about 0.2 kg of 12% NH4OH is added during fermentation
to maintain the pH at 5.0 and approximately 0.20 kg to about 0.24
kg of 12% NH4OH is added during fermentation to maintain the pH at
6.0. Approximately 1.18 kg to about 1.21 kg of feed medium is added
during the feeding phase in a typical fermentation.
[0074] In exemplary embodiments, the glucose concentration was kept
limiting by controlling flow rates of feed medium. A two-phase
feeding strategy can include an initial exponential phase beginning
at 10 hours with a growth rate for example of u=0.12 1/h while
second feeding (or feed phase II) can start at 33 hours with a
constant flow rate for example of 0.180 mls/minute. Feeding can
continue until a final volume of about 1.95 liters can be obtained
by about 120 hours. Other methods of feeding rates for producing
the desired steviol glycosides are described in the application
titled "Fermentation Methods for Producing Steviol Glycosides with
Multi-phase Feeding," U.S. patent application Ser. No. 62/168,372,
and International PCT application titled "Fermentation Methods for
Producing Steviol Glycosides with Multi-phase Feeding," Attorney
Docket No. N00293USP1 (CAR0212/WO) and filed concurrently with the
present application, each application which are hereby incorporated
by reference in their entirety.
[0075] The period of time the engineered yeast are present in the
second medium can include a fermentation period carried out for an
amount of time sufficient to produce a desired amount of steviol
glycosides. For example, the second medium with higher pH can be
formed at a time of 2 hours or later, 10 hours or later, or 24
hours or later, from an initial culturing of the engineered yeast,
and can extend to up to a time of 150 hours, up to 96 hours, or up
to 72 hours, from the initial culturing of the engineered yeast.
Fermentation of the engineered yeast and production of the steviol
glycoside(s) can begin at a point during residence of the
engineered yeast in the second medium. Preferably most of the
steviol glycoside (i.e., greater than 50%) is produced by the
engineered yeast while in the in the second medium at the higher
pH.
[0076] In exemplary modes of practice, fermentation and optionally
growth in the second medium is performed at a temperature in the
range of about 25-35.degree. C., or 28-32.degree. C., and most
preferably at about 30.degree. C. Also, fermentation and optionally
growth of the engineered yeast in the second medium can be
performed with aeration, and with agitation.
[0077] Optionally, the pH in the second medium can be controlled by
monitoring the pH over time, and, if necessary, adjusting the pH in
the second medium such as with a base or a buffer so it remains
within a desired range during fermentation. For example, the pH of
the second medium can also be controlled using a
nitrogen-containing base, such as ammonium hydroxide so the pH is
maintained in at about 5.8 or greater, such as in the range of
about pH 5.8 to about pH 7.5 or greater, about pH 6.0 to about pH
7.0, about 5.8 to about 6.5 or 5.8 to about 6.2. A
nitrogen-containing base used in the second medium can be the same
as the nitrogen-containing base used in the first medium (e.g.,
ammonium hydroxide).
[0078] The engineered yeast can be maintained in the second medium
for a period of time sufficient to produce one or more steviol
glycosides. For example, the engineered yeast can be present in the
second medium for a period up to about 150 hours which can include
growth and fermentation phases of the process. Exemplary periods in
the second medium are in the range of about 20 hours to about 150
hours, about 30 hours to about 120 hours, or about 40 hours to
about 90 hours. The period in the second medium can be most of the
growth phase and all of the fermentation phase, a part of the
growth phase and all of the fermentation phase, all of the
fermentation phase, or most of the fermentation phase.
[0079] In another embodiment, the disclosure provides a method for
producing a steviol glycoside using engineered yeast in which a
higher pH condition is present at an early point in the process,
such as at the outset of the growth phase. For example, both the
growth and fermentation phases of the process can be in a medium
having a pH of 5.8 or greater, about pH 6.0 or greater, such as in
the range of about pH 5.8 to about pH 8.0, pH 5.8 to about pH 7.5
greater, about pH 6.0 to about pH 7.0, about 5.8 to about 6.5 or
about 5.8 to about 6.2. The medium can include a nitrogen source
selected from ammonium hydroxide, urea, and ammonium sulfate. One
or a combination of these compounds can be the primary nitrogen
source during the growth and fermenting stages of the process.
[0080] In a process that starts with a high pH, during the periods
the engineered yeast are growing and fermenting, the pH may
fluctuate. However, the pH is preferably kept at about pH 5.8 or
greater, or about pH 6.0 or greater, such as in the range of about
pH 5.8 to about 8.0, pH 5.8 to about pH 7.5 or greater, or about pH
6.0 to about pH 7.0 during the growth and fermentation period.
During these periods, the pH can be monitored (e.g., periodically
or continuously) and adjustments to the medium can be made if the
pH falls outside a desired range. For example, additional ammonium
hydroxide can be added to the medium if the pH drops below 5.8 or
6.0, so as to adjust the pH to about 5.8 or greater.
[0081] In some modes of practice wherein the engineered yeast are
present in a higher pH medium at the outset or early into the
growth phase, the engineered yeast can be maintained in the medium
for a period of time sufficient to produce one or more steviol
glycosides. For example, the engineered yeast can be present in the
higher pH medium for a period of about 150 hours, or even greater.
Exemplary periods in the second medium are in the range of about 40
hours to about 150 hours, about 50 hours to about 130 hours, or
about 60 hours to about 110 hours.
[0082] During fermentation, the medium at the higher pH can be
monitored for the production of steviol glycosides. Fermentation
can be stopped at a point where there is a desired steviol
glycoside total amount and profile.
[0083] The "total steviol glycosides" (TSG) refers all the steviol
glycosides present in the medium after a period of fermentation,
which includes the amount of steviol glycosides in the liquid
medium and obtainable from the engineered yeast. The steviol
glycoside content can be expressed with regards to a total steviol
glycosides amount in the medium, or the amount of one or more, but
not all, steviol glycosides, in the medium. The amounts of steviol
glycosides in the composition can be expressed in relation to one
another, or to the total amount of steviol glycosides, such as by a
weight percentage of the total amount of steviol glycosides, or a
ratio, or range of ratios, expressed as weight percent, or molar
percent. The sum of the content of all steviol glycosides in a
composition it typically carried out on a dry (anhydrous)
basis.
[0084] The amount of steviol glycosides can also be expressed
relative to a control sample, such as a control sample fermented at
a lower pH. An exemplary comparison is engineered yeast grown at a
pH of about 5.0, and then adjusted to a pH in the range of 5.8 to
7.5 for fermentation, compared to engineered yeast grown and
fermented at a pH of about 5.0, without adjusting to a higher pH.
Another exemplary comparison is engineered yeast grown and
fermented at pH in the range of 5.8 to 7.5 for fermentation,
compared to engineered yeast grown and fermented at a pH of about
5.0.
[0085] For example, engineered yeast that are fermented at the
higher pH condition, or both grown and fermented at the higher pH
condition, can exhibit an increase in total steviol glycoside
amount of about 1.2.times. or greater, about 1.3.times. or greater,
about 1.4.times. or greater, about 1.5.times. or greater, about
1.6.times. or greater, about 1.7.times. or greater, about
1.8.times. or greater, about 1.9.times. or greater, or about
2.0.times. or greater, relative to the engineered yeast strain that
is grown at a lower pH condition (e.g., pH 5.0).
[0086] The production of certain steviol glycosides, such as
rebaudioside D and rebaudioside M, at higher pH conditions can also
be described relative to engineered yeast grown at a lower pH
condition. For example, engineered yeast that are fermented at the
higher pH condition, or both grown and fermented at the higher pH
condition, can exhibit an increase in rebaudioside D amount of
about 1.4.times. or greater, about 1.5.times. or greater, about
1.6.times. or greater, about 1.7.times. or greater, about
1.8.times. or greater, about 1.9.times. or greater, about
2.0.times. or greater, about 2.1.times. or greater, relative to the
engineered yeast strain that is grown at a lower pH condition
(e.g., pH 5.0). Exemplary titers of rebaudioside D in the
fermentation medium are about 1 g/L or greater, about 1.25 g/L or
greater, about 1.5 g/L or greater, about 1.75 g/L or greater, or
about 2.0 g/L or greater.
[0087] As another example, engineered yeast that are fermented at
the higher pH condition, or both grown and fermented at the higher
pH condition, can exhibit an increase in rebaudioside M amount of
about 1.1.times. or greater, about 1.2.times. or greater, about
1.3.times. or greater, about 1.4.times. or greater, about
1.5.times. or greater, or about 1.6.times. or greater, relative to
the engineered yeast strain that is grown at a lower pH condition
(e.g., pH 5.0).
[0088] Engineered yeast fermented at the high pH conditions may
also exhibit a change in the relative amounts of steviol glycosides
produced. For example, at a lower pH, the engineered yeast may
exhibit production of first and second steviol glycosides at a
certain ratio (e.g., X:Y). Upon change to a higher pH fermentation
condition, the engineered yeast may not only be able to produce a
greater amount of the glycosides, including first and second
glycosides, but may also able to produce the first and second
glycosides at a ratio that is different than their production at
the lower pH. In some modes of practice, the first steviol
glycoside has a lower molecular weight than the second steviol
glycoside. For example, with reference to rebaudioside D and
rebaudioside M, fermentation at a higher pH (e.g., in the range of
5.8 to 7.5) can increase the reb D:reb M ratio as compared to the
reb D:reb M ratio grown at a lower pH (e.g., pH 5.0).
[0089] In some modes of practice, the method provides a
fermentation composition wherein the ratio of rebaudioside D to
rebaudioside M in step (b) is about 1:20 or greater, such as in the
range
[0090] of about 1:20 to about 1:1, about 1:5 to about 1:1, about
1:2 to about 1:1, about 1:1.75 to about 1:1, or about 1:1.5 to
about 1:1. For example, engineered yeast that are fermented at the
higher pH condition, or both grown and fermented at the higher pH
condition, can exhibit ratios of rebaudioside D to rebaudioside M
as about 1:20 or greater, such as in the range of about 1:20 to
about 1:1, about 1:10 to about 1:1, about 1:7.5 to about 1:1, about
1:5 to about 1:1, about 1:3 to about 1:1, about 1:2 to about 1:1,
about 1:1.75 to about 1:1, or about 1:1.5 to about 1:1.
[0091] For example, engineered yeast that are fermented at the
higher pH condition, or both grown and fermented at the higher pH
condition, can exhibit an increase in the reb D:reb M ratio of
about 10% or greater, about 20% or greater, about 30% or greater,
or about 40% or greater, relative to the reb D:reb M ratio when the
engineered yeast strain are grown at a lower pH condition (e.g., pH
5.8 or less, or pH 5.0 or less).
[0092] Following the period of fermentation at the higher pH, a
composition containing one or more steviol glycoside(s) can be
obtained from the medium using various techniques. In some
embodiments, a compound such as permeabilizing agent can be added
to the medium to enhance removal of the steviol glycosides from the
cell and into the medium.
[0093] The medium can then be centrifuged or filtered to remove the
engineered cells. The medium can optionally be treated to remove
low molecular weight components (glucose, basic nutrients, and
salts), such as by membrane dialysis. Depending on a desired use, a
composition comprising one or more steviol glycoside compound(s)
can be used.
[0094] After fermentation the engineered yeast can optionally be
treated using a heat treatment method to enhance the recovery of
steviol glycosides. After fermentation, but before any heat
treatment, the medium may contain a suboptimal amount of the
steviol glycosides, with a portion of the desired steviol
glycosides within the engineered yeast. To increase the recovery of
steviol glycosides, in some modes of practice a composition, such
as the medium at the higher pH in which the engineered yeast have
been fermented, is heated to a temperature in the range from
50.degree. C. to 95.degree. C., or 70.degree. C. to 95.degree. C.,
for a period of time in the range of 5 minutes to 48 hours.
[0095] If it is desired to provide a composition with steviol
glycosides in enriched or purified form, or where certain steviol
glycosides are separated from one another, further purification can
be carried out. Such enrichment or purification of steviol
glycoside components can be carried out on the medium in which
fermentation took place, or the medium can then be dried down prior
to purification. For example, medium can be dried down using
lyophilization to form a dry composition (e.g., powder or flakes)
including steviol glycosides that can be subsequently
processed.
[0096] In some modes of practice, dried fermentation broth enriched
for steviol glycosides is used as the starting material for
purification. For example, a solvent or solvent combination can be
added to the dried fermentation broth to dissolve or suspend
material that includes the steviol glycosides. An exemplary
combination for dissolving the steviol glycosides is a mixture of
water and an alcohol (e.g., 50:50 ethanol:water). To facilitate
dissolving or suspending, the dried broth materials can be heated
at a temperature above room temperature, such as in the range of
40.degree. C.-60.degree. C. Mechanical disruption of the dried
broth materials can also be performed, such as by sonication. The
dissolved or suspended broth materials can be filtered using a
micron or sub-micron prior to further purification, such as by
preparative chromatography.
[0097] Dried fermentation broth enriched for steviol glycoside
compounds can be subjected to purification, such as by reverse
phase liquid chromatography. A suitable resin can be used to retain
steviol glycoside compounds in the column, with removal of
hydrophilic compounds which get washed through the column with a
liquid such as water. Elution of steviol glycosides from the column
can be accomplished a suitable solvent or solvent combination such
as acetonitrile or methanol.
[0098] Elution of steviol glycosides from a reverse phase column
can yield a composition which can be useful for any one of a
variety of purposes. For example, a purified steviol glycoside
composition can be used as a sweetener composition for oral
ingestion or oral use. The composition can be defined with regards
to the steviol glycosides in the composition.
[0099] Steviol glycoside-producing S. cerevisiae strains were
constructed using methods as described in WO 2011/153378, WO
2013/022989, WO 2014/122227, and WO 2014/122328, each of which is
incorporated by reference in their entirety. The following
sequences were used for construction of a parent strain (Strain A):
a recombinant gene encoding a Synechococcus sp GGPPS polypeptide
(SEQ ID NO:6), a recombinant gene encoding a truncated Zea mays
CDPS polypeptide (SEQ ID NO:7), a recombinant gene encoding an
Arabidopsis thaliana KS polypeptide (SEQ ID NO:8), a recombinant
gene encoding a recombinant Stevia rebaudiana KO polypeptide (SEQ
ID NO:9, SEQ ID NO:10), a recombinant gene encoding an A. thaliana
ATR2 polypeptide (SEQ ID NO:11, SEQ ID NO:12), a recombinant gene
encoding an Oryza saliva EUGT 11 polypeptide (SEQ ID NO:13), a
recombinant gene encoding an SrKAHe1 polypeptide (SEQ ID NO:14, SEQ
ID NO:15), a recombinant gene encoding an Stevia rebaudiana CPR8
polypeptide (SEQ ID NO:16, SEQ ID NO:17), a recombinant gene
encoding an Stevia rebaudiana UGT85C2 polypeptide (SEQ ID NO:2), a
recombinant gene encoding an Stevia rebaudiana UGT74G1 polypeptide
(SEQ ID NO:1), a recombinant gene encoding an Stevia rebaudiana
UGT76G1 polypeptide (SEQ ID NO:3), and a recombinant gene encoding
an Stevia rebaudiana UGT91D2 variant (or functional homolog),
UGT91D2e-b, (SEQ ID NO:4) polypeptide produced steviol
glycosides.
[0100] The UGT91D2e-b variant of UGT91D2 (SEQ ID NO:5 from
PCT/US2012/050021) includes a substitution of a methionine for
leucine at position 211 and a substitution of an alanine for valine
at position 286. (Additional variants, except T144S, M152L, L213F,
S364P, and G384C variants, described in Table 12 and Example 11 of
PCT/US2012/050021 could be used.) GeneArt codon-optimized sequence
encoding a Stevia rebaudiana UGT91D2e-b with the amino acid
modifications L211M and V286A (SEQ ID NO:4 for amino acid sequence;
codon optimized nucleotide sequence is set forth in SEQ ID
NO:5).
[0101] Strain B is derived from the parent strain described above
and additionally includes a codon-optimized CPR1 from Stevia
rebaudiana (SEQ ID NO:18 corresponding to amino acid SEQ ID
NO:19).
[0102] Strain C is derived from strain B and additionally includes
a gene encoding a KO polypeptide according to SEQ ID NO: 20.
[0103] Strain D is derived from strain C and additionally includes
a gene encoding a KAH polypeptide according to SEQ ID NO: 21.
[0104] Strain E is derived from strain C and additionally includes
a gene encoding a CPR4497 polypeptide according to SEQ ID NO:
22
[0105] Some additional non-limiting embodiments are provided below
to further exemplify the present disclosure:
1. A method for producing steviol glycoside, the method comprising
steps of:
[0106] (a) growing engineered yeast in a first medium at a first
pH, wherein the engineered yeast are capable of producing one or
more steviol glycoside(s);
[0107] (b) adding a composition to the first medium to provide a
second medium with a second pH that is greater than the first pH;
and
[0108] (c) fermenting the engineered yeast to produce the one or
more steviol glycoside(s) in the second medium at the second
pH.
2. The method of embodiment 1 wherein the first pH is less than
6.0. 3. The method of embodiment 2 wherein the first pH is in the
range of 4.0 to 5.5. 4. The method of embodiment 1 wherein the
second pH is greater than 5.0. 5. The method of embodiment 4
wherein the second pH is in the range of 5.5 to 8. 6. The method of
embodiment 5 wherein the second pH is in the range of 5.8 to 7.5.
7. The method of embodiment 1 wherein the composition comprises a
nitrogen-containing compound selected from the group consisting of
ammonium hydroxide, urea, and ammonium sulfate. 8. The method of
embodiment 7 wherein ammonium hydroxide, urea, or ammonium sulfate
is the primary nitrogen source for fermentation of the engineered
yeast in the medium. 9. The method of embodiment 8 wherein ammonium
hydroxide or urea is 90% (wt) or greater, or 95% (wt) or greater,
of the nitrogen source for fermentation of the engineered yeast in
the second medium. 10. The method of embodiment 1, where, in step
(b), the composition comprises a non-nitrogen base. 11. The method
of embodiment 10, wherein the non-nitrogen base is selected from
the group consisting of potassium hydroxide, sodium hydroxide, and
calcium hydroxide. 12. The method of embodiment 8, wherein ammonium
hydroxide, urea, or ammonium sulfate is 90% (wt) or greater, or 95%
(wt) or greater, of the nitrogen source for fermentation of the
engineered yeast in the second medium. 13. The method of embodiment
7 where, in step (a), the first medium comprises ammonium
hydroxide, urea, ammonium sulfate, or any combination thereof, and,
in step (b), the second pH is achieved by adding the composition
comprising ammonium hydroxide, urea, ammonium sulfate, or any
combination thereof, and optionally a non-nitrogen base, to the
first medium. 14. The method of embodiment 1 where the first medium
comprises glucose at a concentration of not greater than 25 g/L.
15. The method of embodiment 1 where the first medium comprises
glucose, a nitrogen source other than ammonium hydroxide or urea, a
potassium source, a magnesium source, trace metals, and vitamins.
16. The method of embodiment 1 where, in step (b), the second
medium comprises glucose at a concentration in the range of 400 g/L
to 750 g/L. 17. The method of embodiment 1 where, in step (b), the
second medium comprises glucose, a nitrogen source, a potassium
source, a magnesium source, a phosphate source, a magnesium source,
trace metals, vitamins, and an antifoam agent. 18. The method of
embodiment 1 where step (b) comprises continuous or batch addition
of additional fermentation material to the second medium comprising
the engineered yeast. 19. The method of embodiment 1 where step (b)
is performed at a time of 2 hours or later from an initial
culturing of the engineered yeast. 20. The method of embodiment 1
where step (b) is performed up to a time of 150 hours from an
initial culturing of the engineered yeast. 21. The method of
embodiment 20 where step (b) is performed at a time of 10 hours or
later, and up to 96 hours, from an initial culturing of the
engineered yeast. 22. The method of embodiment 21 where step (b) is
performed at a time of 24 hours or later, and up to 72 hours, from
an initial culturing of the engineered yeast. 23. The method of
embodiment 1 wherein the engineered yeast produce an amount of the
one or more steviol glycoside(s) in step (b) at the second pH that
is 10% or greater than an amount of the one or more steviol
glycoside(s) produced when engineered yeast are maintained at the
first pH throughout fermentation. 24. The method of embodiment 23
wherein the engineered yeast produce an amount of the one or more
steviol glycoside(s) in step (b) at the second pH that is 20% or
greater than an amount of the one or more steviol glycoside(s)
produced when engineered yeast are maintained at the first pH
throughout fermentation. 25. The method of embodiment 1 wherein the
one or more steviol glycoside(s) comprise rebaudioside M,
rebaudioside D, or both rebaudioside M and rebaudioside D. 26. The
method of embodiment 25 wherein the engineered yeast produce a
ratio of rebaudioside D to rebaudioside M in step (b) at the second
pH that is greater than a ratio of rebaudioside D to rebaudioside M
produced when engineered yeast are maintained at the first pH
throughout fermentation. 27. The method of embodiment 26 wherein
the ratio of rebaudioside D to rebaudioside M in step (b) is 1:20
or greater. 28. The method of embodiment 27 wherein the ratio of
rebaudioside D to rebaudioside M in step (b) is in the range of
1:20 to 1:1. 29. The method of embodiment 1 wherein the engineered
yeast is selected from the group consisting of species of Candida,
Kloeckera (Hanseniaspora), Kluyveromyces, Lipomyces, Pichia
(Hansenula), Rhodotorula, Saccharomycete, Saccharomyces,
Schizosaccharomyces, Torulopsis, Torulaspora, Yarrowia, and
Zygosaccharomyces. 30. The method of embodiment 29 wherein the
engineered yeast is Saccharomyces cerevisiae. 31. The method of
embodiment 1 wherein the engineered yeast expresses one or more
exogenous nucleic acid(s) encoding one or more of the following
proteins heterologous to the yeast: GGPPS polypeptide, an
enf-copalyl diphosphate synthase (CDPS) polypeptide, a kaurene
oxidase (KO) polypeptide, a kaurene synthase (KS) polypeptide; a
steviol synthase (KAH) polypeptide, a cytochrome P450 reductase
(CPR) polypeptide, a UGT74G1 polypeptide, a UGT76G1 polypeptide, a
UGT91 d2 polypeptide, and a EUGT11 polypeptide 32. The method of
embodiment 1 wherein the engineered yeast expresses one or more
exogenous nucleic acid(s) encoding one or more of the following
proteins heterologous to the yeast: a GGPPS polypeptide, a
truncated Zea mays CDPS polypeptide, an A. thaliana KS polypeptide
a S. rebaudiana KO polypeptide, an A. thaliana ATR2 polypeptide, an
O. sativa EUGT 11 polypeptide, a SrKAHe1 polypeptide, a S.
rebaudiana CPRS polypeptide, an S. rebaudiana UGT85C2 polypeptide,
an S. rebaudiana UGT74G1 polypeptide, a S. rebaudiana UGT76G1
polypeptide, a S. rebaudiana UGT91D2 variant or functional homolog,
and a UGT91D2e-b polypeptide. 33. The method of any of the previous
embodiments further comprising a step of providing a seed medium
comprising the engineered yeast, wherein the seed medium is
subsequently used to form the first medium at the first pH. 34. The
method of any of the previous embodiments wherein steps (a)-(c) are
performed in a single vessel. 35. The method of any one of
embodiments 1-33 wherein steps (a)-(c) are performed in a two or
more different vessels. 36. A method for producing steviol
glycoside, the method comprising a step of: fermenting an
engineered yeast at a pH of 6.0 or greater in a fermentation
medium, wherein the engineered yeast to produce one or more steviol
glycoside(s). 37. The method of embodiment 36 wherein the
fermentation medium comprises a nitrogen source selected from
ammonium hydroxide, urea, and ammonium sulfate. 38. The method of
embodiment 37 wherein the pH is in the range of 6.0 to 7.5. 39. The
method of embodiment 38 wherein the pH is in the range of 6.5 to
7.5. 40. The method of embodiment 36 wherein ammonium hydroxide or
urea is 90% (wt) or greater, or 95% (wt) or greater, of the
nitrogen source for fermentation of the engineered yeast in the
medium. 41. The method of embodiment 36 where the medium comprises
glucose, a nitrogen source, a potassium source, a magnesium source,
a phosphate source, a magnesium source, trace metals, vitamins, and
an antifoam agent. 42. The method of embodiment 36 further
comprising continuous or batch addition of additional fermentation
material to the medium comprising the engineered yeast. 43. The
method of embodiment 36 performed for a period of time of up to 150
hours. 44. The method of embodiment 43 performed for a period of
time in the range of 8 to 88 hours. 45. The method of embodiment 44
performed for a period of time in the range of 22 to 48 hours. 46.
The method of embodiment 36 wherein the one or more steviol
glycoside(s) comprise rebaudioside M, rebaudioside D, or both
rebaudioside M and rebaudioside D. 47. The method of embodiment 33
wherein the fermentation media has a biomass in the range of 20-120
g dcw/L. 48. A method for increasing the production of a first,
lower molecular weight steviol glycoside relative to a second,
higher molecular weight steviol glycoside in an engineered yeast,
the method comprising a step of fermenting engineered yeast capable
of producing one or more steviol glycoside(s) at a pH of 6.0 or
greater in a fermentation medium, wherein the engineered yeast to
produce a ratio of the first and second steviol glycosides at pH of
6.0 or greater that is greater than a ratio of the first and second
steviol glycosides produced at a pH that is less than 6.0. 49. The
method of embodiment 48 wherein the ratio of the first and second
steviol glycosides at pH of 6.0 or greater is 10% or greater than
the ratio of the first and second steviol glycosides produced at a
pH that is less than 6.0. 50. The method of embodiment 49 wherein
the ratio of the first and second steviol glycosides at pH of 6.0
or greater is 25% or greater than the ratio of the first and second
steviol glycosides produced at a pH that is less than 6.0. 51. The
method of embodiment 48 wherein the first steviol glycoside is
rebaudioside D and the second steviol glycoside is rebaudioside M.
52. A composition derived from a fermentation method which uses
engineered yeast to produce the one or more steviol glycoside(s),
the composition comprising rebaudioside D and rebaudioside M at a
ratio of 1:20 or greater, respectively. 53. The composition of
embodiment 52 comprising rebaudioside D and rebaudioside M at a
ratio in the range of 1:5 to 1:1, respectively. 54. The composition
of embodiment 53 comprising rebaudioside D and rebaudioside M at a
ratio in the range of 1:1.75 to 1:1, respectively. 55. The
composition of embodiment 54 comprising rebaudioside D and
rebaudioside Mat a ratio in the range of 1:1.5 to 1:1,
respectively. 56. The composition of any one of embodiments 52-55
which is a fermentation medium. 57. The composition of embodiment
56 wherein the fermentation medium has a rebaudioside D
concentration of 1 g/L or greater, 1.25 g/L or greater, 1.5 g/L or
greater, 1.75 g/L or greater, or 2.0 g/L or greater.
EXAMPLE 1
Production of Reb D and Reb M in Fed Batch Fermentation at Higher
pH with Urea or Ammonium Hydroxide as the Primary N Source
[0109] For inoculum preparation, the yeast strains B and strains C
were cultured in 150 mLs of seed flask medium in 1 liter shake
flasks at 250 rpm and 30.degree. C. for 20-24 hours.
TABLE-US-00002 TABLE 1 Seed Flask Medium Component Formula
Concentration Units Biospringer 0251 yeast extract 7.5 g/L Glucose
monohydrate C.sub.6H.sub.12O.sub.6* H.sub.2O 22.0 g/L
[0110] For the fermentation, 75 mLs of seed culture was transferred
into initial fermentation medium (Tables 2, 3 and 4) with a
starting volume of 0.75 liters. Fermentation was carried out in 2 L
New Brunswick BioFlo 310 fermentors. Temperature was maintained at
30.degree. C. throughout. The air flow rate was maintained such
that the dissolved oxygen was less than 20%, and the agitation rate
was automatically controlled to increase in a stepwise manner from
400 to 900 rpm during the fermentation. Glucose concentration was
kept limiting by controlling flow rates of feed medium (Table 5). A
2-phase feeding strategy involved an initial exponential phase
beginning at 10 hours with a growth rate of u=0.12 l/h while the
2nd phase of feeding (or feed phase II) started at 33 hours with a
constant flow rate of 0.180 mls/minute. Feeding was continued until
a final volume of 1.95 liters was obtained by 120 hours.
[0111] In one set of treatments with strain B, pH was controlled at
pH 5 with 12% NH4OH. Then in the 2nd feeding phase, pH was either
controlled at 5 or ramped up to pH 6 or pH 7 with NH4OH. Antifoam
addition was controlled by utilization of foam control probes with
10 wt % antifoam solution (Ivanhoe 1163B). See results in Table
6.
[0112] Results for strain C with pH control at pH 5 with 12% NH4OH
are shown in Table 7.
[0113] In another set of treatments with strain B, fermentation was
controlled at 1) pH 5.0 with 12% NH4OH or 2) with only 6.2N KOH (pH
drifted up to 7) with urea present in the feed. Results are shown
in Table 8.
[0114] The medium was based on Verduyn et al (Verduyn C, Postma E,
Scheffers W A, Van Dijken J P. Yeast. 1992 July; 8(7):501-17) with
modifications as described in Tables 2 through 5. For the urea
treatments, ammonium sulfate was increased to 15 g/L in the initial
fermentation medium and urea was added to 39 g/L in the
fermentation feed medium. KOH was used as the base for pH control
in the urea treatments instead of NH.sub.4OH.
TABLE-US-00003 TABLE 2 Initial Fermentation Medium Component
Formula Concentration Units Glucose monohydrate
C.sub.6H.sub.12O.sub.6* H.sub.2O 22.0 g/L Ammonium sulfate
(NH.sub.4).sub.2SO.sub.4 5.0 g/L Monobasic potassium
KH.sub.2PO.sub.4 3.0 g/L phosphate Magnesium sulfate MgSO.sub.4* 7
H.sub.2O 0.5 g/L heptahydrate Trace metals stock 10.0 ml/L Vitamin
stock 12.0 ml/L
TABLE-US-00004 TABLE 3 Trace Metals Stock Solution Component
Formula Concentration Units Disodium edetate
C.sub.10H.sub.14N.sub.2Na.sub.2O.sub.8 * 2H.sub.2O 15 g/L Zinc
sulfate ZnSO.sub.4 * 7H.sub.2O 4.5 g/L heptahydrate Manganese (II)
MnCl.sub.2 * 4H.sub.2O 1.026 g/L chloride tetrahydrate Cobalt (II)
chloride CoCl.sub.2 * 6H.sub.2O 0.32 g/L hexahydrate Copper (II)
sulfate CuSO.sub.4 * 5H.sub.2O 0.3 g/L heptahydrate Sodium
molybdate Na.sub.2MoO.sub.4 * 2H.sub.2O 0.4 g/L dihydrate Calcium
chloride CaCl.sub.2 * 2H.sub.2O 3 g/L dihydrate Iron (II) sulfate
FeSO.sub.4 * 7H.sub.2O 3 g/L heptahydrate Boric acid
H.sub.3BO.sub.3 1 g/L Potassium iodide KI 0.1 g/L
TABLE-US-00005 TABLE 4 Vitamin Stock Solution Component Formula
Concentration Units d-Biotin C.sub.10H.sub.16N.sub.2O.sub.3S 50
mg/L Calcium pantothenate C.sub.18H.sub.32CaN.sub.2O.sub.10 1000
mg/L Nicotinic acid C.sub.6H.sub.5NO.sub.2 1000 mg/L Thiamine
hydrochloride C.sub.12H.sub.17ClN.sub.4OS.cndot.HCl 1000 mg/L
Pyridoxine hydrochloride C.sub.8H.sub.11NO.sub.3.cndot.HCl 1000
mg/L p-aminobenzoic acid C.sub.7H.sub.7NO.sub.2 200 mg/L
myo-inositol C.sub.6H.sub.12O.sub.6 25000 mg/L
TABLE-US-00006 TABLE 5 Fermentation Feed Medium Component Formula
Concentration Units Glucose monohydrate C.sub.6H.sub.12O.sub.6*
H.sub.2O 660 g/L Urea (in urea treatments only) NH.sub.2CONH.sub.2
33 g/L Antifoam 1.3 g/L Potassium sulfate K.sub.2SO.sub.4 4.2 g/L
Sodium sulfate Na.sub.2SO.sub.4 0.336 g/L Magnesium sulfate
MgSO.sub.4 * 7H.sub.2O 6.12 g/L heptahydrate Monobasic potassium
KH.sub.2PO.sub.4 10.8 g/L phosphate Trace metal stock 14.4 mL/L
Vitamin stock 14.4 mL/L
[0115] Reb DM yields on glucose were calculated based on total
glucose utilized. Yield of Reb DM on biomass was based on cell dry
weight. Reb DM productivity was calculated based on summing Reb D
and Reb M concentrations and dividing by final fermentation time
which was determined as the time the feed medium was emptied.
Biomass determination of cell dry weights was based on the
filtration/oven method common in the art. Quantification of steviol
glycoside can be carried out by high performance liquid
chromatography (HPLC) analysis as described below, and compared
against calibration curves obtained using authentic standards
purchased from Chromadex.
[0116] 100 .mu.L of the fermentation media were pipetted into a 2
mL microcentrifuge tube. 900 .mu.L of 61% methanol (extraction
solvent) was added into the 2 ml microcentrifuge tube and agitated
by placing on a sample rotator for 10 min to extract the steviol
glycosides. The samples were then centrifuged at 10K rpm in a
microcentrifuge for 3 min and the clarified supernatant was
pipetted into an autosampler vial for analysis.
UHPLC Method for Glycoside Separation
[0117] [The steviol glycosides were separated using two Agilent
SB-C18 RRHD columns in series (2.1 mm.times.150 mm, 1.8 um) with a
stem filter assembly from Optimize Technologies installed as a
pre-column filter. The mobile phase used was channel A: 0.01%
trifluroacetic acid (TFA) in water and channel B acetonitrile. The
flow rate was 0.38 mL/min, the column temperature was 65.degree. C.
and the detection was performed at ultraviolet absorption of 210
nm. The gradient elution profile is shown below:
TABLE-US-00007 % % Channel Channel Time A B 0 85 15 0.5 85 15 30 75
25 40 65 35 49 47 53 49.1 0 100 58 0 100 58.1 85 15 62 85 15
[0118] Calibration was performed using Reb A (98.85% purity) from
Cargill, Inc lot 1008-005 in 55% MeOH at the following
concentrations: 0.35, 0.175, 0.07, 0.035, 0.014, 0.007 mg/mL. All
glycosides are quantitated off of the Reb A curve. Experimental
correction factors for Reb D, Reb M, and Reb B were determined
against Reb A while all other analytes are corrected by molecular
weight. Attached is an example of a typical fermentation broth.
TABLE-US-00008 TABLES 6a and 6b SG production for Strain B with
NH.sub.4OH and higher pH setpoints % of Control (pH 5 with NH4OH)
Yield of Yield of Reb DM Reb DM Reb DM Treatment Reb D Reb M on
glucose on biomass Productivity Biomass NH4OH with pH 5 throughout
100.0 100.0 100.0 100.0 100.0 100.0 NH4OH with pH 6 in feed phase
II 207.7 153.1 166.7 173.1 169.6 101.2 NH4OH with pH 7 in feed
phase II 195.6 126.3 144.4 219.2 156.5 68.9
TABLE-US-00009 TABLE 6b % of Control (pH 5 with NH4OH) Reb D Reb M
Yield of Yield of titer titer Reb D to Reb DM Reb DM Reb DM
Treatment (g/L) (g/L) Reb M ratio on glucose on biomass
Productivity Biomass NH4OH with pH 5 throughout 0.91 1.79 1:1.96
100.0 100.0 100.0 100.0 NH4OH with pH 6 in feed phase II 1.89 2.74
1:1.45 166.7 173.1 169.6 101.2 NH4OH with pH 7 in feed phase II
1.78 2.26 1:1.27 144.4 219.2 156.5 68.9
[0119] In Tables 6a and 6b, % Reb DM yield on glucose=Reb DM in
g/L/glucose consumed in g/L*100. % Reb DM yield on biomass=Reb DM
in g/L/biomass produced in g cell dry weight (CDW or DCW)/L*100.
Reb D and Reb M titers originally measured in in g/L. Comparison to
control is the treatment value divided by the control value*100%.
For example, Reb D of treatment in g/L divided by Reb D of
control*100% equals % of control.
Tables 7a and 7b--SG Production for Strain C with NH.sub.4OH and a
High pH Setpoint
TABLE-US-00010 [0120] % of Control (pH 5 with NH.sub.4OH) Strain
Treatment Reb DM C NH.sub.4OH with pH 5 100.0 throughout C
NH.sub.4OH with pH 6 in 135.0 feed phase II
TABLE-US-00011 TABLE 7B Reb D titer Reb M titer Reb D to Strain pH
(g/L) (g/L) Reb M ratio C pH 5 0.74 4.15 1:5.6 throughout C pH 6 in
feed 1.51 5.10 1:3.3 phase II
Tables 8a and 8b--SG Production for Strain B with Urea and Higher
pH
TABLE-US-00012 [0121] % of Control Treatment Yield of Yield of
Primary Reb DM Reb DM Reb DM pH during fermentation N source Reb D
Reb M on glucose on biomass productivity Biomass pH 5 throughout
(control) NH.sub.4OH 100.0 100.0 100.0 100.0 100.0 100.0 pH 5 then
drift to pH 7 Urea 202.7 117.0 145.4 182.3 151.2 79.8 in 2nd feed
phase
TABLE-US-00013 % of Control Treatment Reb D to Yield of Yield of
Primary Reb D Reb M Reb M Reb DM Reb DM Reb DM pH during
fermentation N source (g/L) (g/L) ratio on glucose on biomass
productivity Biomass pH 5 throughout (control) NH.sub.4OH 1.22 2.47
1:2.0 100.0 100.0 100.0 100.0 pH 5 then drift to pH 7 Urea 2.48
2.89 1:1.16 145.4 182.3 151.2 79.8 in 2nd feed phase
[0122] Fermentation of the engineered yeast at the higher pH
provided increased titers, production rates and yields, and
increased specific rates of steviol glycosides Reb D and Reb M. The
higher pH fermentation also provided increased ratios of Reb D:Reb
M. The increased titer of Reb D and Reb M was observed with
multiple strains at the higher pH conditions.
EXAMPLE 2
Production of Reb D and Reb M in Fed Batch Fermentation at Higher
pH
[0123] Fed-batch fermentation was carried out aerobically in 2 L
(working volume) fermenters. 500 mL of initial mineral medium
(Table 9) was inoculated using a seed culture grown in the same
medium to reach an initial OD of 0.2. The culture was run in batch
mode for 18 hours and then operated in fed-batch mode during
.about.110 hours using a 4 phase exponential feed profile and the
feed medium described in Table 2. Glucose was utilized as the
carbon and energy source and its concentration was kept limiting by
controlling flow rates in order to allow for a fully respiratory
metabolism (minimizing ethanol formation). Air flow was kept at
.about.1 vvm during the whole process and stirring set to 800 rpm
during the first 42 hours then increased and maintained at 1200 rpm
for the rest of the process. The temperature was controlled to
30.degree. C. throughout the fermentation.
[0124] In the default fermentation set-up, the pH was controlled at
pH 5.0 using 8% NH4OH during the first 42 hours and then moving to
16% NH4OH for the rest of the process.
[0125] In one set of treatments, fermentation was initially
controlled at pH 5.0 using 8% NH4OH during the first 42 hours and
then ramped up to pH 6.0 in a time interval of 5 h (0.2 pH units
increase per hour) until the end of the process using 16% NH4OH.
See the results shown in Table 13. In a second set of treatments,
the pH of the fermentation was controlled at pH 6.0 throughout the
process using NH4OH during the first 42 hours and 16% NH4OH from
that moment onwards. See the results shown in Table 14.
[0126] In all the sets of conditions described above, 700 mL of
feed medium were employed.
[0127] Whole culture samples (without cell removal) were taken and
boiled in an equal volume of DMSO for Reb D and Reb M levels as
described in Example 1.
TABLE-US-00014 TABLE 9 Initial mineral fermentation medium
Concentration (NH.sub.4).sub.2SO.sub.4 5 g/L KH.sub.2PO.sub.4 3
MgSO.sub.4*7 H2O 0.5 Glucose monohydrate 22 Antifoam 204 0.8 mL/L
Trace metal stock 10 (Table 3) Vitamin stock 12 (Table 4)
TABLE-US-00015 TABLE 10 Fermentation feed medium Concentration
KH.sub.2PO.sub.4 10.8 g/L MgSO.sub.4*7 H.sub.2O 6.12
K.sub.2SO.sub.4 4.2 Na.sub.2SO.sub.4 0.336 Glucose monohydrate 660
Antifoam 204 1 mL/L Trace metal stock 14.4 (Table 3) Vitamin stock
14.4 (Table 4)
TABLE-US-00016 TABLE 11 Trace metal stock solution Concentration
Na.sub.2-EDTA 15 g/L ZnSO.sub.4 * 7H.sub.2O 4.5 MnCl.sub.2 *
2H.sub.2O 0.84 CoCl.sub.2 * 6H.sub.2O 0.32 CuSO.sub.4 * 5H.sub.2O
0.3 Na.sub.2MoO4 * 2H.sub.2O 0.4 CaCl.sub.2 2.265 FeSO4 * 7H.sub.2O
3.0 H.sub.3BO.sub.3 1.0 KI 0.1
TABLE-US-00017 TABLE 12 Vitamin stock solution Concentration
d-biotin 0.05 g/L Calcium pantothenate 1.0 Nicotinic acid 1.0
Thiamine-HCl 1.0 Pyridoxine-HCl 1.0 4-aminobenzoic acid 0.2
Myo-inositol 25.0
[0128] A summary of results for fermentations performed using yeast
strain E at pH 5.0 (control) vs. high pH is shown in Table 13.
Normalized results are presented for all steviol glycosides
measured in the total broth sample.
TABLE-US-00018 TABLE 13 RebD RebM RebD RebM RebD/rebM Strain pH (%)
(%) (g/L) (g/L) ratio E 5.0 100 100 0.73 3.1 0.24 5.0->6.0 189
123 1.38 3.84 0.36
[0129] A summary of results for fermentations performed using yeast
strain D at pH 5.0 (control) vs. high pH is shown in Table 14.
Normalized results for all steviol glycosides measured in the total
broth sample are presented in the table below.
TABLE-US-00019 TABLE 14 RebD RebM RebD RebM RebD/rebM Strain pH (%)
(%) (g/L) (g/L) ratio D 5.0 100 100 1.61 2.47 1.54 throughout 6.0
134 103 2.15 2.54 1.18 throughout
[0130] In this example all of the fermentation elapsed times were
equivalent and the amount of substrate fed (glucose) was the same
in all cases. Therefore, operating the fermentation process at pH
6.0, both from the beginning of the batch phase or performing a
ramp up after the first 42 hours resulted in, increased titers of
Reb D and M with additional strains, increased yields on glucose,
and increased production rates.
Sequence CWU 1
1
221460PRTStevia rebaudiana 1Met Ala Glu Gln Gln Lys Ile Lys Lys Ser
Pro His Val Leu Leu Ile1 5 10 15Pro Phe Pro Leu Gln Gly His Ile Asn
Pro Phe Ile Gln Phe Gly Lys 20 25 30Arg Leu Ile Ser Lys Gly Val Lys
Thr Thr Leu Val Thr Thr Ile His 35 40 45Thr Leu Asn Ser Thr Leu Asn
His Ser Asn Thr Thr Thr Thr Ser Ile 50 55 60Glu Ile Gln Ala Ile Ser
Asp Gly Cys Asp Glu Gly Gly Phe Met Ser65 70 75 80Ala Gly Glu Ser
Tyr Leu Glu Thr Phe Lys Gln Val Gly Ser Lys Ser 85 90 95Leu Ala Asp
Leu Ile Lys Lys Leu Gln Ser Glu Gly Thr Thr Ile Asp 100 105 110Ala
Ile Ile Tyr Asp Ser Met Thr Glu Trp Val Leu Asp Val Ala Ile 115 120
125Glu Phe Gly Ile Asp Gly Gly Ser Phe Phe Thr Gln Ala Cys Val Val
130 135 140Asn Ser Leu Tyr Tyr His Val His Lys Gly Leu Ile Ser Leu
Pro Leu145 150 155 160Gly Glu Thr Val Ser Val Pro Gly Phe Pro Val
Leu Gln Arg Trp Glu 165 170 175Thr Pro Leu Ile Leu Gln Asn His Glu
Gln Ile Gln Ser Pro Trp Ser 180 185 190Gln Met Leu Phe Gly Gln Phe
Ala Asn Ile Asp Gln Ala Arg Trp Val 195 200 205Phe Thr Asn Ser Phe
Tyr Lys Leu Glu Glu Glu Val Ile Glu Trp Thr 210 215 220Arg Lys Ile
Trp Asn Leu Lys Val Ile Gly Pro Thr Leu Pro Ser Met225 230 235
240Tyr Leu Asp Lys Arg Leu Asp Asp Asp Lys Asp Asn Gly Phe Asn Leu
245 250 255Tyr Lys Ala Asn His His Glu Cys Met Asn Trp Leu Asp Asp
Lys Pro 260 265 270Lys Glu Ser Val Val Tyr Val Ala Phe Gly Ser Leu
Val Lys His Gly 275 280 285Pro Glu Gln Val Glu Glu Ile Thr Arg Ala
Leu Ile Asp Ser Asp Val 290 295 300Asn Phe Leu Trp Val Ile Lys His
Lys Glu Glu Gly Lys Leu Pro Glu305 310 315 320Asn Leu Ser Glu Val
Ile Lys Thr Gly Lys Gly Leu Ile Val Ala Trp 325 330 335Cys Lys Gln
Leu Asp Val Leu Ala His Glu Ser Val Gly Cys Phe Val 340 345 350Thr
His Cys Gly Phe Asn Ser Thr Leu Glu Ala Ile Ser Leu Gly Val 355 360
365Pro Val Val Ala Met Pro Gln Phe Ser Asp Gln Thr Thr Asn Ala Lys
370 375 380Leu Leu Asp Glu Ile Leu Gly Val Gly Val Arg Val Lys Ala
Asp Glu385 390 395 400Asn Gly Ile Val Arg Arg Gly Asn Leu Ala Ser
Cys Ile Lys Met Ile 405 410 415Met Glu Glu Glu Arg Gly Val Ile Ile
Arg Lys Asn Ala Val Lys Trp 420 425 430Lys Asp Leu Ala Lys Val Ala
Val His Glu Gly Gly Ser Ser Asp Asn 435 440 445Asp Ile Val Glu Phe
Val Ser Glu Leu Ile Lys Ala 450 455 4602481PRTStevia rebaudiana
2Met Asp Ala Met Ala Thr Thr Glu Lys Lys Pro His Val Ile Phe Ile1 5
10 15Pro Phe Pro Ala Gln Ser His Ile Lys Ala Met Leu Lys Leu Ala
Gln 20 25 30Leu Leu His His Lys Gly Leu Gln Ile Thr Phe Val Asn Thr
Asp Phe 35 40 45Ile His Asn Gln Phe Leu Glu Ser Ser Gly Pro His Cys
Leu Asp Gly 50 55 60Ala Pro Gly Phe Arg Phe Glu Thr Ile Pro Asp Gly
Val Ser His Ser65 70 75 80Pro Glu Ala Ser Ile Pro Ile Arg Glu Ser
Leu Leu Arg Ser Ile Glu 85 90 95Thr Asn Phe Leu Asp Arg Phe Ile Asp
Leu Val Thr Lys Leu Pro Asp 100 105 110Pro Pro Thr Cys Ile Ile Ser
Asp Gly Phe Leu Ser Val Phe Thr Ile 115 120 125Asp Ala Ala Lys Lys
Leu Gly Ile Pro Val Met Met Tyr Trp Thr Leu 130 135 140Ala Ala Cys
Gly Phe Met Gly Phe Tyr His Ile His Ser Leu Ile Glu145 150 155
160Lys Gly Phe Ala Pro Leu Lys Asp Ala Ser Tyr Leu Thr Asn Gly Tyr
165 170 175Leu Asp Thr Val Ile Asp Trp Val Pro Gly Met Glu Gly Ile
Arg Leu 180 185 190Lys Asp Phe Pro Leu Asp Trp Ser Thr Asp Leu Asn
Asp Lys Val Leu 195 200 205Met Phe Thr Thr Glu Ala Pro Gln Arg Ser
His Lys Val Ser His His 210 215 220Ile Phe His Thr Phe Asp Glu Leu
Glu Pro Ser Ile Ile Lys Thr Leu225 230 235 240Ser Leu Arg Tyr Asn
His Ile Tyr Thr Ile Gly Pro Leu Gln Leu Leu 245 250 255Leu Asp Gln
Ile Pro Glu Glu Lys Lys Gln Thr Gly Ile Thr Ser Leu 260 265 270His
Gly Tyr Ser Leu Val Lys Glu Glu Pro Glu Cys Phe Gln Trp Leu 275 280
285Gln Ser Lys Glu Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser Thr
290 295 300Thr Val Met Ser Leu Glu Asp Met Thr Glu Phe Gly Trp Gly
Leu Ala305 310 315 320Asn Ser Asn His Tyr Phe Leu Trp Ile Ile Arg
Ser Asn Leu Val Ile 325 330 335Gly Glu Asn Ala Val Leu Pro Pro Glu
Leu Glu Glu His Ile Lys Lys 340 345 350Arg Gly Phe Ile Ala Ser Trp
Cys Ser Gln Glu Lys Val Leu Lys His 355 360 365Pro Ser Val Gly Gly
Phe Leu Thr His Cys Gly Trp Gly Ser Thr Ile 370 375 380Glu Ser Leu
Ser Ala Gly Val Pro Met Ile Cys Trp Pro Tyr Ser Trp385 390 395
400Asp Gln Leu Thr Asn Cys Arg Tyr Ile Cys Lys Glu Trp Glu Val Gly
405 410 415Leu Glu Met Gly Thr Lys Val Lys Arg Asp Glu Val Lys Arg
Leu Val 420 425 430Gln Glu Leu Met Gly Glu Gly Gly His Lys Met Arg
Asn Lys Ala Lys 435 440 445Asp Trp Lys Glu Lys Ala Arg Ile Ala Ile
Ala Pro Asn Gly Ser Ser 450 455 460Ser Leu Asn Ile Asp Lys Met Val
Lys Glu Ile Thr Val Leu Ala Arg465 470 475 480Asn3458PRTStevia
rebaudiana 3Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg
Ile Ile1 5 10 15Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile
Leu Gln Leu 20 25 30Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr
Ile Phe His Thr 35 40 45Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro
His Phe Thr Phe Arg 50 55 60Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu
Arg Ile Ser Asn Leu Pro65 70 75 80Thr His Gly Pro Leu Ala Gly Met
Arg Ile Pro Ile Ile Asn Glu His 85 90 95Gly Ala Asp Glu Leu Arg Arg
Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110Glu Glu Asp Glu Glu
Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125Phe Ala Gln
Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140Met
Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln145 150
155 160Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu
Glu 165 170 175Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp
Ile Lys Ser 180 185 190Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile
Leu Gly Lys Met Ile 195 200 205Lys Gln Thr Lys Ala Ser Ser Gly Val
Ile Trp Asn Ser Phe Lys Glu 210 215 220Leu Glu Glu Ser Glu Leu Glu
Thr Val Ile Arg Glu Ile Pro Ala Pro225 230 235 240Ser Phe Leu Ile
Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255Leu Leu
Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265
270Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp
275 280 285Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser
Lys Gln 290 295 300Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys
Gly Ser Thr Trp305 310 315 320Val Glu Pro Leu Pro Asp Gly Phe Leu
Gly Glu Arg Gly Arg Ile Val 325 330 335Lys Trp Val Pro Gln Gln Glu
Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350Phe Trp Thr His Ser
Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365Gly Val Pro
Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380Ala
Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn385 390
395 400Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met
Val 405 410 415Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val
Leu Lys Gln 420 425 430Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser
Ser Tyr Glu Ser Leu 435 440 445Glu Ser Leu Val Ser Tyr Ile Ser Ser
Leu 450 4554473PRTStevia rebaudiana 4Met Ala Thr Ser Asp Ser Ile
Val Asp Asp Arg Lys Gln Leu His Val1 5 10 15Ala Thr Phe Pro Trp Leu
Ala Phe Gly His Ile Leu Pro Tyr Leu Gln 20 25 30Leu Ser Lys Leu Ile
Ala Glu Lys Gly His Lys Val Ser Phe Leu Ser 35 40 45Thr Thr Arg Asn
Ile Gln Arg Leu Ser Ser His Ile Ser Pro Leu Ile 50 55 60Asn Val Val
Gln Leu Thr Leu Pro Arg Val Gln Glu Leu Pro Glu Asp65 70 75 80Ala
Glu Ala Thr Thr Asp Val His Pro Glu Asp Ile Pro Tyr Leu Lys 85 90
95Lys Ala Ser Asp Gly Leu Gln Pro Glu Val Thr Arg Phe Leu Glu Gln
100 105 110His Ser Pro Asp Trp Ile Ile Tyr Asp Tyr Thr His Tyr Trp
Leu Pro 115 120 125Ser Ile Ala Ala Ser Leu Gly Ile Ser Arg Ala His
Phe Ser Val Thr 130 135 140Thr Pro Trp Ala Ile Ala Tyr Met Gly Pro
Ser Ala Asp Ala Met Ile145 150 155 160Asn Gly Ser Asp Gly Arg Thr
Thr Val Glu Asp Leu Thr Thr Pro Pro 165 170 175Lys Trp Phe Pro Phe
Pro Thr Lys Val Cys Trp Arg Lys His Asp Leu 180 185 190Ala Arg Leu
Val Pro Tyr Lys Ala Pro Gly Ile Ser Asp Gly Tyr Arg 195 200 205Met
Gly Met Val Leu Lys Gly Ser Asp Cys Leu Leu Ser Lys Cys Tyr 210 215
220His Glu Phe Gly Thr Gln Trp Leu Pro Leu Leu Glu Thr Leu His
Gln225 230 235 240Val Pro Val Val Pro Val Gly Leu Leu Pro Pro Glu
Ile Pro Gly Asp 245 250 255Glu Lys Asp Glu Thr Trp Val Ser Ile Lys
Lys Trp Leu Asp Gly Lys 260 265 270Gln Lys Gly Ser Val Val Tyr Val
Ala Leu Gly Ser Glu Ala Leu Val 275 280 285Ser Gln Thr Glu Val Val
Glu Leu Ala Leu Gly Leu Glu Leu Ser Gly 290 295 300Leu Pro Phe Val
Trp Ala Tyr Arg Lys Pro Lys Gly Pro Ala Lys Ser305 310 315 320Asp
Ser Val Glu Leu Pro Asp Gly Phe Val Glu Arg Thr Arg Asp Arg 325 330
335Gly Leu Val Trp Thr Ser Trp Ala Pro Gln Leu Arg Ile Leu Ser His
340 345 350Glu Ser Val Cys Gly Phe Leu Thr His Cys Gly Ser Gly Ser
Ile Val 355 360 365Glu Gly Leu Met Phe Gly His Pro Leu Ile Met Leu
Pro Ile Phe Gly 370 375 380Asp Gln Pro Leu Asn Ala Arg Leu Leu Glu
Asp Lys Gln Val Gly Ile385 390 395 400Glu Ile Pro Arg Asn Glu Glu
Asp Gly Cys Leu Thr Lys Glu Ser Val 405 410 415Ala Arg Ser Leu Arg
Ser Val Val Val Glu Lys Glu Gly Glu Ile Tyr 420 425 430Lys Ala Asn
Ala Arg Glu Leu Ser Lys Ile Tyr Asn Asp Thr Lys Val 435 440 445Glu
Lys Glu Tyr Val Ser Gln Phe Val Asp Tyr Leu Glu Lys Asn Ala 450 455
460Arg Ala Val Ala Ile Asp His Glu Ser465 47051422DNAArtificial
SequenceSynthetic oligonucleotide 5atggctactt ctgattccat cgttgacgat
agaaagcaat tgcatgttgc tacttttcca 60tggttggctt tcggtcatat tttgccatac
ttgcaattgt ccaagttgat tgctgaaaag 120ggtcacaagg tttcattctt
gtctaccacc agaaacatcc aaagattgtc ctctcatatc 180tccccattga
tcaacgttgt tcaattgact ttgccaagag tccaagaatt gccagaagat
240gctgaagcta ctactgatgt tcatccagaa gatatccctt acttgaaaaa
ggcttccgat 300ggtttacaac cagaagttac tagattcttg gaacaacatt
ccccagattg gatcatctac 360gattatactc attactggtt gccatccatt
gctgcttcat tgggtatttc tagagcccat 420ttctctgtta ctactccatg
ggctattgct tatatgggtc catctgctga tgctatgatt 480aacggttctg
atggtagaac taccgttgaa gatttgacta ctccaccaaa gtggtttcca
540tttccaacaa aagtctgttg gagaaaacac gatttggcta gattggttcc
atacaaagct 600ccaggtattt ctgatggtta cagaatgggt atggttttga
aaggttccga ttgcttgttg 660tctaagtgct atcatgaatt cggtactcaa
tggttgcctt tgttggaaac attgcatcaa 720gttccagttg ttccagtagg
tttgttgcca ccagaaattc caggtgacga aaaagacgaa 780acttgggttt
ccatcaaaaa gtggttggat ggtaagcaaa agggttctgt tgtttatgtt
840gctttgggtt ccgaagcttt ggtttctcaa accgaagttg ttgaattggc
tttgggtttg 900gaattgtctg gtttgccatt tgtttgggct tacagaaaac
ctaaaggtcc agctaagtct 960gattctgttg aattgccaga tggtttcgtt
gaaagaacta gagatagagg tttggtttgg 1020acttcttggg ctccacaatt
gagaattttg tctcatgaat ccgtctgtgg tttcttgact 1080cattgtggtt
ctggttctat cgttgaaggt ttgatgtttg gtcacccatt gattatgttg
1140ccaatctttg gtgaccaacc attgaacgct agattattgg aagataagca
agtcggtatc 1200gaaatcccaa gaaatgaaga agatggttgc ttgaccaaag
aatctgttgc tagatctttg 1260agatccgttg tcgttgaaaa agaaggtgaa
atctacaagg ctaacgctag agaattgtcc 1320aagatctaca acgataccaa
ggtcgaaaaa gaatacgttt cccaattcgt tgactacttg 1380gaaaagaatg
ctagagctgt tgccattgat catgaatctt ga 14226297PRTSynechococcus sp.
6Met Val Ala Gln Thr Phe Asn Leu Asp Thr Tyr Leu Ser Gln Arg Gln1 5
10 15Gln Gln Val Glu Glu Ala Leu Ser Ala Ala Leu Val Pro Ala Tyr
Pro 20 25 30Glu Arg Ile Tyr Glu Ala Met Arg Tyr Ser Leu Leu Ala Gly
Gly Lys 35 40 45Arg Leu Arg Pro Ile Leu Cys Leu Ala Ala Cys Glu Leu
Ala Gly Gly 50 55 60Ser Val Glu Gln Ala Met Pro Thr Ala Cys Ala Leu
Glu Met Ile His65 70 75 80Thr Met Ser Leu Ile His Asp Asp Leu Pro
Ala Met Asp Asn Asp Asp 85 90 95Phe Arg Arg Gly Lys Pro Thr Asn His
Lys Val Phe Gly Glu Asp Ile 100 105 110Ala Ile Leu Ala Gly Asp Ala
Leu Leu Ala Tyr Ala Phe Glu His Ile 115 120 125Ala Ser Gln Thr Arg
Gly Val Pro Pro Gln Leu Val Leu Gln Val Ile 130 135 140Ala Arg Ile
Gly His Ala Val Ala Ala Thr Gly Leu Val Gly Gly Gln145 150 155
160Val Val Asp Leu Glu Ser Glu Gly Lys Ala Ile Ser Leu Glu Thr Leu
165 170 175Glu Tyr Ile His Ser His Lys Thr Gly Ala Leu Leu Glu Ala
Ser Val 180 185 190Val Ser Gly Gly Ile Leu Ala Gly Ala Asp Glu Glu
Leu Leu Ala Arg 195 200 205Leu Ser His Tyr Ala Arg Asp Ile Gly Leu
Ala Phe Gln Ile Val Asp 210 215 220Asp Ile Leu Asp Val Thr Ala Thr
Ser Glu Gln Leu Gly Lys Thr Ala225 230 235 240Gly Lys Asp Gln Ala
Ala Ala Lys Ala Thr Tyr Pro Ser Leu Leu Gly 245 250 255Leu Glu Ala
Ser Arg Gln Lys Ala Glu Glu Leu Ile Gln Ser Ala Lys 260 265 270Glu
Ala Leu Arg Pro Tyr Gly Ser Gln Ala Glu Pro Leu Leu Ala Leu 275 280
285Ala Asp Phe Ile Thr Arg Arg Gln His 290 2957827PRTZea mays 7Met
Val Leu Ser Ser Ser Cys Thr Thr Val Pro His Leu Ser Ser Leu1 5 10
15Ala Val Val Gln Leu Gly Pro Trp Ser Ser Arg Ile Lys Lys Lys Thr
20 25 30Asp Thr Val Ala Val Pro Ala Ala Ala Gly Arg Trp Arg Arg Ala
Leu 35
40 45Ala Arg Ala Gln His Thr Ser Glu Ser Ala Ala Val Ala Lys Gly
Ser 50 55 60Ser Leu Thr Pro Ile Val Arg Thr Asp Ala Glu Ser Arg Arg
Thr Arg65 70 75 80Trp Pro Thr Asp Asp Asp Asp Ala Glu Pro Leu Val
Asp Glu Ile Arg 85 90 95Ala Met Leu Thr Ser Met Ser Asp Gly Asp Ile
Ser Val Ser Ala Tyr 100 105 110Asp Thr Ala Trp Val Gly Leu Val Pro
Arg Leu Asp Gly Gly Glu Gly 115 120 125Pro Gln Phe Pro Ala Ala Val
Arg Trp Ile Arg Asn Asn Gln Leu Pro 130 135 140Asp Gly Ser Trp Gly
Asp Ala Ala Leu Phe Ser Ala Tyr Asp Arg Leu145 150 155 160Ile Asn
Thr Leu Ala Cys Val Val Thr Leu Thr Arg Trp Ser Leu Glu 165 170
175Pro Glu Met Arg Gly Arg Gly Leu Ser Phe Leu Gly Arg Asn Met Trp
180 185 190Lys Leu Ala Thr Glu Asp Glu Glu Ser Met Pro Ile Gly Phe
Glu Leu 195 200 205Ala Phe Pro Ser Leu Ile Glu Leu Ala Lys Ser Leu
Gly Val His Asp 210 215 220Phe Pro Tyr Asp His Gln Ala Leu Gln Gly
Ile Tyr Ser Ser Arg Glu225 230 235 240Ile Lys Met Lys Arg Ile Pro
Lys Glu Val Met His Thr Val Pro Thr 245 250 255Ser Ile Leu His Ser
Leu Glu Gly Met Pro Gly Leu Asp Trp Ala Lys 260 265 270Leu Leu Lys
Leu Gln Ser Ser Asp Gly Ser Phe Leu Phe Ser Pro Ala 275 280 285Ala
Thr Ala Tyr Ala Leu Met Asn Thr Gly Asp Asp Arg Cys Phe Ser 290 295
300Tyr Ile Asp Arg Thr Val Lys Lys Phe Asn Gly Gly Val Pro Asn
Val305 310 315 320Tyr Pro Val Asp Leu Phe Glu His Ile Trp Ala Val
Asp Arg Leu Glu 325 330 335Arg Leu Gly Ile Ser Arg Tyr Phe Gln Lys
Glu Ile Glu Gln Cys Met 340 345 350Asp Tyr Val Asn Arg His Trp Thr
Glu Asp Gly Ile Cys Trp Ala Arg 355 360 365Asn Ser Asp Val Lys Glu
Val Asp Asp Thr Ala Met Ala Phe Arg Leu 370 375 380Leu Arg Leu His
Gly Tyr Ser Val Ser Pro Asp Val Phe Lys Asn Phe385 390 395 400Glu
Lys Asp Gly Glu Phe Phe Ala Phe Val Gly Gln Ser Asn Gln Ala 405 410
415Val Thr Gly Met Tyr Asn Leu Asn Arg Ala Ser Gln Ile Ser Phe Pro
420 425 430Gly Glu Asp Val Leu His Arg Ala Gly Ala Phe Ser Tyr Glu
Phe Leu 435 440 445Arg Arg Lys Glu Ala Glu Gly Ala Leu Arg Asp Lys
Trp Ile Ile Ser 450 455 460Lys Asp Leu Pro Gly Glu Val Val Tyr Thr
Leu Asp Phe Pro Trp Tyr465 470 475 480Gly Asn Leu Pro Arg Val Glu
Ala Arg Asp Tyr Leu Glu Gln Tyr Gly 485 490 495Gly Gly Asp Asp Val
Trp Ile Gly Lys Thr Leu Tyr Arg Met Pro Leu 500 505 510Val Asn Asn
Asp Val Tyr Leu Glu Leu Ala Arg Met Asp Phe Asn His 515 520 525Cys
Gln Ala Leu His Gln Leu Glu Trp Gln Gly Leu Lys Arg Trp Tyr 530 535
540Thr Glu Asn Arg Leu Met Asp Phe Gly Val Ala Gln Glu Asp Ala
Leu545 550 555 560Arg Ala Tyr Phe Leu Ala Ala Ala Ser Val Tyr Glu
Pro Cys Arg Ala 565 570 575Ala Glu Arg Leu Ala Trp Ala Arg Ala Ala
Ile Leu Ala Asn Ala Val 580 585 590Ser Thr His Leu Arg Asn Ser Pro
Ser Phe Arg Glu Arg Leu Glu His 595 600 605Ser Leu Arg Cys Arg Pro
Ser Glu Glu Thr Asp Gly Ser Trp Phe Asn 610 615 620Ser Ser Ser Gly
Ser Asp Ala Val Leu Val Lys Ala Val Leu Arg Leu625 630 635 640Thr
Asp Ser Leu Ala Arg Glu Ala Gln Pro Ile His Gly Gly Asp Pro 645 650
655Glu Asp Ile Ile His Lys Leu Leu Arg Ser Ala Trp Ala Glu Trp Val
660 665 670Arg Glu Lys Ala Asp Ala Ala Asp Ser Val Cys Asn Gly Ser
Ser Ala 675 680 685Val Glu Gln Glu Gly Ser Arg Met Val His Asp Lys
Gln Thr Cys Leu 690 695 700Leu Leu Ala Arg Met Ile Glu Ile Ser Ala
Gly Arg Ala Ala Gly Glu705 710 715 720Ala Ala Ser Glu Asp Gly Asp
Arg Arg Ile Ile Gln Leu Thr Gly Ser 725 730 735Ile Cys Asp Ser Leu
Lys Gln Lys Met Leu Val Ser Gln Asp Pro Glu 740 745 750Lys Asn Glu
Glu Met Met Ser His Val Asp Asp Glu Leu Lys Leu Arg 755 760 765Ile
Arg Glu Phe Val Gln Tyr Leu Leu Arg Leu Gly Glu Lys Lys Thr 770 775
780Gly Ser Ser Glu Thr Arg Gln Thr Phe Leu Ser Ile Val Lys Ser
Cys785 790 795 800Tyr Tyr Ala Ala His Cys Pro Pro His Val Val Asp
Arg His Ile Ser 805 810 815Arg Val Ile Phe Glu Pro Val Ser Ala Ala
Lys 820 8258785PRTArabidopsis thaliana 8Met Ser Ile Asn Leu Arg Ser
Ser Gly Cys Ser Ser Pro Ile Ser Ala1 5 10 15Thr Leu Glu Arg Gly Leu
Asp Ser Glu Val Gln Thr Arg Ala Asn Asn 20 25 30Val Ser Phe Glu Gln
Thr Lys Glu Lys Ile Arg Lys Met Leu Glu Lys 35 40 45Val Glu Leu Ser
Val Ser Ala Tyr Asp Thr Ser Trp Val Ala Met Val 50 55 60Pro Ser Pro
Ser Ser Gln Asn Ala Pro Leu Phe Pro Gln Cys Val Lys65 70 75 80Trp
Leu Leu Asp Asn Gln His Glu Asp Gly Ser Trp Gly Leu Asp Asn 85 90
95His Asp His Gln Ser Leu Lys Lys Asp Val Leu Ser Ser Thr Leu Ala
100 105 110Ser Ile Leu Ala Leu Lys Lys Trp Gly Ile Gly Glu Arg Gln
Ile Asn 115 120 125Lys Gly Leu Gln Phe Ile Glu Leu Asn Ser Ala Leu
Val Thr Asp Glu 130 135 140Thr Ile Gln Lys Pro Thr Gly Phe Asp Ile
Ile Phe Pro Gly Met Ile145 150 155 160Lys Tyr Ala Arg Asp Leu Asn
Leu Thr Ile Pro Leu Gly Ser Glu Val 165 170 175Val Asp Asp Met Ile
Arg Lys Arg Asp Leu Asp Leu Lys Cys Asp Ser 180 185 190Glu Lys Phe
Ser Lys Gly Arg Glu Ala Tyr Leu Ala Tyr Val Leu Glu 195 200 205Gly
Thr Arg Asn Leu Lys Asp Trp Asp Leu Ile Val Lys Tyr Gln Arg 210 215
220Lys Asn Gly Ser Leu Phe Asp Ser Pro Ala Thr Thr Ala Ala Ala
Phe225 230 235 240Thr Gln Phe Gly Asn Asp Gly Cys Leu Arg Tyr Leu
Cys Ser Leu Leu 245 250 255Gln Lys Phe Glu Ala Ala Val Pro Ser Val
Tyr Pro Phe Asp Gln Tyr 260 265 270Ala Arg Leu Ser Ile Ile Val Thr
Leu Glu Ser Leu Gly Ile Asp Arg 275 280 285Asp Phe Lys Thr Glu Ile
Lys Ser Ile Leu Asp Glu Thr Tyr Arg Tyr 290 295 300Trp Leu Arg Gly
Asp Glu Glu Ile Cys Leu Asp Leu Ala Thr Cys Ala305 310 315 320Leu
Ala Phe Arg Leu Leu Leu Ala His Gly Tyr Asp Val Ser Tyr Asp 325 330
335Pro Leu Lys Pro Phe Ala Glu Glu Ser Gly Phe Ser Asp Thr Leu Glu
340 345 350Gly Tyr Val Lys Asn Thr Phe Ser Val Leu Glu Leu Phe Lys
Ala Ala 355 360 365Gln Ser Tyr Pro His Glu Ser Ala Leu Lys Lys Gln
Cys Cys Trp Thr 370 375 380Lys Gln Tyr Leu Glu Met Glu Leu Ser Ser
Trp Val Lys Thr Ser Val385 390 395 400Arg Asp Lys Tyr Leu Lys Lys
Glu Val Glu Asp Ala Leu Ala Phe Pro 405 410 415Ser Tyr Ala Ser Leu
Glu Arg Ser Asp His Arg Arg Lys Ile Leu Asn 420 425 430Gly Ser Ala
Val Glu Asn Thr Arg Val Thr Lys Thr Ser Tyr Arg Leu 435 440 445His
Asn Ile Cys Thr Ser Asp Ile Leu Lys Leu Ala Val Asp Asp Phe 450 455
460Asn Phe Cys Gln Ser Ile His Arg Glu Glu Met Glu Arg Leu Asp
Arg465 470 475 480Trp Ile Val Glu Asn Arg Leu Gln Glu Leu Lys Phe
Ala Arg Gln Lys 485 490 495Leu Ala Tyr Cys Tyr Phe Ser Gly Ala Ala
Thr Leu Phe Ser Pro Glu 500 505 510Leu Ser Asp Ala Arg Ile Ser Trp
Ala Lys Gly Gly Val Leu Thr Thr 515 520 525Val Val Asp Asp Phe Phe
Asp Val Gly Gly Ser Lys Glu Glu Leu Glu 530 535 540Asn Leu Ile His
Leu Val Glu Lys Trp Asp Leu Asn Gly Val Pro Glu545 550 555 560Tyr
Ser Ser Glu His Val Glu Ile Ile Phe Ser Val Leu Arg Asp Thr 565 570
575Ile Leu Glu Thr Gly Asp Lys Ala Phe Thr Tyr Gln Gly Arg Asn Val
580 585 590Thr His His Ile Val Lys Ile Trp Leu Asp Leu Leu Lys Ser
Met Leu 595 600 605Arg Glu Ala Glu Trp Ser Ser Asp Lys Ser Thr Pro
Ser Leu Glu Asp 610 615 620Tyr Met Glu Asn Ala Tyr Ile Ser Phe Ala
Leu Gly Pro Ile Val Leu625 630 635 640Pro Ala Thr Tyr Leu Ile Gly
Pro Pro Leu Pro Glu Lys Thr Val Asp 645 650 655Ser His Gln Tyr Asn
Gln Leu Tyr Lys Leu Val Ser Thr Met Gly Arg 660 665 670Leu Leu Asn
Asp Ile Gln Gly Phe Lys Arg Glu Ser Ala Glu Gly Lys 675 680 685Leu
Asn Ala Val Ser Leu His Met Lys His Glu Arg Asp Asn Arg Ser 690 695
700Lys Glu Val Ile Ile Glu Ser Met Lys Gly Leu Ala Glu Arg Lys
Arg705 710 715 720Glu Glu Leu His Lys Leu Val Leu Glu Glu Lys Gly
Ser Val Val Pro 725 730 735Arg Glu Cys Lys Glu Ala Phe Leu Lys Met
Ser Lys Val Leu Asn Leu 740 745 750Phe Tyr Arg Lys Asp Asp Gly Phe
Thr Ser Asn Asp Leu Met Ser Leu 755 760 765Val Lys Ser Val Ile Tyr
Glu Pro Val Ser Leu Gln Lys Glu Ser Leu 770 775
780Thr78591542DNAArtificial SequenceCodon-optimized KO 9atggatgctg
tgacgggttt gttaactgtc ccagcaaccg ctataactat tggtggaact 60gctgtagcat
tggcggtagc gctaatcttt tggtacctga aatcctacac atcagctaga
120agatcccaat caaatcatct tccaagagtg cctgaagtcc caggtgttcc
attgttagga 180aatctgttac aattgaagga gaaaaagcca tacatgactt
ttacgagatg ggcagcgaca 240tatggaccta tctatagtat caaaactggg
gctacaagta tggttgtggt atcatctaat 300gagatagcca aggaggcatt
ggtgaccaga ttccaatcca tatctacaag gaacttatct 360aaagccctga
aagtacttac agcagataag acaatggtcg caatgtcaga ttatgatgat
420tatcataaaa cagttaagag acacatactg accgccgtct tgggtcctaa
tgcacagaaa 480aagcatagaa ttcacagaga tatcatgatg gataacatat
ctactcaact tcatgaattc 540gtgaaaaaca acccagaaca ggaagaggta
gaccttagaa aaatctttca atctgagtta 600ttcggcttag ctatgagaca
agccttagga aaggatgttg aaagtttgta cgttgaagac 660ctgaaaatca
ctatgaatag agacgaaatc tttcaagtcc ttgttgttga tccaatgatg
720ggagcaatcg atgttgattg gagagacttc tttccatacc taaagtgggt
cccaaacaaa 780aagttcgaaa atactattca acaaatgtac atcagaagag
aagctgttat gaaatcttta 840atcaaagagc acaaaaagag aatagcgtca
ggcgaaaagc taaatagtta tatcgattac 900cttttatctg aagctcaaac
tttaaccgat cagcaactat tgatgtcctt gtgggaacca 960atcattgaat
cttcagatac aacaatggtc acaacagaat gggcaatgta cgaattagct
1020aaaaacccta aattgcaaga taggttgtac agagacatta agtccgtctg
tggatctgaa 1080aagataaccg aagagcatct atcacagctg ccttacatta
cagctatttt ccacgaaaca 1140ctgagaagac actcaccagt tcctatcatt
cctctaagac atgtacatga agataccgtt 1200ctaggcggct accatgttcc
tgctggcaca gaacttgccg ttaacatcta cggttgcaac 1260atggacaaaa
acgtttggga aaatccagag gaatggaacc cagaaagatt catgaaagag
1320aatgagacaa ttgattttca aaagacgatg gccttcggtg gtggtaagag
agtttgtgct 1380ggttccttgc aagccctttt aactgcatct attgggattg
ggagaatggt tcaagagttc 1440gaatggaaac tgaaggatat gactcaagag
gaagtgaaca cgataggcct aactacacaa 1500atgttaagac cattgagagc
tattatcaaa cctaggatct aa 154210513PRTStevia rebaudiana 10Met Asp
Ala Val Thr Gly Leu Leu Thr Val Pro Ala Thr Ala Ile Thr1 5 10 15Ile
Gly Gly Thr Ala Val Ala Leu Ala Val Ala Leu Ile Phe Trp Tyr 20 25
30Leu Lys Ser Tyr Thr Ser Ala Arg Arg Ser Gln Ser Asn His Leu Pro
35 40 45Arg Val Pro Glu Val Pro Gly Val Pro Leu Leu Gly Asn Leu Leu
Gln 50 55 60Leu Lys Glu Lys Lys Pro Tyr Met Thr Phe Thr Arg Trp Ala
Ala Thr65 70 75 80Tyr Gly Pro Ile Tyr Ser Ile Lys Thr Gly Ala Thr
Ser Met Val Val 85 90 95Val Ser Ser Asn Glu Ile Ala Lys Glu Ala Leu
Val Thr Arg Phe Gln 100 105 110Ser Ile Ser Thr Arg Asn Leu Ser Lys
Ala Leu Lys Val Leu Thr Ala 115 120 125Asp Lys Thr Met Val Ala Met
Ser Asp Tyr Asp Asp Tyr His Lys Thr 130 135 140Val Lys Arg His Ile
Leu Thr Ala Val Leu Gly Pro Asn Ala Gln Lys145 150 155 160Lys His
Arg Ile His Arg Asp Ile Met Met Asp Asn Ile Ser Thr Gln 165 170
175Leu His Glu Phe Val Lys Asn Asn Pro Glu Gln Glu Glu Val Asp Leu
180 185 190Arg Lys Ile Phe Gln Ser Glu Leu Phe Gly Leu Ala Met Arg
Gln Ala 195 200 205Leu Gly Lys Asp Val Glu Ser Leu Tyr Val Glu Asp
Leu Lys Ile Thr 210 215 220Met Asn Arg Asp Glu Ile Phe Gln Val Leu
Val Val Asp Pro Met Met225 230 235 240Gly Ala Ile Asp Val Asp Trp
Arg Asp Phe Phe Pro Tyr Leu Lys Trp 245 250 255Val Pro Asn Lys Lys
Phe Glu Asn Thr Ile Gln Gln Met Tyr Ile Arg 260 265 270Arg Glu Ala
Val Met Lys Ser Leu Ile Lys Glu His Lys Lys Arg Ile 275 280 285Ala
Ser Gly Glu Lys Leu Asn Ser Tyr Ile Asp Tyr Leu Leu Ser Glu 290 295
300Ala Gln Thr Leu Thr Asp Gln Gln Leu Leu Met Ser Leu Trp Glu
Pro305 310 315 320Ile Ile Glu Ser Ser Asp Thr Thr Met Val Thr Thr
Glu Trp Ala Met 325 330 335Tyr Glu Leu Ala Lys Asn Pro Lys Leu Gln
Asp Arg Leu Tyr Arg Asp 340 345 350Ile Lys Ser Val Cys Gly Ser Glu
Lys Ile Thr Glu Glu His Leu Ser 355 360 365Gln Leu Pro Tyr Ile Thr
Ala Ile Phe His Glu Thr Leu Arg Arg His 370 375 380Ser Pro Val Pro
Ile Ile Pro Leu Arg His Val His Glu Asp Thr Val385 390 395 400Leu
Gly Gly Tyr His Val Pro Ala Gly Thr Glu Leu Ala Val Asn Ile 405 410
415Tyr Gly Cys Asn Met Asp Lys Asn Val Trp Glu Asn Pro Glu Glu Trp
420 425 430Asn Pro Glu Arg Phe Met Lys Glu Asn Glu Thr Ile Asp Phe
Gln Lys 435 440 445Thr Met Ala Phe Gly Gly Gly Lys Arg Val Cys Ala
Gly Ser Leu Gln 450 455 460Ala Leu Leu Thr Ala Ser Ile Gly Ile Gly
Arg Met Val Gln Glu Phe465 470 475 480Glu Trp Lys Leu Lys Asp Met
Thr Gln Glu Glu Val Asn Thr Ile Gly 485 490 495Leu Thr Thr Gln Met
Leu Arg Pro Leu Arg Ala Ile Ile Lys Pro Arg 500 505
510Ile112139DNAArabidopsis thaliana 11atgtcttcct cttcctcttc
cagtacctct atgattgatt tgatggctgc tattattaaa 60ggtgaaccag ttatcgtctc
cgacccagca aatgcctctg cttatgaatc agttgctgca 120gaattgtctt
caatgttgat cgaaaacaga caattcgcca tgatcgtaac tacatcaatc
180gctgttttga tcggttgtat tgtcatgttg gtatggagaa gatccggtag
tggtaattct 240aaaagagtcg aacctttgaa accattagta attaagccaa
gagaagaaga aatagatgac 300ggtagaaaga aagttacaat atttttcggt
acccaaactg gtacagctga aggttttgca 360aaagccttag gtgaagaagc
taaggcaaga tacgaaaaga ctagattcaa gatagtcgat 420ttggatgact
atgccgctga tgacgatgaa tacgaagaaa agttgaagaa agaagatgtt
480gcatttttct ttttggcaac ctatggtgac ggtgaaccaa ctgacaatgc
agccagattc 540tacaaatggt ttacagaggg taatgatcgt ggtgaatggt
tgaaaaactt aaagtacggt 600gttttcggtt tgggtaacag acaatacgaa
catttcaaca aagttgcaaa ggttgtcgac 660gatattttgg tcgaacaagg
tgctcaaaga ttagtccaag taggtttggg tgacgatgac 720caatgtatag
aagatgactt tactgcctgg agagaagctt tgtggcctga attagacaca
780atcttgagag aagaaggtga caccgccgtt gctaccccat atactgctgc
agtattagaa 840tacagagttt ccatccatga tagtgaagac gcaaagttta
atgatatcac tttggccaat 900ggtaacggtt atacagtttt cgatgcacaa
cacccttaca aagctaacgt tgcagtcaag 960agagaattac atacaccaga
atccgacaga agttgtatac acttggaatt tgatatcgct 1020ggttccggtt
taaccatgaa gttgggtgac catgtaggtg ttttatgcga caatttgtct
1080gaaactgttg atgaagcatt gagattgttg gatatgtccc ctgacactta
ttttagtttg 1140cacgctgaaa aagaagatgg tacaccaatt tccagttctt
taccacctcc attccctcca 1200tgtaacttaa gaacagcctt gaccagatac
gcttgcttgt tatcatcccc taaaaagtcc 1260gccttggttg ctttagccgc
tcatgctagt gatcctactg aagcagaaag attgaaacac 1320ttagcatctc
cagccggtaa agatgaatat tcaaagtggg tagttgaatc tcaaagatca
1380ttgttagaag ttatggcaga atttccatct gccaagcctc cattaggtgt
cttctttgct 1440ggtgtagcac ctagattgca accaagattc tactcaatca
gttcttcacc taagatcgct 1500gaaactagaa ttcatgttac atgtgcatta
gtctacgaaa agatgccaac cggtagaatt 1560cacaagggtg tatgctctac
ttggatgaaa aatgctgttc cttacgaaaa atcagaaaag 1620ttgttcttag
gtagaccaat cttcgtaaga caatcaaact tcaagttgcc ttctgattca
1680aaggttccaa taatcatgat aggtcctggt acaggtttag ccccattcag
aggtttcttg 1740caagaaagat tggctttagt tgaatctggt gtcgaattag
gtccttcagt tttgttcttt 1800ggttgtagaa acagaagaat ggatttcatc
tatgaagaag aattgcaaag attcgtcgaa 1860tctggtgcat tggccgaatt
atctgtagct ttttcaagag aaggtccaac taaggaatac 1920gttcaacata
agatgatgga taaggcatcc gacatatgga acatgatcag tcaaggtgct
1980tatttgtacg tttgcggtga cgcaaagggt atggccagag atgtccatag
atctttgcac 2040acaattgctc aagaacaagg ttccatggat agtaccaaag
ctgaaggttt cgtaaagaac 2100ttacaaactt ccggtagata cttgagagat
gtctggtga 213912712PRTArabidopsis thaliana 12Met Ser Ser Ser Ser
Ser Ser Ser Thr Ser Met Ile Asp Leu Met Ala1 5 10 15Ala Ile Ile Lys
Gly Glu Pro Val Ile Val Ser Asp Pro Ala Asn Ala 20 25 30Ser Ala Tyr
Glu Ser Val Ala Ala Glu Leu Ser Ser Met Leu Ile Glu 35 40 45Asn Arg
Gln Phe Ala Met Ile Val Thr Thr Ser Ile Ala Val Leu Ile 50 55 60Gly
Cys Ile Val Met Leu Val Trp Arg Arg Ser Gly Ser Gly Asn Ser65 70 75
80Lys Arg Val Glu Pro Leu Lys Pro Leu Val Ile Lys Pro Arg Glu Glu
85 90 95Glu Ile Asp Asp Gly Arg Lys Lys Val Thr Ile Phe Phe Gly Thr
Gln 100 105 110Thr Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Gly Glu
Glu Ala Lys 115 120 125Ala Arg Tyr Glu Lys Thr Arg Phe Lys Ile Val
Asp Leu Asp Asp Tyr 130 135 140Ala Ala Asp Asp Asp Glu Tyr Glu Glu
Lys Leu Lys Lys Glu Asp Val145 150 155 160Ala Phe Phe Phe Leu Ala
Thr Tyr Gly Asp Gly Glu Pro Thr Asp Asn 165 170 175Ala Ala Arg Phe
Tyr Lys Trp Phe Thr Glu Gly Asn Asp Arg Gly Glu 180 185 190Trp Leu
Lys Asn Leu Lys Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln 195 200
205Tyr Glu His Phe Asn Lys Val Ala Lys Val Val Asp Asp Ile Leu Val
210 215 220Glu Gln Gly Ala Gln Arg Leu Val Gln Val Gly Leu Gly Asp
Asp Asp225 230 235 240Gln Cys Ile Glu Asp Asp Phe Thr Ala Trp Arg
Glu Ala Leu Trp Pro 245 250 255Glu Leu Asp Thr Ile Leu Arg Glu Glu
Gly Asp Thr Ala Val Ala Thr 260 265 270Pro Tyr Thr Ala Ala Val Leu
Glu Tyr Arg Val Ser Ile His Asp Ser 275 280 285Glu Asp Ala Lys Phe
Asn Asp Ile Thr Leu Ala Asn Gly Asn Gly Tyr 290 295 300Thr Val Phe
Asp Ala Gln His Pro Tyr Lys Ala Asn Val Ala Val Lys305 310 315
320Arg Glu Leu His Thr Pro Glu Ser Asp Arg Ser Cys Ile His Leu Glu
325 330 335Phe Asp Ile Ala Gly Ser Gly Leu Thr Met Lys Leu Gly Asp
His Val 340 345 350Gly Val Leu Cys Asp Asn Leu Ser Glu Thr Val Asp
Glu Ala Leu Arg 355 360 365Leu Leu Asp Met Ser Pro Asp Thr Tyr Phe
Ser Leu His Ala Glu Lys 370 375 380Glu Asp Gly Thr Pro Ile Ser Ser
Ser Leu Pro Pro Pro Phe Pro Pro385 390 395 400Cys Asn Leu Arg Thr
Ala Leu Thr Arg Tyr Ala Cys Leu Leu Ser Ser 405 410 415Pro Lys Lys
Ser Ala Leu Val Ala Leu Ala Ala His Ala Ser Asp Pro 420 425 430Thr
Glu Ala Glu Arg Leu Lys His Leu Ala Ser Pro Ala Gly Lys Asp 435 440
445Glu Tyr Ser Lys Trp Val Val Glu Ser Gln Arg Ser Leu Leu Glu Val
450 455 460Met Ala Glu Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe
Phe Ala465 470 475 480Gly Val Ala Pro Arg Leu Gln Pro Arg Phe Tyr
Ser Ile Ser Ser Ser 485 490 495Pro Lys Ile Ala Glu Thr Arg Ile His
Val Thr Cys Ala Leu Val Tyr 500 505 510Glu Lys Met Pro Thr Gly Arg
Ile His Lys Gly Val Cys Ser Thr Trp 515 520 525Met Lys Asn Ala Val
Pro Tyr Glu Lys Ser Glu Lys Leu Phe Leu Gly 530 535 540Arg Pro Ile
Phe Val Arg Gln Ser Asn Phe Lys Leu Pro Ser Asp Ser545 550 555
560Lys Val Pro Ile Ile Met Ile Gly Pro Gly Thr Gly Leu Ala Pro Phe
565 570 575Arg Gly Phe Leu Gln Glu Arg Leu Ala Leu Val Glu Ser Gly
Val Glu 580 585 590Leu Gly Pro Ser Val Leu Phe Phe Gly Cys Arg Asn
Arg Arg Met Asp 595 600 605Phe Ile Tyr Glu Glu Glu Leu Gln Arg Phe
Val Glu Ser Gly Ala Leu 610 615 620Ala Glu Leu Ser Val Ala Phe Ser
Arg Glu Gly Pro Thr Lys Glu Tyr625 630 635 640Val Gln His Lys Met
Met Asp Lys Ala Ser Asp Ile Trp Asn Met Ile 645 650 655Ser Gln Gly
Ala Tyr Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala 660 665 670Arg
Asp Val His Arg Ser Leu His Thr Ile Ala Gln Glu Gln Gly Ser 675 680
685Met Asp Ser Thr Lys Ala Glu Gly Phe Val Lys Asn Leu Gln Thr Ser
690 695 700Gly Arg Tyr Leu Arg Asp Val Trp705 71013462PRTOryza
sativa 13Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly
Met His1 5 10 15Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu
Pro Cys Leu 20 25 30Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg
Val Ser Phe Val 35 40 45Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro
Val Arg Pro Ala Leu 50 55 60Ala Pro Leu Val Ala Phe Val Ala Leu Pro
Leu Pro Arg Val Glu Gly65 70 75 80Leu Pro Asp Gly Ala Glu Ser Thr
Asn Asp Val Pro His Asp Arg Pro 85 90 95Asp Met Val Glu Leu His Arg
Arg Ala Phe Asp Gly Leu Ala Ala Pro 100 105 110Phe Ser Glu Phe Leu
Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp 115 120 125Val Phe His
His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro 130 135 140Cys
Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala145 150
155 160Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala
Gly 165 170 175Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala
Arg Met Lys 180 185 190Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser
Leu Ala Glu Arg Phe 195 200 205Ser Leu Thr Leu Ser Arg Ser Ser Leu
Val Val Gly Arg Ser Cys Val 210 215 220Glu Phe Glu Pro Glu Thr Val
Pro Leu Leu Ser Thr Leu Arg Gly Lys225 230 235 240Pro Ile Thr Phe
Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg 245 250 255Glu Asp
Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala 260 265
270Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val
275 280 285Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly
Thr Arg 290 295 300Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser
Asp Ala Asp Leu305 310 315 320Leu Pro Ala Gly Phe Glu Glu Arg Thr
Arg Gly Arg Gly Val Val Ala 325 330 335Thr Arg Trp Val Pro Gln Met
Ser Ile Leu Ala His Ala Ala Val Gly 340 345 350Ala Phe Leu Thr His
Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met 355 360 365Phe Gly His
Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln Gly Pro 370 375 380Asn
Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg385 390
395 400Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala
Ile 405 410 415Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe
Gln Ala Lys 420 425 430Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met
Ala Cys His Glu Arg 435 440 445Tyr Ile Asp Gly Phe Ile Gln Gln Leu
Arg Ser Tyr Lys Asp 450 455 460141503DNAArtificial
SequenceCodon-optimized stevia rebaudiana 14atggaagcct cttacctata
catttctatt ttgcttttac tggcatcata cctgttcacc 60actcaactta gaaggaagag
cgctaatcta ccaccaaccg tgtttccatc aataccaatc 120attggacact
tatacttact caaaaagcct ctttatagaa ctttagcaaa aattgccgct
180aagtacggac caatactgca attacaactc ggctacagac gtgttctggt
gatttcctca 240ccatcagcag cagaagagtg ctttaccaat aacgatgtaa
tcttcgcaaa tagacctaag 300acattgtttg gcaaaatagt gggtggaaca
tcccttggca gtttatccta cggcgatcaa 360tggcgtaatc taaggagagt
agcttctatc gaaatcctat cagttcatag gttgaacgaa 420tttcatgata
tcagagtgga tgagaacaga ttgttaatta gaaaacttag aagttcatct
480tctcctgtta ctcttataac agtcttttat gctctaacat tgaacgtcat
tatgagaatg 540atctctggca aaagatattt cgacagtggg gatagagaat
tggaggagga aggtaagaga 600tttcgagaaa tcttagacga aacgttgctt
ctagccggtg cttctaatgt tggcgactac 660ttaccaatat tgaactggtt
gggagttaag tctcttgaaa agaaattgat cgctttgcag 720aaaaagagag
atgacttttt ccagggtttg attgaacagg ttagaaaatc tcgtggtgct
780aaagtaggca aaggtagaaa aacgatgatc gaactcttat tatctttgca
agagtcagaa 840cctgagtact atacagatgc tatgataaga tcttttgtcc
taggtctgct ggctgcaggt 900agtgatactt cagcgggcac tatggaatgg
gccatgagct tactggtcaa tcacccacat 960gtattgaaga aagctcaagc
tgaaatcgat agagttatcg gtaataacag attgattgac 1020gagtcagaca
ttggaaatat cccttacatc gggtgtatta tcaatgaaac tctaagactc
1080tatccagcag ggccattgtt gttcccacat gaaagttctg ccgactgcgt
tatttccggt 1140tacaatatac ctagaggtac aatgttaatc gtaaaccaat
gggcgattca tcacgatcct 1200aaagtctggg atgatcctga aacctttaaa
cctgaaagat ttcaaggatt agaaggaact 1260agagatggtt tcaaacttat
gccattcggt tctgggagaa gaggatgtcc aggtgaaggt 1320ttggcaataa
ggctgttagg gatgacacta ggctcagtga tccaatgttt tgattgggag
1380agagtaggag atgagatggt tgacatgaca gaaggtttgg gtgtcacact
tcctaaggcc 1440gttccattag ttgccaaatg taagccacgt tccgaaatga
ctaatctcct atccgaactt 1500taa 150315500PRTStevia rebaudiana 15Met
Glu Ala Ser Tyr Leu Tyr Ile Ser Ile Leu Leu Leu Leu Ala Ser1 5 10
15Tyr Leu Phe Thr Thr Gln Leu Arg Arg Lys Ser Ala Asn Leu Pro Pro
20 25 30Thr Val Phe Pro Ser Ile Pro Ile Ile Gly His Leu Tyr Leu Leu
Lys 35 40 45Lys Pro Leu Tyr Arg Thr Leu Ala Lys Ile Ala Ala Lys Tyr
Gly Pro 50 55 60Ile Leu Gln Leu Gln Leu Gly Tyr Arg Arg Val Leu Val
Ile Ser Ser65 70 75 80Pro Ser Ala Ala Glu Glu Cys Phe Thr Asn Asn
Asp Val Ile Phe Ala 85 90 95Asn Arg Pro Lys Thr Leu Phe Gly Lys Ile
Val Gly Gly Thr Ser Leu 100 105 110Gly Ser Leu Ser Tyr Gly Asp Gln
Trp Arg Asn Leu Arg Arg Val Ala 115 120 125Ser Ile Glu Ile Leu Ser
Val His Arg Leu Asn Glu Phe His Asp Ile 130 135 140Arg Val Asp Glu
Asn Arg Leu Leu Ile Arg Lys Leu Arg Ser Ser Ser145 150 155 160Ser
Pro Val Thr Leu Ile Thr Val Phe Tyr Ala Leu Thr Leu Asn Val 165 170
175Ile Met Arg Met Ile Ser Gly Lys Arg Tyr Phe Asp Ser Gly Asp Arg
180 185 190Glu Leu Glu Glu Glu Gly Lys Arg Phe Arg Glu Ile Leu Asp
Glu Thr 195 200 205Leu Leu Leu Ala Gly Ala Ser Asn Val Gly Asp Tyr
Leu Pro Ile Leu 210 215 220Asn Trp Leu Gly Val Lys Ser Leu Glu Lys
Lys Leu Ile Ala Leu Gln225 230 235 240Lys Lys Arg Asp Asp Phe Phe
Gln Gly Leu Ile Glu Gln Val Arg Lys 245 250 255Ser Arg Gly Ala Lys
Val Gly Lys Gly Arg Lys Thr Met Ile Glu Leu 260 265 270Leu Leu Ser
Leu Gln Glu Ser Glu Pro Glu Tyr Tyr Thr Asp Ala Met 275 280 285Ile
Arg Ser Phe Val Leu Gly Leu Leu Ala Ala Gly Ser Asp Thr Ser 290 295
300Ala Gly Thr Met Glu Trp Ala Met Ser Leu Leu Val Asn His Pro
His305 310 315 320Val Leu Lys Lys Ala Gln Ala Glu Ile Asp Arg Val
Ile Gly Asn Asn 325 330 335Arg Leu Ile Asp Glu Ser Asp Ile Gly Asn
Ile Pro Tyr Ile Gly Cys 340 345 350Ile Ile Asn Glu Thr Leu Arg Leu
Tyr Pro Ala Gly Pro Leu Leu Phe 355 360 365Pro His Glu Ser Ser Ala
Asp Cys Val Ile Ser Gly Tyr Asn Ile Pro 370 375 380Arg Gly Thr Met
Leu Ile Val Asn Gln Trp Ala Ile His His Asp Pro385 390 395 400Lys
Val Trp Asp Asp Pro Glu Thr Phe Lys Pro Glu Arg Phe Gln Gly 405 410
415Leu Glu Gly Thr Arg Asp Gly Phe Lys Leu Met Pro Phe Gly Ser Gly
420 425 430Arg Arg Gly Cys Pro Gly Glu Gly Leu Ala Ile Arg Leu Leu
Gly Met 435 440 445Thr Leu Gly Ser Val Ile Gln Cys Phe Asp Trp Glu
Arg Val Gly Asp 450 455 460Glu Met Val Asp Met Thr Glu Gly Leu Gly
Val Thr Leu Pro Lys Ala465 470 475 480Val Pro Leu Val Ala Lys Cys
Lys Pro Arg Ser Glu Met Thr Asn Leu 485 490 495Leu Ser Glu Leu
500162124DNAStevia rebaudiana 16atgcaatcta actccgtgaa gatttcgccg
cttgatctgg taactgcgct gtttagcggc 60aaggttttgg acacatcgaa cgcatcggaa
tcgggagaat ctgctatgct gccgactata 120gcgatgatta tggagaatcg
tgagctgttg atgatactca caacgtcggt tgctgtattg 180atcggatgcg
ttgtcgtttt ggtgtggcgg agatcgtcta cgaagaagtc ggcgttggag
240ccaccggtga ttgtggttcc gaagagagtg caagaggagg aagttgatga
tggtaagaag 300aaagttacgg ttttcttcgg cacccaaact ggaacagctg
aaggcttcgc taaggcactt 360gttgaggaag ctaaagctcg atatgaaaag
gctgtcttta aagtaattga tttggatgat 420tatgctgctg atgacgatga
gtatgaggag aaactaaaga aagaatcttt ggcctttttc 480tttttggcta
cgtatggaga tggtgagcca acagataatg ctgccagatt ttataaatgg
540tttactgagg gagatgcgaa aggagaatgg cttaataagc ttcaatatgg
agtatttggt 600ttgggtaaca gacaatatga acattttaac aagatcgcaa
aagtggttga tgatggtctt 660gtagaacagg gtgcaaagcg tcttgttcct
gttggacttg gagatgatga tcaatgtatt 720gaagatgact tcaccgcatg
gaaagagtta gtatggccgg agttggatca attacttcgt 780gatgaggatg
acacaactgt tgctactcca tacacagctg ctgttgcaga atatcgcgtt
840gtttttcatg aaaaaccaga cgcgctttct gaagattata gttatacaaa
tggccatgct 900gttcatgatg ctcaacatcc atgcagatcc aacgtggctg
tcaaaaagga acttcatagt 960cctgaatctg accggtcttg cactcatctt
gaatttgaca tctcgaacac cggactatca 1020tatgaaactg gggaccatgt
tggagtttac tgtgaaaact tgagtgaagt tgtgaatgat 1080gctgaaagat
tagtaggatt accaccagac acttactcct ccatccacac tgatagtgaa
1140gacgggtcgc cacttggcgg agcctcattg ccgcctcctt tcccgccatg
cactttaagg 1200aaagcattga cgtgttatgc tgatgttttg agttctccca
agaagtcggc tttgcttgca 1260ctagctgctc atgccaccga tcccagtgaa
gctgatagat tgaaatttct tgcatccccc 1320gccggaaagg atgaatattc
tcaatggata gttgcaagcc aaagaagtct ccttgaagtc 1380atggaagcat
tcccgtcagc taagccttca cttggtgttt tctttgcatc tgttgccccg
1440cgcttacaac caagatacta ctctatttct tcctcaccca agatggcacc
ggataggatt 1500catgttacat gtgcattagt ctatgagaaa acacctgcag
gccgcatcca caaaggagtt 1560tgttcaactt ggatgaagaa cgcagtgcct
atgaccgaga gtcaagattg cagttgggcc 1620ccaatatacg tccgaacatc
caatttcaga ctaccatctg accctaaggt cccggttatc 1680atgattggac
ctggcactgg tttggctcct tttagaggtt tccttcaaga
gcggttagct 1740ttaaaggaag ccggaactga cctcggttta tccattttat
tcttcggatg taggaatcgc 1800aaagtggatt tcatatatga aaacgagctt
aacaactttg tggagactgg tgctctttct 1860gagcttattg ttgctttctc
ccgtgaaggc ccgactaagg aatatgtgca acacaagatg 1920agtgagaagg
cttcggatat ctggaacttg ctttctgaag gagcatattt atacgtatgt
1980ggtgatgcca aaggcatggc caaagatgta catcgaaccc tccacacaat
tgtgcaagaa 2040cagggatctc ttgactcgtc aaaggcagaa ctctacgtga
agaatctaca aatgtcagga 2100agatacctcc gtgacgtttg gtaa
212417707PRTStevia rebaudiana 17Met Gln Ser Asn Ser Val Lys Ile Ser
Pro Leu Asp Leu Val Thr Ala1 5 10 15Leu Phe Ser Gly Lys Val Leu Asp
Thr Ser Asn Ala Ser Glu Ser Gly 20 25 30Glu Ser Ala Met Leu Pro Thr
Ile Ala Met Ile Met Glu Asn Arg Glu 35 40 45Leu Leu Met Ile Leu Thr
Thr Ser Val Ala Val Leu Ile Gly Cys Val 50 55 60Val Val Leu Val Trp
Arg Arg Ser Ser Thr Lys Lys Ser Ala Leu Glu65 70 75 80Pro Pro Val
Ile Val Val Pro Lys Arg Val Gln Glu Glu Glu Val Asp 85 90 95Asp Gly
Lys Lys Lys Val Thr Val Phe Phe Gly Thr Gln Thr Gly Thr 100 105
110Ala Glu Gly Phe Ala Lys Ala Leu Val Glu Glu Ala Lys Ala Arg Tyr
115 120 125Glu Lys Ala Val Phe Lys Val Ile Asp Leu Asp Asp Tyr Ala
Ala Asp 130 135 140Asp Asp Glu Tyr Glu Glu Lys Leu Lys Lys Glu Ser
Leu Ala Phe Phe145 150 155 160Phe Leu Ala Thr Tyr Gly Asp Gly Glu
Pro Thr Asp Asn Ala Ala Arg 165 170 175Phe Tyr Lys Trp Phe Thr Glu
Gly Asp Ala Lys Gly Glu Trp Leu Asn 180 185 190Lys Leu Gln Tyr Gly
Val Phe Gly Leu Gly Asn Arg Gln Tyr Glu His 195 200 205Phe Asn Lys
Ile Ala Lys Val Val Asp Asp Gly Leu Val Glu Gln Gly 210 215 220Ala
Lys Arg Leu Val Pro Val Gly Leu Gly Asp Asp Asp Gln Cys Ile225 230
235 240Glu Asp Asp Phe Thr Ala Trp Lys Glu Leu Val Trp Pro Glu Leu
Asp 245 250 255Gln Leu Leu Arg Asp Glu Asp Asp Thr Thr Val Ala Thr
Pro Tyr Thr 260 265 270Ala Ala Val Ala Glu Tyr Arg Val Val Phe His
Glu Lys Pro Asp Ala 275 280 285Leu Ser Glu Asp Tyr Ser Tyr Thr Asn
Gly His Ala Val His Asp Ala 290 295 300Gln His Pro Cys Arg Ser Asn
Val Ala Val Lys Lys Glu Leu His Ser305 310 315 320Pro Glu Ser Asp
Arg Ser Cys Thr His Leu Glu Phe Asp Ile Ser Asn 325 330 335Thr Gly
Leu Ser Tyr Glu Thr Gly Asp His Val Gly Val Tyr Cys Glu 340 345
350Asn Leu Ser Glu Val Val Asn Asp Ala Glu Arg Leu Val Gly Leu Pro
355 360 365Pro Asp Thr Tyr Ser Ser Ile His Thr Asp Ser Glu Asp Gly
Ser Pro 370 375 380Leu Gly Gly Ala Ser Leu Pro Pro Pro Phe Pro Pro
Cys Thr Leu Arg385 390 395 400Lys Ala Leu Thr Cys Tyr Ala Asp Val
Leu Ser Ser Pro Lys Lys Ser 405 410 415Ala Leu Leu Ala Leu Ala Ala
His Ala Thr Asp Pro Ser Glu Ala Asp 420 425 430Arg Leu Lys Phe Leu
Ala Ser Pro Ala Gly Lys Asp Glu Tyr Ser Gln 435 440 445Trp Ile Val
Ala Ser Gln Arg Ser Leu Leu Glu Val Met Glu Ala Phe 450 455 460Pro
Ser Ala Lys Pro Ser Leu Gly Val Phe Phe Ala Ser Val Ala Pro465 470
475 480Arg Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Lys Met
Ala 485 490 495Pro Asp Arg Ile His Val Thr Cys Ala Leu Val Tyr Glu
Lys Thr Pro 500 505 510Ala Gly Arg Ile His Lys Gly Val Cys Ser Thr
Trp Met Lys Asn Ala 515 520 525Val Pro Met Thr Glu Ser Gln Asp Cys
Ser Trp Ala Pro Ile Tyr Val 530 535 540Arg Thr Ser Asn Phe Arg Leu
Pro Ser Asp Pro Lys Val Pro Val Ile545 550 555 560Met Ile Gly Pro
Gly Thr Gly Leu Ala Pro Phe Arg Gly Phe Leu Gln 565 570 575Glu Arg
Leu Ala Leu Lys Glu Ala Gly Thr Asp Leu Gly Leu Ser Ile 580 585
590Leu Phe Phe Gly Cys Arg Asn Arg Lys Val Asp Phe Ile Tyr Glu Asn
595 600 605Glu Leu Asn Asn Phe Val Glu Thr Gly Ala Leu Ser Glu Leu
Ile Val 610 615 620Ala Phe Ser Arg Glu Gly Pro Thr Lys Glu Tyr Val
Gln His Lys Met625 630 635 640Ser Glu Lys Ala Ser Asp Ile Trp Asn
Leu Leu Ser Glu Gly Ala Tyr 645 650 655Leu Tyr Val Cys Gly Asp Ala
Lys Gly Met Ala Lys Asp Val His Arg 660 665 670Thr Leu His Thr Ile
Val Gln Glu Gln Gly Ser Leu Asp Ser Ser Lys 675 680 685Ala Glu Leu
Tyr Val Lys Asn Leu Gln Met Ser Gly Arg Tyr Leu Arg 690 695 700Asp
Val Trp705182133DNAArtificial SequenceCodon optimized 18atgcaatcag
attcagtcaa agtctctcca tttgatttgg tttccgctgc tatgaatggc 60aaggcaatgg
aaaagttgaa cgctagtgaa tctgaagatc caacaacatt gcctgcacta
120aagatgctag ttgaaaatag agaattgttg acactgttca caacttcctt
cgcagttctt 180attgggtgtc ttgtatttct aatgtggaga cgttcatcct
ctaaaaagct ggtacaagat 240ccagttccac aagttatcgt tgtaaagaag
aaagagaagg agtcagaggt tgatgacggg 300aaaaagaaag tttctatttt
ctacggcaca caaacaggaa ctgccgaagg ttttgctaaa 360gcattagtcg
aggaagcaaa agtgagatat gaaaagacct ctttcaaggt tatcgatcta
420gatgactacg ctgcagatga tgatgaatat gaggaaaaac tgaaaaagga
atccttagcc 480ttcttcttct tggccacata cggtgatggt gaacctactg
ataatgctgc taacttctac 540aagtggttca cagaaggcga cgataaaggt
gaatggctga aaaagttaca atacggagta 600tttggtttag gtaacagaca
atatgaacat ttcaacaaga tcgctattgt agttgatgat 660aaacttactg
aaatgggagc caaaagatta gtaccagtag gattagggga tgatgatcag
720tgtatagaag atgacttcac cgcctggaag gaattggtat ggccagaatt
ggatcaactt 780ttaagggacg aagatgatac ttctgtgact accccataca
ctgcagccgt attggagtac 840agagtggttt accatgataa accagcagac
tcatatgctg aagatcaaac ccatacaaac 900ggtcatgttg ttcatgatgc
acagcatcct tcaagatcta atgtggcttt caaaaaggaa 960ctacacacct
ctcaatcaga taggtcttgt actcacttag aattcgatat ttctcacaca
1020ggactgtctt acgaaactgg cgatcacgtt ggcgtttatt ccgagaactt
gtccgaagtt 1080gtcgatgaag cactaaaact gttagggtta tcaccagaca
catacttctc agtccatgct 1140gataaggagg atgggacacc tatcggtggt
gcttcactac caccaccttt tcctccttgc 1200acattgagag acgctctaac
cagatacgca gatgtcttat cctcacctaa aaaggtagct 1260ttgctggcat
tggctgctca tgctagtgat cctagtgaag ccgataggtt aaagttcctg
1320gcttcaccag ccggaaaaga tgaatatgca caatggatcg tcgccaacca
acgttctttg 1380ctagaagtga tgcaaagttt tccatctgcc aagcctccat
taggtgtgtt cttcgcagca 1440gtagctccac gtttacaacc aagatactac
tctatcagtt catctcctaa gatgtctcct 1500aacagaatac atgttacatg
tgctttggtg tacgagacta ctccagcagg cagaattcac 1560agaggattgt
gttcaacctg gatgaaaaat gctgtccctt taacagagtc acctgattgc
1620tctcaagcat ccattttcgt tagaacatca aatttcagac ttccagtgga
tccaaaagtt 1680ccagtcatta tgataggacc aggcactggt cttgccccat
tcaggggctt tcttcaagag 1740agattggcct tgaaggaatc tggtacagaa
ttgggttctt ctatcttttt ctttggttgc 1800cgtaatagaa aagttgactt
tatctacgag gacgagctta acaattttgt tgagacagga 1860gcattgtcag
aattgatcgt cgcattttca agagaaggga ctgccaaaga gtacgttcag
1920cacaagatga gtcaaaaagc ctccgatata tggaaacttc taagtgaagg
tgcctatctt 1980tatgtctgtg gcgatgcaaa gggcatggcc aaggatgtcc
atagaactct gcatacaatt 2040gttcaggaac aagggagtct ggattcttcc
aaggctgaat tgtacgtcaa aaacttacag 2100atgtctggaa gatacttaag
agatgtttgg taa 213319710PRTStevia rebaudiana 19Met Gln Ser Asp Ser
Val Lys Val Ser Pro Phe Asp Leu Val Ser Ala1 5 10 15Ala Met Asn Gly
Lys Ala Met Glu Lys Leu Asn Ala Ser Glu Ser Glu 20 25 30Asp Pro Thr
Thr Leu Pro Ala Leu Lys Met Leu Val Glu Asn Arg Glu 35 40 45Leu Leu
Thr Leu Phe Thr Thr Ser Phe Ala Val Leu Ile Gly Cys Leu 50 55 60Val
Phe Leu Met Trp Arg Arg Ser Ser Ser Lys Lys Leu Val Gln Asp65 70 75
80Pro Val Pro Gln Val Ile Val Val Lys Lys Lys Glu Lys Glu Ser Glu
85 90 95Val Asp Asp Gly Lys Lys Lys Val Ser Ile Phe Tyr Gly Thr Gln
Thr 100 105 110Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Val Glu Glu
Ala Lys Val 115 120 125Arg Tyr Glu Lys Thr Ser Phe Lys Val Ile Asp
Leu Asp Asp Tyr Ala 130 135 140Ala Asp Asp Asp Glu Tyr Glu Glu Lys
Leu Lys Lys Glu Ser Leu Ala145 150 155 160Phe Phe Phe Leu Ala Thr
Tyr Gly Asp Gly Glu Pro Thr Asp Asn Ala 165 170 175Ala Asn Phe Tyr
Lys Trp Phe Thr Glu Gly Asp Asp Lys Gly Glu Trp 180 185 190Leu Lys
Lys Leu Gln Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln Tyr 195 200
205Glu His Phe Asn Lys Ile Ala Ile Val Val Asp Asp Lys Leu Thr Glu
210 215 220Met Gly Ala Lys Arg Leu Val Pro Val Gly Leu Gly Asp Asp
Asp Gln225 230 235 240Cys Ile Glu Asp Asp Phe Thr Ala Trp Lys Glu
Leu Val Trp Pro Glu 245 250 255Leu Asp Gln Leu Leu Arg Asp Glu Asp
Asp Thr Ser Val Thr Thr Pro 260 265 270Tyr Thr Ala Ala Val Leu Glu
Tyr Arg Val Val Tyr His Asp Lys Pro 275 280 285Ala Asp Ser Tyr Ala
Glu Asp Gln Thr His Thr Asn Gly His Val Val 290 295 300His Asp Ala
Gln His Pro Ser Arg Ser Asn Val Ala Phe Lys Lys Glu305 310 315
320Leu His Thr Ser Gln Ser Asp Arg Ser Cys Thr His Leu Glu Phe Asp
325 330 335Ile Ser His Thr Gly Leu Ser Tyr Glu Thr Gly Asp His Val
Gly Val 340 345 350Tyr Ser Glu Asn Leu Ser Glu Val Val Asp Glu Ala
Leu Lys Leu Leu 355 360 365Gly Leu Ser Pro Asp Thr Tyr Phe Ser Val
His Ala Asp Lys Glu Asp 370 375 380Gly Thr Pro Ile Gly Gly Ala Ser
Leu Pro Pro Pro Phe Pro Pro Cys385 390 395 400Thr Leu Arg Asp Ala
Leu Thr Arg Tyr Ala Asp Val Leu Ser Ser Pro 405 410 415Lys Lys Val
Ala Leu Leu Ala Leu Ala Ala His Ala Ser Asp Pro Ser 420 425 430Glu
Ala Asp Arg Leu Lys Phe Leu Ala Ser Pro Ala Gly Lys Asp Glu 435 440
445Tyr Ala Gln Trp Ile Val Ala Asn Gln Arg Ser Leu Leu Glu Val Met
450 455 460Gln Ser Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe Phe
Ala Ala465 470 475 480Val Ala Pro Arg Leu Gln Pro Arg Tyr Tyr Ser
Ile Ser Ser Ser Pro 485 490 495Lys Met Ser Pro Asn Arg Ile His Val
Thr Cys Ala Leu Val Tyr Glu 500 505 510Thr Thr Pro Ala Gly Arg Ile
His Arg Gly Leu Cys Ser Thr Trp Met 515 520 525Lys Asn Ala Val Pro
Leu Thr Glu Ser Pro Asp Cys Ser Gln Ala Ser 530 535 540Ile Phe Val
Arg Thr Ser Asn Phe Arg Leu Pro Val Asp Pro Lys Val545 550 555
560Pro Val Ile Met Ile Gly Pro Gly Thr Gly Leu Ala Pro Phe Arg Gly
565 570 575Phe Leu Gln Glu Arg Leu Ala Leu Lys Glu Ser Gly Thr Glu
Leu Gly 580 585 590Ser Ser Ile Phe Phe Phe Gly Cys Arg Asn Arg Lys
Val Asp Phe Ile 595 600 605Tyr Glu Asp Glu Leu Asn Asn Phe Val Glu
Thr Gly Ala Leu Ser Glu 610 615 620Leu Ile Val Ala Phe Ser Arg Glu
Gly Thr Ala Lys Glu Tyr Val Gln625 630 635 640His Lys Met Ser Gln
Lys Ala Ser Asp Ile Trp Lys Leu Leu Ser Glu 645 650 655Gly Ala Tyr
Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Lys Asp 660 665 670Val
His Arg Thr Leu His Thr Ile Val Gln Glu Gln Gly Ser Leu Asp 675 680
685Ser Ser Lys Ala Glu Leu Tyr Val Lys Asn Leu Gln Met Ser Gly Arg
690 695 700Tyr Leu Arg Asp Val Trp705 71020511PRTRubus suavissimus
20Met Ala Thr Leu Leu Glu His Phe Gln Ala Met Pro Phe Ala Ile Pro1
5 10 15Ile Ala Leu Ala Ala Leu Ser Trp Leu Phe Leu Phe Tyr Ile Lys
Val 20 25 30Ser Phe Phe Ser Asn Lys Ser Ala Gln Ala Lys Leu Pro Pro
Val Pro 35 40 45Val Val Pro Gly Leu Pro Val Ile Gly Asn Leu Leu Gln
Leu Lys Glu 50 55 60Lys Lys Pro Tyr Gln Thr Phe Thr Arg Trp Ala Glu
Glu Tyr Gly Pro65 70 75 80Ile Tyr Ser Ile Arg Thr Gly Ala Ser Thr
Met Val Val Leu Asn Thr 85 90 95Thr Gln Val Ala Lys Glu Ala Met Val
Thr Arg Tyr Leu Ser Ile Ser 100 105 110Thr Arg Lys Leu Ser Asn Ala
Leu Lys Ile Leu Thr Ala Asp Lys Cys 115 120 125Met Val Ala Ile Ser
Asp Tyr Asn Asp Phe His Lys Met Ile Lys Arg 130 135 140Tyr Ile Leu
Ser Asn Val Leu Gly Pro Ser Ala Gln Lys Arg His Arg145 150 155
160Ser Asn Arg Asp Thr Leu Arg Ala Asn Val Cys Ser Arg Leu His Ser
165 170 175Gln Val Lys Asn Ser Pro Arg Glu Ala Val Asn Phe Arg Arg
Val Phe 180 185 190Glu Trp Glu Leu Phe Gly Ile Ala Leu Lys Gln Ala
Phe Gly Lys Asp 195 200 205Ile Glu Lys Pro Ile Tyr Val Glu Glu Leu
Gly Thr Thr Leu Ser Arg 210 215 220Asp Glu Ile Phe Lys Val Leu Val
Leu Asp Ile Met Glu Gly Ala Ile225 230 235 240Glu Val Asp Trp Arg
Asp Phe Phe Pro Tyr Leu Arg Trp Ile Pro Asn 245 250 255Thr Arg Met
Glu Thr Lys Ile Gln Arg Leu Tyr Phe Arg Arg Lys Ala 260 265 270Val
Met Thr Ala Leu Ile Asn Glu Gln Lys Lys Arg Ile Ala Ser Gly 275 280
285Glu Glu Ile Asn Cys Tyr Ile Asp Phe Leu Leu Lys Glu Gly Lys Thr
290 295 300Leu Thr Met Asp Gln Ile Ser Met Leu Leu Trp Glu Thr Val
Ile Glu305 310 315 320Thr Ala Asp Thr Thr Met Val Thr Thr Glu Trp
Ala Met Tyr Glu Val 325 330 335Ala Lys Asp Ser Lys Arg Gln Asp Arg
Leu Tyr Gln Glu Ile Gln Lys 340 345 350Val Cys Gly Ser Glu Met Val
Thr Glu Glu Tyr Leu Ser Gln Leu Pro 355 360 365Tyr Leu Asn Ala Val
Phe His Glu Thr Leu Arg Lys His Ser Pro Ala 370 375 380Ala Leu Val
Pro Leu Arg Tyr Ala His Glu Asp Thr Gln Leu Gly Gly385 390 395
400Tyr Tyr Ile Pro Ala Gly Thr Glu Ile Ala Ile Asn Ile Tyr Gly Cys
405 410 415Asn Met Asp Lys His Gln Trp Glu Ser Pro Glu Glu Trp Lys
Pro Glu 420 425 430Arg Phe Leu Asp Pro Lys Phe Asp Pro Met Asp Leu
Tyr Lys Thr Met 435 440 445Ala Phe Gly Ala Gly Lys Arg Val Cys Ala
Gly Ser Leu Gln Ala Met 450 455 460Leu Ile Ala Cys Pro Thr Ile Gly
Arg Leu Val Gln Glu Phe Glu Trp465 470 475 480Lys Leu Arg Asp Gly
Glu Glu Glu Asn Val Asp Thr Val Gly Leu Thr 485 490 495Thr His Lys
Arg Tyr Pro Met His Ala Ile Leu Lys Pro Arg Ser 500 505
51021523PRTRubus suavissimus 21Met Glu Val Thr Val Ala Ser Ser Val
Ala Leu Ser Leu Val Phe Ile1 5 10 15Ser Ile Val Val Arg Trp Ala Trp
Ser Val Val Asn Trp Val Trp Phe 20 25 30Lys Pro Lys Lys Leu Glu Arg
Phe Leu Arg Glu Gln Gly Leu Lys Gly 35 40 45Asn Ser Tyr Arg Phe Leu
Tyr Gly Asp Met Lys Glu Asn Ser Ile Leu 50 55 60Leu Lys Gln Ala Arg
Ser Lys Pro Met Asn Leu Ser Thr Ser His Asp65 70 75 80Ile Ala Pro
Gln Val Thr Pro Phe Val Asp Gln Thr Val Lys Ala Tyr 85 90 95Gly Lys
Asn Ser Phe Asn Trp Val Gly Pro Ile
Pro Arg Val Asn Ile 100 105 110Met Asn Pro Glu Asp Leu Lys Asp Val
Leu Thr Lys Asn Val Asp Phe 115 120 125Val Lys Pro Ile Ser Asn Pro
Leu Ile Lys Leu Leu Ala Thr Gly Ile 130 135 140Ala Ile Tyr Glu Gly
Glu Lys Trp Thr Lys His Arg Arg Ile Ile Asn145 150 155 160Pro Thr
Phe His Ser Glu Arg Leu Lys Arg Met Leu Pro Ser Phe His 165 170
175Gln Ser Cys Asn Glu Met Val Lys Glu Trp Glu Ser Leu Val Ser Lys
180 185 190Glu Gly Ser Ser Cys Glu Leu Asp Val Trp Pro Phe Leu Glu
Asn Met 195 200 205Ser Ala Asp Val Ile Ser Arg Thr Ala Phe Gly Thr
Ser Tyr Lys Lys 210 215 220Gly Gln Lys Ile Phe Glu Leu Leu Arg Glu
Gln Val Ile Tyr Val Thr225 230 235 240Lys Gly Phe Gln Ser Phe Tyr
Ile Pro Gly Trp Arg Phe Leu Pro Thr 245 250 255Lys Met Asn Lys Arg
Met Asn Glu Ile Asn Glu Glu Ile Lys Gly Leu 260 265 270Ile Arg Gly
Ile Ile Ile Asp Arg Glu Gln Ile Ile Lys Ala Gly Glu 275 280 285Glu
Thr Asn Asp Asp Leu Leu Gly Ala Leu Met Glu Ser Asn Leu Lys 290 295
300Asp Ile Arg Glu His Gly Lys Asn Asn Lys Asn Val Gly Met Ser
Ile305 310 315 320Glu Asp Val Ile Gln Glu Cys Lys Leu Phe Tyr Phe
Ala Gly Gln Glu 325 330 335Thr Thr Ser Val Leu Leu Ala Trp Thr Met
Val Leu Leu Gly Gln Asn 340 345 350Gln Asn Trp Gln Asp Arg Ala Arg
Gln Glu Val Leu Gln Val Phe Gly 355 360 365Ser Ser Lys Pro Asp Phe
Asp Gly Leu Ala His Leu Lys Val Val Thr 370 375 380Met Ile Leu Leu
Glu Val Leu Arg Leu Tyr Pro Pro Val Ile Glu Leu385 390 395 400Ile
Arg Thr Ile His Lys Lys Thr Gln Leu Gly Lys Leu Ser Leu Pro 405 410
415Glu Gly Val Glu Val Arg Leu Pro Thr Leu Leu Ile His His Asp Lys
420 425 430Glu Leu Trp Gly Asp Asp Ala Asn Gln Phe Asn Pro Glu Arg
Phe Ser 435 440 445Glu Gly Val Ser Lys Ala Thr Lys Asn Arg Leu Ser
Phe Phe Pro Phe 450 455 460Gly Ala Gly Pro Arg Ile Cys Ile Gly Gln
Asn Phe Ser Met Met Glu465 470 475 480Ala Lys Leu Ala Leu Ala Leu
Ile Leu Gln His Phe Thr Phe Glu Leu 485 490 495Ser Pro Ser His Ala
His Ala Pro Ser His Arg Ile Thr Leu Gln Pro 500 505 510Gln Tyr Gly
Val Arg Ile Ile Leu His Arg Arg 515 52022701PRTSiraitia grosvenorii
22Met Lys Val Ser Pro Phe Glu Phe Met Ser Ala Ile Ile Lys Gly Arg1
5 10 15Met Asp Pro Ser Asn Ser Ser Phe Glu Ser Thr Gly Glu Val Ala
Ser 20 25 30Val Ile Phe Glu Asn Arg Glu Leu Val Ala Ile Leu Thr Thr
Ser Ile 35 40 45Ala Val Met Ile Gly Cys Phe Val Val Leu Met Trp Arg
Arg Ala Gly 50 55 60Ser Arg Lys Val Lys Asn Val Glu Leu Pro Lys Pro
Leu Ile Val His65 70 75 80Glu Pro Glu Pro Glu Val Glu Asp Gly Lys
Lys Lys Val Ser Ile Phe 85 90 95Phe Gly Thr Gln Thr Gly Thr Ala Glu
Gly Phe Ala Lys Ala Leu Ala 100 105 110Asp Glu Ala Lys Ala Arg Tyr
Glu Lys Ala Thr Phe Arg Val Val Asp 115 120 125Leu Asp Asp Tyr Ala
Ala Asp Asp Asp Gln Tyr Glu Glu Lys Leu Lys 130 135 140Asn Glu Ser
Phe Ala Val Phe Leu Leu Ala Thr Tyr Gly Asp Gly Glu145 150 155
160Pro Thr Asp Asn Ala Ala Arg Phe Tyr Lys Trp Phe Ala Glu Gly Lys
165 170 175Glu Arg Gly Glu Trp Leu Gln Asn Leu His Tyr Ala Val Phe
Gly Leu 180 185 190Gly Asn Arg Gln Tyr Glu His Phe Asn Lys Ile Ala
Lys Val Ala Asp 195 200 205Glu Leu Leu Glu Ala Gln Gly Gly Asn Arg
Leu Val Lys Val Gly Leu 210 215 220Gly Asp Asp Asp Gln Cys Ile Glu
Asp Asp Phe Ser Ala Trp Arg Glu225 230 235 240Ser Leu Trp Pro Glu
Leu Asp Met Leu Leu Arg Asp Glu Asp Asp Ala 245 250 255Thr Thr Val
Thr Thr Pro Tyr Thr Ala Ala Val Leu Glu Tyr Arg Val 260 265 270Val
Phe His Asp Ser Ala Asp Val Ala Ala Glu Asp Lys Ser Trp Ile 275 280
285Asn Ala Asn Gly His Ala Val His Asp Ala Gln His Pro Phe Arg Ser
290 295 300Asn Val Val Val Arg Lys Glu Leu His Thr Ser Ala Ser Asp
Arg Ser305 310 315 320Cys Ser His Leu Glu Phe Asn Ile Ser Gly Ser
Ala Leu Asn Tyr Glu 325 330 335Thr Gly Asp His Val Gly Val Tyr Cys
Glu Asn Leu Thr Glu Thr Val 340 345 350Asp Glu Ala Leu Asn Leu Leu
Gly Leu Ser Pro Glu Thr Tyr Phe Ser 355 360 365Ile Tyr Thr Asp Asn
Glu Asp Gly Thr Pro Leu Gly Gly Ser Ser Leu 370 375 380Pro Pro Pro
Phe Pro Ser Cys Thr Leu Arg Thr Ala Leu Thr Arg Tyr385 390 395
400Ala Asp Leu Leu Asn Ser Pro Lys Lys Ser Ala Leu Leu Ala Leu Ala
405 410 415Ala His Ala Ser Asn Pro Val Glu Ala Asp Arg Leu Arg Tyr
Leu Ala 420 425 430Ser Pro Ala Gly Lys Asp Glu Tyr Ala Gln Ser Val
Ile Gly Ser Gln 435 440 445Lys Ser Leu Leu Glu Val Met Ala Glu Phe
Pro Ser Ala Lys Pro Pro 450 455 460Leu Gly Val Phe Phe Ala Ala Val
Ala Pro Arg Leu Gln Pro Arg Phe465 470 475 480Tyr Ser Ile Ser Ser
Ser Pro Arg Met Ala Pro Ser Arg Ile His Val 485 490 495Thr Cys Ala
Leu Val Tyr Asp Lys Met Pro Thr Gly Arg Ile His Lys 500 505 510Gly
Val Cys Ser Thr Trp Met Lys Asn Ser Val Pro Met Glu Lys Ser 515 520
525His Glu Cys Ser Trp Ala Pro Ile Phe Val Arg Gln Ser Asn Phe Lys
530 535 540Leu Pro Ala Glu Ser Lys Val Pro Ile Ile Met Val Gly Pro
Gly Thr545 550 555 560Gly Leu Ala Pro Phe Arg Gly Phe Leu Gln Glu
Arg Leu Ala Leu Lys 565 570 575Glu Ser Gly Val Glu Leu Gly Pro Ser
Ile Leu Phe Phe Gly Cys Arg 580 585 590Asn Arg Arg Met Asp Tyr Ile
Tyr Glu Asp Glu Leu Asn Asn Phe Val 595 600 605Glu Thr Gly Ala Leu
Ser Glu Leu Val Ile Ala Phe Ser Arg Glu Gly 610 615 620Pro Thr Lys
Glu Tyr Val Gln His Lys Met Ala Glu Lys Ala Ser Asp625 630 635
640Ile Trp Asn Leu Ile Ser Glu Gly Ala Tyr Leu Tyr Val Cys Gly Asp
645 650 655Ala Lys Gly Met Ala Lys Asp Val His Arg Thr Leu His Thr
Ile Met 660 665 670Gln Glu Gln Gly Ser Leu Asp Ser Ser Lys Ala Glu
Ser Met Val Lys 675 680 685Asn Leu Gln Met Asn Gly Arg Tyr Leu Arg
Asp Val Trp 690 695 700
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