U.S. patent application number 13/133242 was filed with the patent office on 2011-10-06 for microorganism expressing xylose isomerase.
This patent application is currently assigned to TERRANOL A/S. Invention is credited to Thomas Hvid Andersen, Birgitte Ronnow, Ole Sibbesen.
Application Number | 20110244525 13/133242 |
Document ID | / |
Family ID | 40326210 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110244525 |
Kind Code |
A1 |
Ronnow; Birgitte ; et
al. |
October 6, 2011 |
MICROORGANISM EXPRESSING XYLOSE ISOMERASE
Abstract
The present invention relates to a transformed microorganism
capable of (a) a higher xylose isomerase activity than the
equivalent microorganism prior to transformation; and/or (b) a
higher growth rate in or on a growth medium comprising xylose than
the equivalent microorganism prior to transformation; and/or (c) a
faster metabolism of xylose than the equivalent microorganism prior
to transformation; and/or (d) a higher production of ethanol when
grown anaerobically on xylose as the carbon source than the
equivalent microorganism prior to transformation.
Inventors: |
Ronnow; Birgitte;
(Copenhagenk, DK) ; Andersen; Thomas Hvid;
(Frederiksberg, DK) ; Sibbesen; Ole; (Bagsard,
DK) |
Assignee: |
TERRANOL A/S
LYNGBY
DK
|
Family ID: |
40326210 |
Appl. No.: |
13/133242 |
Filed: |
December 10, 2009 |
PCT Filed: |
December 10, 2009 |
PCT NO: |
PCT/IB09/55652 |
371 Date: |
June 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61138293 |
Dec 17, 2008 |
|
|
|
Current U.S.
Class: |
435/94 ; 435/110;
435/115; 435/126; 435/128; 435/132; 435/139; 435/140; 435/142;
435/144; 435/145; 435/147; 435/148; 435/156; 435/161; 435/166;
435/167; 435/171; 435/252; 435/254.21; 435/471; 44/452;
568/840 |
Current CPC
Class: |
Y02E 50/10 20130101;
Y02E 50/17 20130101; C12N 9/90 20130101; C12N 15/81 20130101; Y02P
20/52 20151101; C12P 7/06 20130101; C12P 19/24 20130101; C12N 9/92
20130101 |
Class at
Publication: |
435/94 ;
435/254.21; 435/471; 435/252; 435/171; 435/161; 435/139; 435/145;
435/140; 435/167; 435/147; 435/166; 435/148; 435/128; 435/126;
435/142; 435/110; 435/144; 435/156; 435/115; 435/132; 568/840;
44/452 |
International
Class: |
C12P 19/24 20060101
C12P019/24; C12N 1/19 20060101 C12N001/19; C12N 15/81 20060101
C12N015/81; C12N 1/22 20060101 C12N001/22; C12P 1/02 20060101
C12P001/02; C12P 7/06 20060101 C12P007/06; C12P 7/56 20060101
C12P007/56; C12P 7/46 20060101 C12P007/46; C12P 7/54 20060101
C12P007/54; C12P 5/02 20060101 C12P005/02; C12P 7/24 20060101
C12P007/24; C12P 5/00 20060101 C12P005/00; C12P 7/26 20060101
C12P007/26; C12P 13/00 20060101 C12P013/00; C12P 17/04 20060101
C12P017/04; C12P 7/44 20060101 C12P007/44; C12P 13/14 20060101
C12P013/14; C12P 7/48 20060101 C12P007/48; C12P 7/22 20060101
C12P007/22; C12P 13/08 20060101 C12P013/08; C12P 7/00 20060101
C12P007/00; C07C 31/08 20060101 C07C031/08; C10L 1/02 20060101
C10L001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2008 |
GB |
GB 0822937.9 |
Claims
1. A transformed microorganism capable of (a) a higher xylose
isomerase activity than the equivalent microorganism prior to
transformation; and/or (b) a higher growth rate in or on a growth
medium comprising xylose than the equivalent microorganism prior to
transformation; and/or (c) a faster metabolism of xylose than the
equivalent microorganism prior to transformation; and/or (d) a
higher production of ethanol when grown anaerobically on xylose as
the carbon source than the equivalent microorganism prior to
transformation; wherein said microorganism has been transformed
with a nucleotide sequence encoding a xylose isomerase wherein said
xylose isomerase is an exogenous xylose isomerase and wherein said
xylose isomerase is an exogenous xylose isomerase derived from a
Lactococcus species; and wherein said transformed microorganism is
a transformed yeast wherein said transformed yeast is
Saccharomyces.
2. The microorganism according to claim 1 wherein said
microorganism has been transformed with a nucleotide sequence
encoding the amino acid sequence shown as SEQ ID No 14, SEQ ID No
11, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No 20, or a
variant, homologue or derivative thereof encoding an amino acid
sequence having at least 75% identity to the amino acid sequence
shown as SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID No 13,
SEQ ID No 19 or SEQ ID No 20.
3. The microorganism according to claim 1 wherein said
microorganism has been transformed with the nucleotide sequence
shown as SEQ ID No 1, SEQ ID No 10, SEQ ID 17, SEQ ID No 12, SEQ ID
No 27 or SEQ ID No 28, or a variant, homologue or derivative
thereof having at least 75% identity to the nucleotide sequence
shown as SEQ ID No 1, SEQ ID No 10, SEQ ID 17, SEQ ID No 12, SEQ ID
No 27 or SEQ ID No 28.
4. An inoculum comprising a microorganism according to claim 1.
5. A culture medium comprising a microorganism according to claim
1.
6. The culture medium according to claim 5 wherein said culture
medium comprises a source of xylose.
7. The culture medium according to claim 5 wherein said culture
medium comprises material derived from lignocellulosic
material.
8. A method for preparing a transformed microorganism, said method
comprising the step of transforming a microorganism such that said
transformed microorganism is capable of: (a) a higher xylose
isomerase activity than the microorganism prior to transformation;
and/or (b) a higher growth rate in or on a growth medium comprising
xylose than the microorganism prior to transformation; and/or (c) a
faster metabolism of xylose than the microorganism prior to
transformation; and/or (d) a higher production of ethanol when
grown anaerobically on xylose as the carbon source than the
microorganism prior to transformation; wherein said microorganism
is transformed with a nucleotide sequence encoding a xylose
isomerase wherein said xylose isomerase is an exogenous xylose
isomerase and wherein said xylose isomerase is an exogenous xylose
isomerase derived from a Lactococcus species; and wherein said
transformed microorganism is a transformed yeast wherein said
transformed yeast is Saccharomyces.
9. The method according to claim 8 wherein said nucleotide sequence
encoding a xylose isomerase comprises the nucleotide sequence shown
as SEQ ID No 1, SEQ ID No 10, SEQ ID No 17, SEQ ID No 12, SEQ ID No
27 or SEQ ID No 28, or a variant, homologue or derivative thereof
having at least 75% identity to the nucleotide sequence shown as
SEQ ID No 1, SEQ ID No 10, SEQ ID No 17, SEQ ID No 12, SEQ ID No 27
or SEQ ID No 28.
10. The method according to claim 8 wherein said nucleotide
sequence encoding a xylose isomerase is in an expression vector
encoding same.
11. A fermentation method comprising culturing in a culture medium
a microorganism according to claim 1.
12. A method for producing a product derived from xylose comprising
culturing in a culture medium a microorganism according to claim
1.
13. A method for producing a biofuel wherein said method comprises
the step of culturing in a culture medium a microorganism according
to claim 1.
14. The method according to claim 13 wherein said method further
comprises the step of obtaining the biofuel from the culture
medium.
15. A biofuel obtained by the method according to claim 13.
16. (canceled)
17. The method according to claim 12 wherein said product derived
from xylose is selected from the group consisting of xylulose,
xylulose-5-phosphate, ethanol, aromatic amino acids, lactic acid,
succinic acid, acetic acid, acetaldehyde, furfural, itaconic acid,
glutamic acid, citric acid, cresol, lysine, 3-hydroxypropionic
acid, poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene.
18. (canceled)
19. A fermentation method comprising culturing in a culture medium
a microorganism according to a microorganism prepared by the method
claim 8.
20. A method for producing a product derived from xylose comprising
culturing in a culture medium a microorganism according to a
microorganism prepared by the method of claim 8.
21. A method for producing a biofuel wherein said method comprises
the step of culturing in a culture medium a microorganism according
to a microorganism prepared by the method claim 8.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a microorganism.
[0002] In particular, the present invention relates to a
transformed microorganism capable of: a higher xylose isomerase
activity than the equivalent microorganism prior to transformation;
and/or a higher growth rate in or on a growth medium comprising
xylose than the equivalent microorganism prior to transformation;
and/or a faster metabolism of xylose than the equivalent
microorganism prior to transformation; and/or a higher production
of ethanol when grown anaerobically on xylose as the carbon source
than the equivalent microorganism prior to transformation.
[0003] The present invention further relates to a method for
preparing transformed microorganisms capable of: a higher xylose
isomerase activity than the microorganism prior to transformation;
and/or a higher growth rate in or on a growth medium comprising
xylose than the microorganism prior to transformation; and/or a
faster metabolism of xylose than the microorganism prior to
transformation; and/or a higher production of ethanol when grown
anaerobically on xylose as the carbon source than the microorganism
prior to transformation.
[0004] Further, the present invention relates to inocula and
culture media comprising a microorganism according to the present
invention or a microorganism prepared by a method according to the
present invention.
[0005] In another aspect, the present invention relates to a
fermentation method comprising culturing in a culture medium a
microorganism according to the present invention or a microorganism
prepared by the method of the present invention.
[0006] The present invention additionally relates to methods for
producing a biofuel and/or a product derived from xylose comprising
culturing in a culture medium a microorganism of the present
invention or a microorganism prepared by the method of the present
invention.
[0007] In addition the present invention relates to a biofuel
and/or a product derived from xylose obtained by a method of the
present invention.
[0008] Further, the present invention relates to the use of a
microorganism according to the present invention or a microorganism
prepared by the method of the present invention for the production
of a product derived from xylose and/or a biofuel.
BACKGROUND
[0009] Ethanol is considered an attractive alternative
transportation fuel that may be used as an additive to, a
supplement to or a part replacement for the fossil fuel gasoline,
which is an ultimately limited resource.
[0010] Ethanol may be used in smaller concentrations instead of
Methyl tert-butyl ether (MTBE) as a cleaner octane booster or
oxygenating additive to gasoline, or it may be used for blending
into gasoline in higher amounts from 10 to 85%, where the mixtures
can be used in only slightly modified automobile engines as we know
them today. It therefore has a tremendous advantage over other
alternative transportation fuels. Any fractional substitution can
be done with maximum flexibility, and this without drastic changes
in transportation engine technology as we know it today.
[0011] Ethanol has the advantage of being a renewable resource, as
it can be produced in very large amounts from plant material. From
this also follows the advantage of having a low net contribution to
the release of carbon dioxide to the atmosphere as the carbon
dioxide released by the use of the ethanol is reabsorbed by the
necessary production of the plant material to regenerate the used
ethanol.
[0012] In traditional ethanol production by fermentation, the plant
material used is 6-carbon sugar, either directly extracted from
plants containing free monosaccharides or disaccharides, or
generated by the hydrolysis of starch from starch containing plant
parts. These sugars are from plant parts that are also used for
human or for animal consumption. Traditional ethanol production is,
therefore, competing directly for plant material which would
otherwise be used for food or feed. The conversion of food into
fuel raises ethical concerns in a world where millions of people
are starving; calculations show that the amount of corn necessary
to produce a tank-full of fuel for one very common off-roader type
car in the USA is approximately the amount needed to feed one
otherwise starving person for one full year.
[0013] An alternative solution is to use lignocellulosic plant
material that is otherwise regarded as waste for generation of
energy and fuel. This is, in general, regarded in a very positive
way, and the development of ways to use lignocellulosic plant
material for ethanol production has, therefore, in many countries
attracted strong support, both politically and by the general
public. Another advantage is the abundance of lignocellulosic
material, actually making the substitution of 50% or more of the
current gasoline consumption with ethanol theoretically
feasible.
[0014] There are numerous sources of lignocellulosic material that
may be used for ethanol production--provided that an efficient and
economical collection and conversion process can be established. A
few examples, where the collection is already being done today,
include sugar cane bagasse, wood chips, corn stover and wheat
straw.
[0015] The two primary technical problems encountered in the use of
lignocellulosic material for ethanol production through
fermentation are: [0016] 1. the difficulties in hydrolysing the
6-carbon sugar containing cellulose into glucose without generating
side-products that hamper or prevent further microbial conversion
of the glucose into ethanol; and [0017] 2. the lack of an organism
that is capable of efficient conversion of 5-carbon sugars (e.g.
xylose and arabinose) present in the hemicellulose part of the
lignocellulosic material into ethanol.
[0018] Saccharomyces cerevisiae has for more than 6000 years been
the preferred microorganism for conversion of 6-carbon sugars
derived from plant material into ethanol. This is due to a
favourable combination of high ethanol yield, high specific
productivity and high ethanol tolerance together with the ability
to grow and ferment at low pH, where other competing microorganisms
have trouble just surviving. When the difficulties in cellulose
hydrolysis has been overcome, then S. cerevisiae will most likely
continue to be the preferred organism to be used for generating
ethanol by fermentation of the released 6-carbon sugar monomers.
But, unfortunately, S. cerevisiae is unable to metabolize 5-carbon
sugars that constitute up to one-third of the lignocellulosic sugar
material.
[0019] Work has consequently been made during the last two decades
to seek ways to genetically engineer S. cerevisiae in order to
introduce 5-carbon sugar fermenting capacity. This work has
primarily focused on xylose fermentation, as xylose constitutes the
dominant part of 5-carbon sugars in lignocellulose. Although S.
cerevisiae cannot metabolize xylose, it is capable of metabolizing
the isomer xylulose (Wang and Schneider, 1980), which is the
metabolite constituting an entry point into the pentose phosphate
pathway. So in principle, the problem can be solved by providing S.
cerevisiae with the ability to convert xylose into xylulose.
[0020] Two different, existing biochemical pathways have been
successfully introduced into S. cerevisiae in order to channel
xylose into the pentose phosphate pathway through xylulose. In
natural xylose-metabolizing fungi, xylose is converted into
xylulose in a two-step process (see FIG. 1). Xylose is first
reduced to xylitol by a xylose reductase (XR--EC 1.1.1.21). Xylitol
is then dehydrogenated to xylulose by xylitol dehydrogenase
(XDH--EC 1.1.1.9). The reductase and the dehydrogenase are not
present in S. cerevisiae, but it has been possible to transfer and
express genes encoding those two enzymes from Pichia stipitis into
S. cerevisiae, and the resulting, modified S. cerevisiae strain is
able to metabolize xylose.
[0021] The activity of the two enzymes requires NADPH and NAD+ and
generates NADP+ and NADH, so it is necessary for the organism to
regenerate the NAD+-NADH and the NADP+-NADPH balance by redox
processes elsewhere in the metabolism, otherwise the xylose
metabolism will cease to function when NADPH and NAD+ has been
depleted. A fairly low rate of xylose metabolism and the generation
of significant amounts of xylitol by S. cerevisiae strains modified
this way has been attributed to this inherent problem in the xylose
reductase--xylitol dehydrogenase pathway.
[0022] In xylose metabolizing bacteria, conversion of xylose into
xylulose is different: a single enzyme, xylose isomerase (EC
5.3.1.5), converts xylose directly into xylulose (see FIG. 1). This
appears to be simpler, and it does not generate the NAD+-NADPH
imbalance as mentioned above. It was therefore also the first
strategy to be proposed for conferring xylose metabolism ability
into S. cerevisiae. But it has proven difficult to make this
strategy work. Expression of most xylose isomerase genes from
bacteria does not result in the presence of an active xylose
isomerase in S. cerevisiae, and the exact reason for this is still
not known. Various studies have revealed that often a protein
produced from expression of a bacterial gene can be detected, but
the expressed protein apparently fails to fold into an active
enzyme in S. cerevisiae.
[0023] Heat stable enzymes are often also more stable in other
types of extreme conditions, such as high salt and extreme pH
values. This may indicate that heat stable enzymes are more likely
to maintain (and probably also to obtain) a correct fold. And it
has indeed been possible to find examples of bacterial xylose
isomerase genes isolated from thermophilic organisms that express
active xylose isomerases in S. cerevisiae (see: Walfridsson et al,
1996; and Bao et al, 1999). These genes have been shown to enable
S. cerevisiae to metabolize xylose, but at a very low rate. The low
rate has been attributed to the fact that the heat-stable xylose
isomerases has an optimal activity at 80-90.degree. C. but the
temperatures that permit the survival of most S. cerevisiae strains
are 30-35.degree. C.
STATEMENTS OF THE INVENTION
[0024] The present invention relates to a transformed microorganism
capable of: [0025] (a) a higher xylose isomerase activity than the
equivalent microorganism prior to transformation; and/or [0026] (b)
a higher growth rate in or on a growth medium comprising xylose
than the equivalent microorganism prior to transformation; and/or
[0027] (c) a faster metabolism of xylose than the equivalent
microorganism prior to transformation; and/or [0028] (d) a higher
production of ethanol when grown anaerobically on xylose as the
carbon source than the equivalent microorganism prior to
transformation.
[0029] In another aspect, the present invention relates to an
inoculum comprising a microorganism according to the present
invention.
[0030] The present invention relates to, in another aspect, a
culture medium comprising a microorganism according to the present
invention.
[0031] In a further aspect, the present invention relates to a
method for preparing a transformed microorganism, said method
comprising the step of transforming a microorganism such that said
transformed microorganism is capable of: [0032] (a) a higher xylose
isomerase activity than the microorganism prior to transformation;
and/or [0033] (b) a higher growth rate in or on a growth medium
comprising xylose than the microorganism prior to transformation;
and/or [0034] (c) a faster metabolism of xylose than the
microorganism prior to transformation; and/or [0035] (d) a higher
production of ethanol when grown anaerobically on xylose as the
carbon source than the microorganism prior to transformation.
[0036] In another aspect, the present invention relates to a
fermentation method comprising culturing in a culture medium a
microorganism according to the present invention or a microorganism
prepared by the method of the present invention.
[0037] The present invention provides, in a further aspect, a
method for producing a product derived from xylose comprising
culturing in a culture medium a microorganism according to the
present invention or a microorganism prepared by the method of the
present invention.
[0038] The present invention provides, in another aspect, a method
for producing a biofuel wherein said method comprises the step of
culturing in a culture medium a microorganism of the present
invention or a microorganism prepared by the method of the present
invention.
[0039] There is provided, in another aspect of the present
invention, a biofuel obtained by a method of the present
invention.
[0040] There is provided, in a further aspect of the present
invention, a product derived from xylose obtained by a method of
the present invention.
[0041] There is provided in a further aspect of the present
invention, the use of a microorganism according to the present
invention or a microorganism prepared by the method of the present
invention for the production of a product derived from xylose.
[0042] Further, the present invention provides the use of a
microorganism according to the present invention or a microorganism
prepared by the method of the present invention for the production
of a biofuel.
[0043] In another aspect, the present invention provides a
transformed microorganism wherein said transformed microorganism
comprises an exogenous nucleotide sequence encoding a xylose
isomerase.
DISCUSSION
[0044] Earlier efforts to find bacterial xylose isomerase genes of
mesophilic origin with high activity when expressed in, for
example, Saccharomyces cerevisiae have been unsuccessful.
Unexpectedly, as disclosed herein, the inventors have now succeeded
in identifying bacteria, in particular mesophilic bacteria, that
contain nucleotide sequences encoding xylose isomerase which upon
expression in, for example, S. cerevisiae, give rise to a high
intracellular xylose isomerase activity in the organism. Such
sequences may be used, for example, to engineer strains of the
Saccharomyces family to enable the yeast to convert xylose into
xylulose and thereby facilitate the efficient production and/or
metabolism of xylose into ethanol. FIG. 4 details the metabolism of
xylose into ethanol; xylulose-5-phosphate--an intermediate which
may be derived from D-xylose--enters into the pentose phosphate
pathway and is further metabolized into ethanol under anaerobic
conditions. But in addition to that, such sequences may also be
used to engineer strains of for example, S. cerevisiae to be
efficient producers of other compounds derived from xylose through
the pentose phosphate pathway. Examples of such compounds include,
but are not limited to, aromatic amino acids, lactic acid, succinic
acid, acetic acid, acetaldehyde, furfural, itaconic acid, glutamic
acid, citric acid, cresol, lysine, 3-hydroxypropionic acid,
poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene.
SOME ADVANTAGES
[0045] Advantageously, the present invention relates to the
transformation (engineering) of microorganisms, such as those of
the Saccharomyces family, to enable the microorganisms to
metabolise xylose. This, in turn, advantageously increases the
efficiency of the microorganisms--in particular Saccharomyces
strains--to produce ethanol from xylose-containing feedstock (such
as lignocellulosic material). In addition, it advantageously
increases the efficiency of microorganisms to produce other natural
or engineered metabolites derived from intermediates in the pentose
phosphate pathway such as aromatic amino acids, lactic acid,
succinic acid, acetic acid, acetaldehyde, furfural, itaconic acid,
glutamic acid, citric acid, cresol, lysine, 3-hydroxypropionic
acid, poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene. Further,
it facilitates the general use of xylose-containing material as
feedstock for the growth of strains of the Saccharomyces family for
industrial purposes.
[0046] A further advantage is that the nucleotide sequence encoding
xylose isomerase is derived from a mesophilic organism and/or is
capable of high activity at mesophilic temperatures.
[0047] Another advantage is that the microorganisms according to
the present invention produce an active xylose isomerase.
[0048] Advantageously, by using the microorganisms according to the
present invention, biofuels (such as ethanol) can be produced.
[0049] More advantageously, by using the microorganisms according
to the present invention, biofuels can be produced from waste
materials such as agricultural wastes (including cereal straw--such
as wheat straw; sugar beet pulp; sugar cane bagasse; stovers--such
as sorghum, Soya beans, maize or corn stovers; and wood chips).
With the present invention there is no need (or there is a reduced
need) to use materials (such as sugar cane extract, sugar beet
extract, sorghum starch, maize starch, wheat starch or corn starch)
which could otherwise be used as a food source for humans and/or as
an animal feed.
[0050] Advantageously, the microorganisms according to the present
invention enable the optimum use of the sugars released by
hydrolysis of lignocellulosic material by the fermentation of
pentose sugars--in particular xylose.
[0051] The present invention enables the production of a biofuel
which is more CO.sub.2 neutral in comparison with petroleum based
transportation fuel. With the present invention, CO.sub.2 emissions
will be low (even lower) when producing the biofuel according to
the present invention than compared to the production of a typical
petroleum based transportation fuel (fossil fuels).
FIGURES
[0052] FIG. 1. A schematic representation of the metabolic pathways
detailing the metabolism of the two aldopentoses, D-xylose and
L-arabinose. Both aldopentoses are converted into a ketopentose,
and further into D-xylulose 5-phosphate. Without wishing to be
bound by theory, as indicated on the figure, one type of pathway
(aldose reductase type) is found in fungi whereas the other type of
pathway (isomerase type) is found in bacteria. In the fungal
pathway type, the first enzymes may be called "D-xylose reductase"
and "L-arabinose reductase", but often the same enzyme can reduce
both D-xylose and L-arabinose and may then serve both pathways and
be referred to by the less specific name "aldose reductase".
[0053] FIG. 2. Without wishing to be bound by theory, FIG. 2 shows
a schematic representation of fungal and bacterial types of
metabolic pathways of some less abundant pentoses (D- and L-lyxose,
D-ribose) detailing the initial metabolism until the entry into the
pentose phosphate pathway.
[0054] FIG. 3. A schematic representation of the non-oxidative part
of the pentose phosphate pathway (PPP).
[0055] FIG. 4. A schematic representation of the metabolic
conversion of xylose into ethanol. Here, ketopentose
xylulose-5-phosphate, which may be derived from D-xylose, is
further metabolized into ethanol under anaerobic conditions. XI is
xylose isomerase and XK is xylulokinase. The net input of xylose
into the process and the net output of ethanol from the process is
shown (nettoprocess).
[0056] FIG. 5. Xylose isomerase (XI) activity in Saccharomyces
cerevisiae transformed with a nucleotide sequence encoding a
bacterial xylose isomerase. The bacterial xylose isomerase is
derived from (i) Pseudomonas syringae, (ii) Thermoanaerobacter
thermohydrosulfuricus, (iii) Thermoanaerobacter
thermohydrosulfurigenes or (iv) Lactococcus lactis subsp. lactis
(Lactobacillus xyloses).
[0057] FIG. 6. The growth on xylose of Saccharomyces cerevisiae
transformed with a nucleotide sequences encoding and expressing a
bacterial xylose isomerase and a nucleotide sequence encoding and
expressing a xylulose kinase cloned from Pichia stipitis.
[0058] FIG. 7. The specific xylose isomerase activity of
Saccharomyces cerevisiae transformed with a nucleotide sequence
encoding and expressing a variant Lactococcus xylose isomerase. The
variant Lactococcus xylose isomerase (XI) has the amino sequence
SEQ ID No 14, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No
20.
DESCRIPTION OF THE INVENTION
[0059] As used herein the phrases "a higher xylose isomerase
activity than the equivalent microorganism prior to transformation"
and "a higher xylose isomerase activity than the microorganism
prior to transformation" refer to a transformed microorganism which
has an xylose isomerase activity of at least 0.16, 0.2, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9 or 1.0 xylose isomerase units per mg
microorganism protein, when cultured under suitable culture
conditions permitting at least the maintenance of said
microorganism; for instance, said culture medium may comprise one
or more substrates (e.g. xylose) capable of activating the
expression of the nucleotide sequence encoding the xylose isomerase
but said culture medium does not comprise any substrate which is
capable of inhibiting the expression of the nucleotide sequence
encoding the xylose isomerase. One example of suitable culture
conditions is described in Example 5. Said xylose isomerase
activity is measured using the cysteine-carbazole assay described
by Dische and Borenfreund (1951). The xylose isomerase activity of
the transformed microorganism is higher than that of the equivalent
microorganism prior to transformation, or the microorganism prior
to transformation, when cultured under the same culture conditions
as the transformed microorganism.
[0060] As used herein the phrases "a higher growth rate in or on a
growth medium comprising xylose than the equivalent microorganism
prior to transformation" and "a higher growth rate in or on a
growth medium comprising xylose than the microorganism prior to
transformation" refer to a transformed microorganism capable of an
increased rate of growth such that the time taken for a doubling in
the number of microorganisms is at least 10%, 15%, 20% or 25% lower
than that of the equivalent microorganism prior to transformation,
or the microorganism prior to transformation, when cultured under
the same conditions.
[0061] The phrases "a faster metabolism of xylose than the
equivalent microorganism prior to transformation" and "a faster
metabolism of xylose than the equivalent microorganism prior to
transformation", as used herein, refer to a transformed
microorganism which is capable of metabolising xylose such that the
consumed amount of xylose in the culture medium is at least 10%,
20%, 25%, 30% or 35% higher per cell than that of the equivalent
microorganism prior to transformation, or the microorganism prior
to transformation, when cultured under the same conditions for a
given time period within the exponential growth phase. Said
transformed microorganism is cultured under suitable culture
conditions permitting at least the maintenance of said
microorganism; for instance, said culture medium may comprise one
or more substrates (e.g. xylose) capable of activating the
expression of the nucleotide sequence encoding the xylose isomerase
but said culture medium does not comprise any substrate which is
capable of inhibiting the expression of the nucleotide sequence
encoding the xylose isomerase.
[0062] As used herein the phrases "a higher production of ethanol
when grown anaerobically on xylose as the carbon source than the
equivalent microorganism prior to transformation" and "a higher
production of ethanol when grown anaerobically on xylose as the
carbon source than the microorganism prior to transformation" refer
to a transformed microorganism which is capable of producing at
least 10%, 20% or 30% more ethanol per cell than that of the
equivalent microorganism prior to transformation when cultured
under anaerobic conditions, with xylose as the carbon source, for a
given time period.
[0063] The term "equivalent microorganism prior to transformation"
as used herein includes references to the microorganism prior to
transformation with a nucleotide sequence that encodes xylose
isomerase or prior to transformation with a nucleotide sequence
that causes the upregulation (e.g. overexpression) of xylose
isomerase.
[0064] In one embodiment the microorganism according to the present
invention has been transformed with a nucleotide sequence that
causes the microorganism to overexpress xylose isomerase. For
example, a promoter is inserted into the genome of a microorganism
which enables the microorganism to overexpress an endogenous
nucleotide sequence encoding xylose isomerase.
[0065] In another embodiment the microorganism according to the
present invention has been transformed with a nucleotide sequence
encoding xylose isomerase. For example, the microorganism is
transformed with an expression vector comprising a nucleotide
sequence encoding xylose isomerase operably linked to a regulatory
sequence.
[0066] Preferably, the microorganism according to the present
invention has been transformed with a nucleotide sequence encoding
a xylose isomerase.
[0067] Preferably, the microorganism according to the present
invention has been transformed with a nucleotide sequence encoding
an exogenous xylose isomerase.
[0068] Preferably, the microorganism according to the present
invention has been transformed with a nucleotide sequence encoding
an exogenous xylose isomerase derived from a Lactococcus
species.
[0069] In a preferred embodiment, the microorganism according to
the present invention has been transformed with a nucleotide
sequence encoding the amino acid sequence shown as SEQ ID No 14,
SEQ ID No 11, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No
20, or a variant, homologue or derivative thereof.
[0070] In a more preferred embodiment, the microorganism according
to the present invention has been transformed with a nucleotide
sequence encoding the amino acid sequence shown as SEQ ID No 19 or
SEQ ID No 20, or a variant, homologue or derivative thereof.
[0071] In a preferred embodiment, the microorganism according to
the present invention has been transformed with the nucleotide
sequence shown as SEQ ID No 1, SEQ ID No 10, SEQ ID No 17, SEQ ID
No 12, SEQ ID No 27 or SEQ ID No 28, or a variant, homologue or
derivative thereof.
[0072] In a more preferred embodiment, the microorganism according
to the present invention has been transformed with the nucleotide
sequence shown as SEQ ID No 27 or SEQ ID No 28, or a variant,
homologue or derivative thereof.
[0073] In one aspect, the microorganism according to the present
invention has been transformed with two or more nucleotide
sequences encoding xylose isomerase.
[0074] Preferably the nucleotide sequence encoding xylose isomerase
mentioned herein is in an expression vector encoding same.
[0075] Preferably the expression vector mentioned herein comprises
a promoter capable of overexpressing the nucleotide sequence
encoding xylose isomerase. Examples of such promoters include the
GPD promoter, the TEF promoter and the ADP promoter. Preferred
promoters which may be used to overexpress xylose isomerase can be
any of the regulatory elements controlling the expression of
nucleotide sequences encoding proteins involved in glycolysis and
glucose fermentation.
[0076] In one embodiment, in the method according to the present
invention, the microorganism is transformed with a nucleotide
sequence that causes the microorganism to overexpress xylose
isomerase. For example, a promoter is inserted into the genome of a
microorganism which enables the microorganism to overexpress an
endogenous nucleotide sequence encoding xylose isomerase. In a
further example, a promoter is inserted into the genome of a
microorganism which enables the microorganism to constitutively
express an endogenous nucleotide sequence encoding xylose
isomerase.
[0077] In another embodiment in the method according to the present
invention, the microorganism is transformed with a nucleotide
sequence encoding xylose isomerase. For example, the microorganism
is transformed with an expression vector comprising a nucleotide
sequence encoding xylose isomerase operably linked to a regulatory
sequence. In a further example, the microorganism is transformed
with an expression vector comprising a nucleotide sequence encoding
xylose isomerase operably linked to a regulatory sequence wherein
said regulatory sequence enables the constitutive expression of the
nucleotide sequence encoding xylose isomerase.
[0078] Preferably, in the method according to the present
invention, the microorganism is transformed with a nucleotide
sequence encoding a xylose isomerase.
[0079] Preferably, in the method according to the present
invention, the microorganism is transformed with a nucleotide
sequence encoding an exogenous xylose isomerase.
[0080] In a preferred embodiment, in the method according to the
present invention the microorganism is transformed with a
nucleotide sequence, encoding a xylose isomerase, comprising the
nucleotide sequence shown as SEQ ID No 1, SEQ ID No 10, SEQ ID No
17, SEQ ID No 12, SEQ ID No 27 or SEQ ID No 28, or a variant,
homologue or derivative thereof.
[0081] In a preferred embodiment, in the method according to the
present invention the microorganism is transformed with a
nucleotide sequence encoding a xylose isomerase comprising the
amino acid sequence shown as SEQ ID No 14, SEQ ID No 11, SEQ ID No
18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No 20, or a variant,
homologue or derivative thereof.
[0082] In one aspect, in the method according to the present
invention, the microorganism is transformed with two or more
nucleotide sequences encoding xylose isomerase.
[0083] In the method according to the present invention, preferably
the nucleotide sequence encoding a xylose isomerase is in an
expression vector encoding same.
[0084] As used herein the term "fermentation method" refers to
culturing a microorganism or microorganisms under aerobic and
anaerobic conditions.
[0085] In one embodiment, the culture medium comprises xylose
and/or a source of xylose.
[0086] In one embodiment, the culture medium comprises a pentose
sugar and/or a source of pentose sugar. Preferably, the pentose
sugar is xylose. In one embodiment, the pentose sugar is derived
and/or derivable from lignocellulosic material.
[0087] Alternatively, or in addition, the culture medium comprises
material derived from lignocellulosic material.
[0088] Preferably, the method according to the present invention
further comprises the step of obtaining the biofuel from the
culture medium.
[0089] In one embodiment the product derived from xylose is
selected from the group consisting of xylulose,
xylulose-5-phosphate, ethanol, aromatic amino acids, lactic acid,
succinic acid, acetic acid, acetaldehyde, furfural, itaconic acid,
glutamic acid, citric acid, cresol, lysine, 3-hydroxypropionic
acid, poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene.
[0090] Preferably, the product derived from xylose is selected from
the group consisting of xylulose, xylulose-5-phosphate, ethanol,
aromatic amino acids, lactic acid, succinic acid, acetic acid,
acetaldehyde, furfural, itaconic acid, glutamic acid, citric acid,
cresol, lysine, 3-hydroxypropionic acid and
poly-3-hydroxyalkanoates.
[0091] In a highly preferred embodiment, the product derived from
xylose is ethanol.
[0092] As used herein, the term "overexpress" in the phrase "a
nucleotide sequence that causes the microorganism to overexpress
xylose isomerase" and "a promoter capable of overexpressing the
nucleotide sequence encoding xylose isomerase" refers to an
increase in expression from zero to a level of expression or going
from a lower level of expression to a higher level of expression
(e.g. upregulation) when the transformed microorganism is compared
to the equivalent microorganism prior to transformation.
Microorganisms overexpressing xylose isomerase have an increased
ability to catalyse the conversion of xylose to xylulose. The
ability to convert xylose to xylulose may be confirmed by
determining the ability of cell lysate to produce xylulose from
xylose by assaying the produced xylulose using, for example, a
ketopentose assay as described by Dische and Borenfreund (Dische
and Borenfreund, 1951), also described in the example section
herein.
[0093] Preferably the transformed microorganism which overexpresses
xylose isomerase is capable of: [0094] (a) a higher xylose
isomerase activity than the equivalent microorganism prior to
transformation; and/or [0095] (b) a higher growth rate in or on a
growth medium comprising xylose than the equivalent microorganism
prior to transformation; and/or [0096] (c) a faster metabolism of
xylose than the equivalent microorganism prior to transformation;
and/or [0097] (d) a higher production of ethanol when grown
anaerobically on xylose as the carbon source than the equivalent
microorganism prior to transformation.
[0098] Examples of microorganisms overexpressing xylose isomerase
include: (i) microorganisms transformed with an expression vector
encoding xylose isomerase (prior to transformation said
microorganism was not capable of expressing xylose isomerase); and
(ii) microorganisms transformed to upregulate the expression of an
endogenous xylose isomerase (prior to transformation said
microorganism was capable of expressing said xylose isomerase for a
given set of culture conditions during exponential growth but after
transformation said microorganism is capable of expressing said
xylose isomerase at a higher level, in the same culture conditions,
during exponential growth).
[0099] The term "a nucleotide sequence encoding xylose isomerase"
as used herein encompasses nucleotide sequences comprising
regulatory sequences enabling the expression of the nucleotide
sequence encoding xylose isomerase such as promoters and enhancers
which may be natively or non-natively associated with the
nucleotide sequence encoding xylose isomerase.
[0100] In one aspect, the present invention provides a genetically
modified yeast cell having a functional exogenous xylose isomerase
gene derived from a microorganism of the Lactococcus family,
wherein the exogenous xylose isomerase gene is operatively linked
to promoter and terminator sequences that are functional in the
yeast.
[0101] In another aspect, the present invention provides a
fermentation process in which a microorganism as described herein
is cultured under fermentation conditions in a fermentation broth
that includes material derived from lignocellulosic material.
[0102] In yet another aspect, the present invention provides a
fermentation process utilising a microorganism according to the
present invention wherein the fermentation broth (i.e. the culture
medium) comprises a pentose sugar.
Transformed Microorganism
[0103] As mentioned herein, the term "transformed microorganism"
refers to a microorganism that has been genetically altered by
recombinant DNA technology. The term "transformed" as used herein
is synonymous with terms such as "transfected", "recombinant",
"genetically engineered" and "genetically modified".
[0104] The term "transformed microorganism" in relation to the
present invention includes any microorganism that comprises an
expression vector(s) comprising the nucleotide sequence(s)
mentioned herein and/or a promoter(s) that is capable of allowing
the expression (in particular overexpression i.e. upregulation) of
the nucleotide sequence(s) mentioned herein. In one embodiment the
nucleotide sequence(s) is incorporated in the genome of the
microorganism. In another embodiment, the promoter is incorporated
in the genome of the microorganism. These features enable the
transformed microorganism (when compared to equivalent
microorganism prior to transformation) to have (a) a higher xylose
isomerase activity; and/or (b) a higher growth rate in or on a
growth medium comprising xylose; and/or (c) a faster metabolism of
xylose; and/or (d) a higher production of ethanol when grown
anaerobically on xylose as the carbon source.
[0105] The term "transformed microorganism" does not cover native
nucleotide coding sequences in their natural environment when they
are under the control of their native promoter which is also in its
natural environment.
[0106] Therefore, the transformed microorganism of the present
invention includes a microorganism comprising any one of, or
combinations of, the nucleotide sequences coding for the enzymes
mentioned herein, constructs comprising said nucleotide sequences,
vectors comprising said nucleotide sequences, plasmids comprising
said nucleotide sequences and expression vectors comprising said
nucleotide sequences.
[0107] Thus, a further embodiment of the present invention provides
microorganisms transformed or transfected with a nucleotide
sequence(s) that expresses the enzyme(s) mentioned herein. The
microorganism will be chosen to be compatible with the vector and
may be, for example, bacterial, fungal or yeast cells.
[0108] Examples of suitable bacterial host organisms are gram
positive or gram negative bacterial species.
[0109] Depending on the nature of the nucleotide sequence(s)
encoding the enzyme(s) mentioned herein, eukaryotic hosts such as
yeasts or other fungi may be preferred. In general, yeast cells are
preferred over fungal cells because they are easier to
manipulate.
[0110] The use of suitable microorganisms--such as yeast and fungal
host cells--may provide for post-translational modifications (e.g.
myristoylation, glycosylation, truncation, lipidation and tyrosine,
serine or threonine phosphorylation) as may be needed to confer
optimal biological activity on recombinant expression products
mentioned herein.
[0111] Suitable microorganisms include bacteria, fungi and yeasts.
Preferably the microorganism is a yeast.
[0112] Preferably said transformed microorganism is a transformed
yeast. Preferably said transformed yeast is derived from the genus
Saccharomyces. More preferably said transformed yeast is
Saccharomyces cerevisiae.
[0113] In one embodiment, the transformed microorganism described
herein is capable of a higher xylose isomerase activity than the
equivalent microorganism prior to transformation.
[0114] In another aspect, the transformed microorganism described
herein is capable of a higher growth rate in or on a growth medium
comprising xylose than the equivalent microorganism prior to
transformation.
[0115] In a further aspect, the transformed microorganism described
herein is capable of a faster metabolism of xylose than the
equivalent microorganism prior to transformation.
[0116] In another aspect, the transformed microorganism described
herein is capable of a higher production of ethanol when grown
anaerobically on xylose as the carbon source than the equivalent
microorganism prior to transformation.
[0117] The microorganism may be transformed using techniques which
are routine in the art such as electroporation (Sambrook et al
1989). Further, the presence of a sequence in a transformed
microorganism may be determined by growth selection on suitable
media which select for the growth of the transformed microorganism.
Alternatively or in addition, the presence of inserted,
heterologous DNA sequences may be determined by direct colony PCR
using primers specifically designed for the inserted sequence. Such
techniques are well known and routine in the art (see, for example,
Sambrook et al 1989 and Ausubel et al 1995).
[0118] The transformed microorganisms according to the present
invention may be used in combination with one or more further
microorganisms. For example, one or more transformed microorganisms
according to the present invention may cultured in combination with
at least one microorganism capable of producing, under certain
culture conditions, one of more components selected from the list
consisting of: ethanol, aromatic amino acids, lactic acid, succinic
acid, acetic acid, acetaldehyde, furfural, itaconic acid, glutamic
acid, citric acid, cresol, lysine, 3-hydroxypropionic acid,
poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene.
[0119] In a further aspect, there is provided a combination of (i)
one or more transformed microorganisms according to the present
invention and (ii) at least one further microorganism capable of
producing, under certain culture conditions, one of more components
selected from the list consisting of: ethanol, aromatic amino
acids, lactic acid, succinic acid, acetic acid, acetaldehyde,
furfural, itaconic acid, glutamic acid, citric acid, cresol,
lysine, 3-hydroxypropionic acid, poly-3-hydroxyalkanoates,
protocatechuic acid, pyrocatechol, guaiacol, veratrol, resveratrol,
vanillin, vanillic acid, vanillyl alcohol, muconic acid, adipic
acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde,
4-methoxybenzoic acid, 4-aminobenzoate, 4-hydroxyaniline,
4-methoxyaniline, quinol, anisole, phenol, anthranilic acid,
3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid, 2-aminophenol,
1,4-cyclohexanedione, isoprene and styrene.
[0120] Additionally, the present invention provides an inoculum
comprising a combination of a transformed microorganism according
to the present invention and one or more further
microorganisms.
[0121] Further, there is provided a culture medium comprising a
combination of a transformed microorganism according to the present
invention and one or more further microorganisms.
[0122] In addition, the present invention provides a kit comprising
an inoculum comprising one or more microorganisms according to the
present invention.
[0123] In addition, the present invention provides a kit comprising
(i) an inoculum comprising one or more microorganisms according to
the present invention and (ii) an inoculum comprising one or more
further microorganisms.
Transformed Yeast
[0124] In a preferred embodiment, the transgenic microorganism is a
yeast.
[0125] A review of the principles of heterologous gene expression
in yeast are provided in, for example, Methods Mol Biol (1995),
49:341-54, and Curr Opin Biotechnol (1997) October; 8(5):554-60
[0126] In this regard, yeast--such as the species Saccharomyces
cerevisi or Pichia pastoris (see FEMS Microbiol Rev (2000
24(1):45-66), may be used as a vehicle for heterologous gene
expression.
[0127] A review of the principles of heterologous gene expression
in Saccharomyces cerevisiae and secretion of gene products is given
by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the
expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose
and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
[0128] For the transformation of yeast, several transformation
protocols have been developed. For example, a transgenic
Saccharomyces according to the present invention can be prepared by
following the teachings of Hinnen et al., (1978, Proceedings of the
National Academy of Sciences of the USA 75, 1929); Beggs, J D
(1978, Nature, London, 275, 104); and Ito, H et al (1983, J
Bacteriology 153, 163-168).
[0129] The transformed yeast cells may be selected using various
selective markers--such as auxotrophic markers dominant antibiotic
resistance markers.
Expression Vector
[0130] The term "expression vector" means a construct capable of in
vivo or in vitro expression.
[0131] In one aspect, the expression vector is incorporated into
the genome of a suitable microorganism. The term "incorporated"
preferably covers stable incorporation into the genome.
[0132] The nucleotide sequences mentioned herein may be present in
a vector in which the nucleotide sequence is operably linked to
regulatory sequences capable of providing for the expression of the
nucleotide sequence by a suitable host microorganism.
[0133] The vectors are transformed into a suitable host
microorganism as described herein.
[0134] The choice of vector e.g. a plasmid, cosmid, or phage vector
will often depend on the microorganism into which it is to be
introduced.
[0135] The vectors for use herein may contain one or more
selectable marker nucleotide sequences--such as a nucleotide
sequence which confers antibiotic resistance e.g. ampicillin,
kanamycin, chloramphenicol or tetracyclin resistance.
Alternatively, the selection may be accomplished by
co-transformation (as described in WO91/17243).
[0136] Vectors may be used in vitro, for example to transfect,
transform, transduce or infect a host microorganism.
[0137] The vector may further comprise a nucleotide sequence
enabling the vector to replicate in the host microorganism in
question. Examples of such sequences are the origins of replication
of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0138] In a preferred aspect, the microorganism capable of
converting xylose to xylulose as mentioned herein comprises a
nucleotide sequence encoding xylose isomerase.
[0139] Preferably an expression vector as mentioned herein
comprises a nucleotide sequence encoding xylose isomerase.
[0140] In a further aspect, preferably the microorganism capable of
converting xylose to xylulose as mentioned herein comprises at
least one expression vector encoding xylose isomerase.
[0141] Preferably, in another aspect, the microorganism capable of
converting xylose to xylulose as mentioned herein may further
comprise at least one expression vector encoding one or more
enzymes selected from the group consisting of xylulokinase,
D-ribulokinase, ribose-5-phosphate isomerase, ribulose-5-phosphate
epimerase, transaldolase, transketolase and any other enzyme of the
pentose phosphate pathway. More preferably, said microorganism
capable of converting an aldopentose to a ketopentose as mentioned
herein further comprises at least one expression vector encoding
xylulokinase.
[0142] In one aspect, an expression vector as mentioned herein, may
further encode one or more enzymes selected from the group
consisting of an aldose-1-epimerase, xylose reductase, D-xylulose
reductase, arabinose reductase, L-arabitol 4-dehydrogenase,
L-xylulose reductase, L-arabinose isomerase, ribulokinase, ribulose
phosphate 4-epimerase, D-lyxose isomerase, D-ribose isomerase,
xylulokinase, D-ribulokinase, ribulose-5-phosphate epimerase,
ribose-5-phosphate isomerase, transaldolase, and transketolase.
[0143] In one aspect, an expression vector as mentioned herein, may
further encode one or more enzymes selected from the group
consisting of aldose-1-epimerase, xylulokinase, D-ribulokinase,
ribose-5-phosphate isomerase, D-ribulose-5-phosphate epimerase,
transaldolase, transketolase and any other enzyme of the pentose
phosphate pathway. Preferably, said expression vector as mentioned
herein further encodes xylulokinase.
[0144] In a preferred aspect, the microorganism capable of
converting xylose to xylulose as mentioned herein further comprises
at least one expression vector encoding an aldose-1-epimerase.
[0145] Preferably an expression vector as mentioned herein further
comprises a nucleotide sequence encoding an aldose-1-epimerase.
Regulatory Sequences
[0146] In some applications, the nucleotide sequence(s) mentioned
herein is operably linked to a regulatory sequence which is capable
of providing for the expression of the nucleotide sequence, such as
by the chosen microorganism. By way of example, the present
invention covers the use of a vector comprising the nucleotide
sequence(s) mentioned herein operably linked to such a regulatory
sequence, i.e. the vector is an expression vector.
[0147] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A regulatory sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0148] The term "regulatory sequences" includes promoters and
enhancers and other expression regulation signals.
[0149] In one embodiment, the regulatory sequence enables the
nucleotide sequence(s) operably linked to the regulatory sequence
to be constitutively expressed in a host cell.
[0150] As used herein the terms "constitutively expressed" and
"constitutive expression" refer to the continual transcription of a
nucleotide sequence operably linked to the regulatory sequence
(such as a constitutive promoter). Thus, for example, the culture
medium does not need to comprise a substrate in order to activate
the expression of the nucleotide sequence encoding the xylose
isomerase. In another example, the culture medium could not
comprise a substrate which inhibits the regulatory sequence and,
therefore, inhibits the expression of the nucleotide sequence
encoding the xylose isomerase.
[0151] The term "promoter" is used in the normal sense of the art,
e.g. an RNA polymerase binding site.
[0152] Enhanced expression of the nucleotide sequence(s) encoding
the enzyme(s) mentioned herein may also be achieved by the
selection of heterologous regulatory regions, e.g. promoter,
secretion leader and terminator regions.
[0153] Preferably, the nucleotide sequence(s) mentioned herein is
operably linked to at least a promoter.
[0154] Other promoters may even be used to direct expression of the
polypeptide(s) mentioned herein.
[0155] Examples of suitable promoters for directing the
transcription of the nucleotide sequence in a bacterial, fungal or
yeast cell are well known in the art.
[0156] The promoter can additionally include features to ensure or
to increase expression in a suitable host. For example, the
features can be conserved regions such as a Pribnow Box or a TATA
box.
[0157] In one embodiment, the promoter enables the nucleotide
sequence(s) operably linked to the promoter to be constitutively
expressed in a host cell.
Constructs
[0158] The term "construct"--which is synonymous with terms such as
"conjugate", "cassette" and "hybrid"--includes a nucleotide
sequence mentioned herein directly or indirectly attached to a
promoter.
[0159] An example of an indirect attachment is the provision of a
suitable spacer group such as an intron sequence, such as the
Sh1-intron or the ADH intron, intermediate the promoter and the
nucleotide sequence(s) mentioned herein. The same is true for the
term "fused" in relation to the present invention which includes
direct or indirect attachment. In some cases, the terms do not
cover the natural combination of the nucleotide sequence coding for
the protein ordinarily associated with the wild type gene promoter
and when they are both in their natural environment.
[0160] The construct may even contain or express a marker, which
allows for the selection of the genetic construct.
[0161] For some applications, preferably the construct comprises at
least the nucleotide sequence(s) mentioned herein operably linked
to a promoter.
Promoters
[0162] As mentioned herein, in one aspect the present invention
relates to a microorganism that has been transformed with a
nucleotide sequence, such as a promoter, that causes the
microorganism to overexpress xylose isomerase.
[0163] For instance, the promoter is inserted into the genome of a
microorganism which enables the microorganism to overexpress (e.g.
upregulate) an endogenous nucleotide sequence encoding xylose
isomerase.
[0164] In one embodiment, the promoter is inserted into the genome
of a microorganism which enables the microorganism to
constitutively express an endogenous nucleotide sequence encoding
xylose isomerase.
[0165] In another aspect, the promoter is operably linked to a
nucleotide sequence in, for example, an expression vector.
[0166] In one embodiment, the promoter enables the nucleotide
sequence(s) operably linked to the promoter to be constitutively
expressed in a host cell.
[0167] In another aspect, the promoter is not repressed by the
presence of glucose.
[0168] Examples of suitable promoters that could be used in
microorganisms according to the present invention, such as
Saccharomyces cerevisiae, include: the promoter of the
glyceraldehyde-3-phosphate dehydrogenase (GPD) gene; the promoter
of the alcohol dehydrogenase (ADH) gene; and the promoter of the
Thyrotrophic embryonic factor (TEF) gene.
[0169] Preferred promoters which may be used to overexpress xylose
isomerase can be any of the regulatory elements controlling the
expression of nucleotide sequences encoding proteins involved in
glycolysis and glucose fermentation in yeast, in particular
Saccharomyces such as S. cerevisiae. Examples of these are the
glucokinase (GLK1) promoter, the phosphoglucose isomerase (PGI1)
promoter, the phosphofructokinase (PFK1) promoter and the
glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter.
Biofuel
[0170] As used herein, the term "biofuel" refers to a fuel (e.g. a
liquid fuel) suitable for use in (for example) combustion engines.
Said biofuel is derived from biological matter comprising pentose
sugars and/or from which pentose sugars can be derived by
hydrolysis by enzymatic means and/or by acidic treatment.
Preferably said pentose sugar is the aldopentose xylose.
[0171] Plant materials--including plant waste comprising
lignocellulosic material (for instance: cereal straw, such as wheat
straw; sugar beet pulp; sugar cane bagasse; sorghum stover; Soya
bean stover; maize stover; corn stover; wood-chips; and paper-pulp)
and whole plants (such as those which are grown for energy purposes
e.g. switchgrass)--are suitable sources for pentose sugars, in
particular aldopentose sugars (such as xylose), for the present
invention. Other suitable sources of plant material include
non-waste products (in other words, food and feed sources) such as
sugar cane extract, sugar beet extract, sorghum, Soya beans, wheat
starch and corn starch.
[0172] Preferably the biofuel mentioned herein comprises at least
one alcohol.
[0173] In a preferred aspect, the alcohol is selected from the
group consisting of methanol, ethanol, propanol and butanol. More
preferably the biofuel comprises ethanol.
[0174] Preferably, said biofuel is obtained (or obtainable)--in
other words, extracted (or extractable)--from the culture medium in
which one or more transformed microorganisms according to the
present invention have been cultured under suitable conditions.
Said biofuel may be obtained (or obtainable) from the culture
medium using techniques which are routine in the art such as the
removal of microorganism by centrifugation, isolation of the
supernatant followed by distillation, and a further dehydration
step to yield a 99.5% pure alcohol such as ethanol.
[0175] The biofuel may comprise one or more further biofuel
components such as butanol.
[0176] The one or more further biofuel components may be admixed
with the biofuel before and/or after the biofuel is obtained or
extracted (obtainable or extractable) from a culture.
[0177] Alternatively or in addition, one or more further biofuel
components may be produced by culturing a microorganism in a
culture medium before and/or after and/or at the same time as a
transformed microorganism according to the present invention is/has
been cultured in a culture medium in order to produce the
biofuel.
[0178] The present invention further provides a transportation fuel
which comprises a biofuel produced using the microorganisms
according to the present invention.
[0179] Ethanol used as a transportation fuel may serve two
different purposes:
(i) it can act as an oxygenated additive that raises the octane
value and reduces emission in ReFormulated Gasoline (RFG)
(tetraethyl lead or MTBE replacement); (ii) it can act as a partial
or full substitute for Regular Gasoline (RG) to reduce dependency
on gasoline supply.
[0180] Anhydrous ethanol has an octane value of 130, and can be
added in concentrations of 5-10% (depending on the season) to
Regular Gasoline obtained directly from refineries. Traditionally,
tetra-ethyl lead has been used for octane boosting however, due to
health issues, the use of lead has been banned almost worldwide.
The addition of oxygenates to Regular Gasoline lowers the carbon
monoxide emissions as well as other particles contributing to air
pollution. Methyl tert-butyl ether (MTBE) was initially used as a
oxygenate additive, however the occurrence of MTBE contamination in
drinking water aquifers has prompted some states to ban the use of
this oxygenate. Ethanol is increasingly used worldwide as a
replacement for MTBE as an oxygenate additive for the manufacturing
of RFG.
[0181] Apart from serving as an oxygenate additive in the
production of ReFormulated Gasoline, ethanol can be used as a
general substitute for regular gasoline. Cars can use E10 blends
(10% added ethanol) without any modification of the engine.
[0182] Further, vehicles have been manufactured which can run on
100% ethanol--in other words, there is no requirement for a fossil
based fuel.
[0183] The transformed microorganisms according to the present
invention or the microorganisms prepared by a method according to
the present invention are capable of producing a biofuel at a
higher rate than the equivalent microorganism prior to
transformation. As used here, the term "higher rate" refers to a
transformed microorganism which is capable of producing in the
culture medium at least 5%, 10%, 20%, 25%, 30% or 35% more biofuel
(such as bioethanol) per cell than that of the equivalent
microorganism prior to transformation when cultured under the same
culture conditions for a given time period within the exponential
growth phase.
Xylose Derived Product
[0184] As used herein, the term "xylose derived product" or
"product derived from xylose" refers to any compound derived from
xylose.
[0185] Examples of products derived from xylose include, but are
not limited to: ethanol, aromatic amino acids, lactic acid,
succinic acid, acetic acid, acetaldehyde, furfural, itaconic acid,
glutamic acid, citric acid, cresol, lysine, 3-hydroxypropionic
acid, poly-3-hydroxyalkanoates, protocatechuic acid, pyrocatechol,
guaiacol, veratrol, resveratrol, vanillin, vanillic acid, vanillyl
alcohol, muconic acid, adipic acid, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, 4-methoxybenzoic acid, 4-aminobenzoate,
4-hydroxyaniline, 4-methoxyaniline, quinol, anisole, phenol,
anthranilic acid, 3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid,
2-aminophenol, 1,4-cyclohexanedione, isoprene and styrene.
[0186] A product derived from xylose may be converted to another
product. For example, the ketopentose D-xylulose, derived from the
aldopentose D-xylose, may be converted via the pentose phosphate
pathway into ethanol.
[0187] Preferably said product derived from xylose is one or more
selected from the group consisting of ethanol, aromatic amino
acids, lactic acid, succinic acid, acetic acid, acetaldehyde,
furfural, itaconic acid, glutamic acid, citric acid, cresol,
lysine, 3-hydroxypropionic acid, poly-3-hydroxyalkanoates,
protocatechuic acid, pyrocatechol, guaiacol, veratrol, resveratrol,
vanillin, vanillic acid, vanillyl alcohol, muconic acid, adipic
acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde,
4-methoxybenzoic acid, 4-aminobenzoate, 4-hydroxyaniline,
4-methoxyaniline, quinol, anisole, phenol, anthranilic acid,
3-hydroxyanthranilate, 2,3-dihydroxybenzoic acid, 2-aminophenol,
1,4-cyclohexanedione, isoprene and styrene.
[0188] More preferably said product derived from xylose is
ethanol.
Culture Medium
[0189] In one embodiment, the culture medium comprises xylose
and/or a source of xylose.
[0190] In addition, the culture medium may comprise at least one
further pentose. In a preferred aspect the culture medium further
comprises at least one aldopentose.
[0191] Preferably the transformed microorganisms are grown in the
optimal culture medium for said microorganism. Using routine
techniques, the optimal culture medium can be determined; in
addition, the optimal growth conditions can be determined.
[0192] In one embodiment, the culture medium comprises one or more
substrates capable of activating the expression of a nucleotide
sequence encoding the xylose isomerase in a microorganism according
to the present invention but said culture medium does not comprise
any substrate which is capable of inhibiting the expression of a
nucleotide sequence encoding the xylose isomerase in a
microorganism according to the present invention. For example: if
the nucleotide sequence encoding the xylose isomerase comprises a
xylose inducible promoter then the medium comprises xylose.
[0193] In one aspect said culture medium comprises about 1%, about
2%, about 4%, about 8%, about 15% or about 25% xylose before
inoculation with the microorganism (i.e. at time zero).
[0194] Preferably said culture medium comprises the optimal amounts
of salts, vitamins and other nutrients necessary for the
microorganism.
[0195] The microorganisms are preferably cultured at their optimal
growth temperature. The skilled person would have readily been able
to determine the optimal temperature at which to culture
microorganisms mentioned herein.
[0196] In one embodiment the microorganisms are cultured at about
20.degree. C., 25.degree. C., 30.degree. C., 35.degree. C., or
37.degree. C.
[0197] In one embodiment the microorganisms are cultured at about
35.degree. C. to 39.degree. C., preferably about 36.degree. C. to
38.degree. C., more preferably at about 35.5.degree. C. to
37.5.degree. C.
[0198] In one embodiment the microorganisms are cultured for about
3 to 96 hours; preferably about 3 to 48 hours, about 3 to 24 hours,
about 3 to 15 hours and about 3 to 6 hours.
[0199] Preferably the microorganisms are cultured for about 3
hours, about 6 hours, about 15 hours, about 24 hours, about 48
hours or about 96 hours.
[0200] In one aspect, the microorganism, in particular the
transformed microorganism, is alcohol tolerant and/or acid
tolerant.
[0201] The term "alcohol tolerant" in relation to the present
invention refers to microorganisms which are capable of growth in a
culture medium which comprises at least 2%, 5%, 10% or 15%
alcohol.
[0202] As mentioned herein, the term "acid tolerant" refers to
microorganisms which are capable of growth in a culture medium
which has a pH equal to or less than 6.5, 6.0, 5.0, 4.0 or 3.0.
[0203] In a preferred aspect, the culture medium is inoculated with
at least 5.times.10.sup.7 to 5.times.10.sup.11 cells per kg of
culture medium, preferably 5.times.10.sup.8 to 5.times.10.sup.10
cells per kg of culture medium, preferably 1.times.10.sup.9 to
1.times.10.sup.10 cells per kg of culture medium and more
preferably about 5.times.10.sup.9 cells per kg of culture
medium.
[0204] The terms "inoculum" and "starter culture" are
interchangeable.
[0205] The culture conditions permit, at least, the maintenance of
the microorganism according to the present invention or the
microorganism prepared by a method according to the present
invention. The culture conditions may optionally permit the growth
of the microorganism according to the present invention or the
microorganism prepared by a method according to the present
invention.
Sources of Xylose
[0206] Xylose is an aldopentose. Xylose may be derived from: plant
materials typically used as food or feed sources (such as: sugar
cane, sugar beet, sorghum, wheat and corn--which are starch-rich
and sugar-rich plant materials); whole plants (such as those which
are grown for energy purposes e.g. switchgrass); and, in
particular, waste agricultural (plant) materials (such as: cereal
straw, for instance, wheat straw; sugar beet pulp; bagasse, for
instance, sugar cane bagasse; stovers, for instance, sorghum, Soya
bean, maize or corn stovers; and wood chips).
[0207] Sources of xylose for the culture medium described herein
include lignocellulosic materials normally regarded as agricultural
waste material. Stems, stalks and leaves contain lignocellulosic
material and are, therefore, sources of lignocellulosic material.
Sugar cane bagasses, corn stovers and wood chips (hemicellulose
only) are three easily accessible sources of lignocellulosic
material as these are already collected or stocked in large amounts
for various reasons.
[0208] Lignocellulosic material consists primarily of long sugar
chains. On average, two thirds of these sugars are hexose sugars,
which are mainly present in cellulose, and one third of the sugars
are pentose sugars present mainly in arabinoxylan polymers.
[0209] A significant amount of hemicellulose derived pentose sugar
is xylose.
[0210] Lignocellulosic materials can be hydrolysed in order to
release the hexose and/or pentose sugars in the long-chain sugars
of the cellulose, hemicellulose and lignin.
[0211] Hydrolysis of lignocellulosic materials can be carried out
by acidic treatment at elevated temperature. However, this
treatment may generate sugar derived by-products that are toxic to
the majority of microorganisms and prevent the conversion of the
sugars to ethanol. Such toxic by-products (if generated) can be
removed but this is generally uneconomical.
[0212] Alternatively, lignocellulosic materials can be hydrolysed
using cellulose and hemicellulose hydrolyzing enzymes.
Advantageously, this process avoids the generation of toxic
side-products.
[0213] In a preferred aspect, the culture medium comprises material
derived from one or more lignocellulosic materials which have been
treated (examples of such treatment techniques include: steam
treatment, steam explosion, wet oxidation, acid hydrolysis,
alkaline wet oxidation and ammonia fibre expansion) to release
xylose. Preferably the lignocellulosic material is treated by an
enzymatic hydrolysis process. Said hydrolysed lignocellulosic
material may be further treated in order to extract the sugars
before the use of said extract in a culture medium.
Hydrolysis of Lignocellulosic Material
Initial Mechanical Treatment:
[0214] The lignocellulosic material is chopped into smaller pieces
as and when deemed necessary. For example, wheat straw is cut into
pieces of approximately 5 cm in length.
Subsequent Hydrothermal Pretreatment:
[0215] The hydrothermal pretreatment of the lignocellulosic
material may be carried out as a steam pretreatment followed by a
washing step, thereby producing a fibre fraction and a liquid
fraction. The fibre fraction contains more than 90% of the
cellulose, the lignin originally present in the cellulosic
material, and some of the hemicelluloses. The liquid fraction
contains sugars from the hemicelluloses (C5 sugars), more than 90%
of alkali chlorides comprised in the lignocellulosic biomass, and
the majority of fermentation inhibitors arising from pretreatment
of lignocellulosic feedstock.
[0216] Typically, wheat straw is heated by steam to a temperature
between 180 and 200.degree. C. with a residence time of 5-15 min.
The pretreated biomass is unloaded from the pressure reactor and
washed and pressed. Released steam is collected and reused for
evaporation of the liquid fraction to feed molasses.
Enzymatic Hydrolysis:
[0217] Subsequent hydrolysis of sugar polymers may be carried out
by the addition of cellulases and hemicellulases, either prior to
fermentation or during fermentation or both inter alia a
simultaneous saccharification and fermentation process.
Hexose
[0218] Hexose sugars have 6 carbon atoms. Aldohexoses have an
aldehyde at position 1, and ketohexoses having a ketone at position
2. Glucose is an example of an aldohexose. Fructose is an example
of a ketohexose.
Pentose
[0219] Pentose sugars have 5 carbon atoms. Pentoses either have an
aldehyde functional group in position 1 (aldopentoses) or a ketone
functional group in position 2 (ketopentoses). Xylose, arabinose,
ribose, lyxose, xylulose and ribulose are examples of pentoses.
Aldopentose
[0220] Xylose, arabinose, ribose and lyxose are examples of
aldopentoses.
[0221] In one aspect, preferably said aldopentose is xylose.
[0222] Xylose may be L-xylose or D-xylose.
[0223] Preferably said xylose is D-xylose.
Ketopentose
[0224] Xylulose and ribulose are examples of ketopentoses.
[0225] In one aspect said ketopentose is xylulose or ribulose.
[0226] In one preferred aspect said ketopentose is xylulose.
[0227] Xylulose may be L-xylulose or D-xylulose.
[0228] Preferably said xylulose is D-xylulose.
[0229] In an alternative embodiment, said ketopentose is ribulose.
More preferably said ribulose is D-ribulose.
Enzymes
[0230] The enzyme nomenclature numbers (EC numbers) mentioned
herein refer to the recommendations of the Nomenclature Committee
of the International Union of Biochemistry and Molecular Biology on
the nomenclature and classification of enzymes published in 1992
(ISBN 0-12-227165-3).
[0231] The enzymes mentioned herein can be produced by nucleotide
sequences derived from a wide variety of sources. In one aspect,
the nucleotide sequences encoding the enzymes mentioned herein may
be derived or derivable from Lactococcus species (such as
Lactococcus lactis and Lactococcus lactis subsp. lactis),
Geobacillus stearothermophilus, Enterococcus faecalis, Piromyces
sp, Thermoanaerobacter species (such as Thermoanaerobacter
thermohydrosulfuricus and Thermoanaerobacter thermosulphurigenes),
Pichia stipitis, or Saccharomyces cerevisiae. Preferably the
nucleotide sequences encoding the enzymes mentioned herein are
derived or derivable from Lactococcus species (such as Lactococcus
lactis and Lactococcus lactis subsp. lactis).
Xylose Isomerase (EC 5.3.1.5)
[0232] Xylose isomerase has the EC nomenclature number EC 5.3.1.5.
Xylose isomerase may be referred to as D-xylose isomerase, D-xylose
ketoisomerase or D-xylose ketol-isomerase.
[0233] The term xylose isomerase refers to an enzyme which is
capable of converting D-xylose to D-xylulose and vice versa.
[0234] A xylose isomerase mentioned herein is capable of acting on
D-xylose.
[0235] Xylose isomerase may be derived from a bacterial species
such as Lactococcus species. In particular, xylose isomerase may be
derived from Lactococcus lactis subsp. lactis; Lactococcus lactis
subsp. lactis strain NRRL B-449; Lactococcus lactis subsp. lactis
strain KF 147; Lactococcus lactis subsp. lactis strain DSM20175;
and Lactococcus lactis subsp. lactis strain IO-1.
[0236] Examples of xylose isomerases suitable for use as described
herein include xylose isomerases comprising: the amino acid
sequence shown as SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID
No 13, SEQ ID No 19 or SEQ ID No 20, or a variant, homologue or
derivative thereof; the amino acid sequence shown as GenBank
accession number ABX75758; or the amino acid sequence shown as
GenBank accession number AAD20249.
[0237] Examples of xylose isomerases suitable for use as described
herein include xylose isomerase encoded by: the nucleotide sequence
shown as SEQ ID No 1, SEQ ID No 10, SEQ ID No 17, SEQ ID No 12, SEQ
ID No 27 or SEQ ID No 28, or a variant, homologue or derivative
thereof; a nucleotide sequence encoding the amino acid sequence
shown as SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID No 13,
SEQ ID No 19 or SEQ ID No 20, or a variant, homologue or derivative
thereof.
Aldose-1-Epimerase (EC 5.1.3.3)
[0238] An aldose-1-epimerase mentioned herein is capable of acting
on the aldopentose.
[0239] Aldose-1-epimerase has the EC nomenclature number 5.1.3.3.
Aldose-1-epimerase may be referred to as a mutarotase or an aldose
mutarotase.
[0240] The term aldose-1-epimerase refers to an enzyme which is
capable of converting an .alpha.-aldopentose to a
.beta.-aldopentose and vice versa.
[0241] In a preferred embodiment the aldose-1-epimerase is encoded
by a nucleotide sequence selected from the group consisting of:
AAD20257, ABX75760, AAK05605, AAD20245, AAD20251, ABJ73095,
ABI49935 and AAO80762 (NCBI accession numbers). More preferably the
aldose-1-epimerase is selected from the group consisting of:
AAD20257, ABX75760, AAK05605, AAD20245 and AAD20251.
[0242] Examples of aldose-1-epimerases suitable for use as
described herein include aldose-1-epimerase encoded by: the
nucleotide sequence of the Lactococcus lactis aldose-1-epimerase
gene (NCBI accession number AAD20245); the nucleotide sequence of
the Saccharomyces cerevisiae GAL10 gene (in particular, the part
encoding an amino acid sequence having mutarotase activity); and
the nucleotide sequence of the Saccharomyces cerevisiae strain
D0002 GAL10 gene (in particular, the part encoding an amino acid
sequence having mutarotase activity).
Aldose Reductase (EC 1.1.1.21)
[0243] Aldose reductase has the EC nomenclature number 1.1.1.21.
Aldose reductase may be referred to as: polyol dehydrogenase,
aldehyde reductase, ALR2, NADPH-aldopentose reductase, NADPH-aldose
reductase, alditol:NADP oxidoreductase or alditol:NADP.sup.+
1-oxidoreductase.
[0244] The term aldose reductase refers to an enzyme which is
capable of converting an alditol to an aldose and vice versa.
[0245] An aldose reductase may reduce more than one type of aldose.
For example, the same enzyme may be capable of reducing both
D-xylose and L-arabinose such an enzyme may thus be called aldose
reductase or, it may be called more specifically after one of the
substrates, e.g. xylose reductase.
Xylose Reductase (EC 1.1.1.21)
[0246] In one embodiment, the aldose reductase is a xylose
reductase. Xylose reductase has the EC nomenclature number
1.1.1.21.
[0247] The term xylose reductase refers to an enzyme which is
capable of converting D-xylose to xylitol and vice versa.
[0248] A xylose reductase mentioned herein is capable of acting on
D-xylose.
[0249] Examples of xylose reductases suitable for use as described
herein include xylose reductase encoded by: the nucleotide sequence
of Pichia stipitis xylose reductase gene (PsXR); the nucleotide
sequence of Pichia stipitis strain DSM3651 xylose reductase gene
(PsXR)--NCBI accession number X59465; the nucleotide sequence of
Candida tenuis (said nucleotide sequence encoding xylose reductase
can be obtained as described by Kavanagh et al, 2003); and the
nucleotide sequence of Neurospora crassa (said nucleotide sequence
encoding xylose reductase can be obtained as described by Woodyer
et al, 2005).
Arabinose Reductase (EC 1.1.1.21)
[0250] In another embodiment, the aldose reductase is an arabinose
reductase. Arabinose reductase has the EC nomenclature number
1.1.1.21.
[0251] The term arabinose reductase refers to an enzyme which is
capable of converting L-arabinose to L-arabitol and vice versa.
[0252] An arabinose reductase mentioned herein is capable of acting
on L-arabinose.
[0253] D-xylose reductases currently known in the art may also act
on L-arabinose as a substrate with similar activity. Hence, the
term L-arabinose reductase may also refer to enzymes which are
classified as being D-xylose reductases, and the xylose reductases
mentioned herein as suitable for introducing xylose metabolism are
similarly suitable for use in introducing arabinose metabolism to a
microorganism.
[0254] In a further embodiment, the aldose reductase may be capable
of converting L-lyxose to L-arabitol and vice versa. In another
embodiment, the aldose reductase may be capable of converting
D-lyxose to D-arabitol and vice versa.
[0255] In another embodiment, the aldose reductase may be capable
of converting ribose to ribitol (in particular D-ribose to
D-ribitol) and vice versa.
Xylulose Reductase (EC 1.1.1.9 and EC 1.1.1.10)
[0256] The term xylulose reductase encompasses D-xylulose reductase
and L-xylulose reductase.
D-Xylulose Reductase (EC 1.1.1.9)
[0257] D-xylulose reductase has the EC nomenclature number 1.1.1.9.
D-xylulose reductase may be referred to as xylitol dehydrogenase,
xylitol-2-dehydrogenase, 2,3-cis-polyol(DPN)dehydrogenase (C3-5),
NAD-dependent xylitol dehydrogenase, erythritol dehydrogenase or
pentitol-DPN dehydrogenase.
[0258] The term D-xylulose reductase refers to an enzyme which is
capable of converting xylitol to D-xylulose and vice versa.
[0259] A D-xylulose reductase mentioned herein is capable of acting
on xylitol.
[0260] Examples of D-xylulose reductases suitable for use as
described herein include D-xylulose reductase encoded by: the
nucleotide sequence of Pichia stipitis D-xylulose gene (PsXDH); and
the nucleotide sequence of Pichia stipitis strain DSM3651
D-xylulose reductase gene (PsXDH)--NCBE accession number
X55392.
L-Xylulose Reductase (EC 1.1.1.10)
[0261] L-xylulose reductase has the EC nomenclature number
1.1.1.10. L-xylulose reductase may be referred to as L xylitol
dehydrogenase.
[0262] The term L-xylulose reductase refers to an enzyme which is
capable of converting L-xylulose to xylitol and vice versa.
[0263] An L-xylulose reductase mentioned herein is capable of
acting on L-xylulose.
[0264] A nucleotide sequence encoding L-xylulose reductase may be
obtained from Aspergillus niger as described by Witteveen et al
(1994) or from the yeast Ambrosiozyma monospora (Verho et al,
2004).
Xylulokinase (EC 2.7.1.17)
[0265] Xylulokinase has the EC nomenclature number 2.7.1.17.
Xylulokinase may be referred to as D-xylulokinase.
[0266] The term xylulokinase refers to an enzyme which is capable
of converting D-xylulose to D-xylulose 5-phosphate and vice
versa.
[0267] A xylulokinase mentioned herein is capable of acting on
D-xylulose.
[0268] Examples of xylulokinases suitable for use as described
herein include xylulokinase encoded by: the nucleotide sequence of
Pichia stipitis xylulokinase gene (PsXKS); the nucleotide sequence
of Pichia stipitis strain DSM3651 xylulokinase gene (PsXKS)--NCBI
accession number AF127802; the nucleotide sequence of S. cerevisiae
xylulokinase gene (ScXKS); and the nucleotide sequence of S.
cerevisiae strain D0002 xylulokinase gene (ScXKS)--NCBI accession
number X61377.
D-Arabinitol 4-Dehydrogenase (EC 1.1.1.11)
[0269] D-arabinitol 4-dehydrogenase has the EC nomenclature number
1.1.1.11. D-arabinitol 4-dehydrogenase may be referred to as
D-arabitol dehydrogenase or arabitol dehydrogenase.
[0270] The term D-arabinitol 4-dehydrogenase refers to an enzyme
which is capable of converting D-arabinitol to D-xylulose and vice
versa.
[0271] A D-arabinitol 4-dehydrogenase mentioned herein is capable
of acting on D-arabinitol.
[0272] A suitable D-arabinitol 4-dehydrogenase and the
corresponding gene is described by Cheng et al 2005.
L-Arabinitol 4-Dehydrogenase (EC 1.1.1.12)
[0273] L-arabinitol 4-dehydrogenase has the EC nomenclature number
1.1.1.12. L-arabinitol 4-dehydrogenase may be referred to as
L-arabitol 4-dehydrogenase or pentitol-DPN dehydrogenase.
[0274] The term L-arabinitol 4-dehydrogenase refers to an enzyme
which is capable of converting L-arabinitol to L-xylulose and vice
versa.
[0275] An L-arabinitol 4-dehydrogenase mentioned herein is capable
of acting on L-arabinitol.
[0276] A suitable L-arabinitol 4-dehydrogenase and the
corresponding gene is described in Richard et al (2001).
L-Arabinose Isomerase (EC 5.3.1.4)
[0277] L-arabinose isomerase has the EC nomenclature number
5.3.1.4. L-arabinose isomerase may be referred to as L-arabinose
ketol-isomerase.
[0278] The term L-arabinose isomerase refers to an enzyme which is
capable of converting L-arabinose to L-ribulose and vice versa.
[0279] An L-arabinose isomerase mentioned herein is capable of
acting on L-arabinose.
[0280] An example of a nucleotide sequence encoding L-arabinose
isomerase is the nucleotide sequence which may be obtained from
Lactobacillus plantarum strain NCIMB8826 (ATCC 14917) (gene
described in NCBI accession code NC.sub.--004567).
Ribulokinase (EC 2.7.1.16)
[0281] Ribulokinase has the EC nomenclature number 2.7.1.16.
Ribulokinase may be referred to as L-ribulokinase.
[0282] The term ribulokinase refers to an enzyme which is capable
of converting (i) L-ribulose to L-ribulose 5-phosphate and vice
versa; and/or (ii) D-ribulose to D-ribulose 5-phosphate and vice
versa.
[0283] A ribulokinase mentioned herein is capable of acting on
L-ribulose and/or D-ribulose.
[0284] A suitable nucleotide sequence encoding ribulokinase may be
obtained from Lactobacillus plantarum strain NCIMB8826 (ATCC 14917)
(gene described in NCBI accession code NC 004567).
D-Ribulokinase (EC 2.7.1.47)
[0285] D-ribulokinase has the EC nomenclature number 2.7.1.47.
[0286] The term ribulokinase refers to an enzyme which is capable
of converting D-ribulose to D-ribulose 5-phosphate and vice
versa.
[0287] An example of a nucleotide sequence encoding D-ribulokinase
may be obtained from Botryotinia fuckeliana (GenBank accession code
CH476984). GenBank accession number EDN20859 details the amino acid
sequence of a D-ribulokinase.
L-Ribulose Phosphate 4-Epimerase (EC 5.1.3.4)
[0288] L-ribulose phosphate 4-epimerase has the EC nomenclature
number 5.1.3.4. L-ribulose phosphate 4-epimerase may be referred to
as ribulose phosphate 4-epimerase, phosphoribulose isomerase,
L-ribulose 5-phosphate 4-epimerase, AraD or L-Ru5P.
[0289] The term L-ribulose phosphate 4-epimerase refers to an
enzyme which is capable of converting L-ribulose 5-phosphate to
D-xylulose 5-phosphate and vice versa.
[0290] An L-ribulose phosphate 4-epimerase mentioned herein is
capable of acting on L-ribulose 5-phosphate.
[0291] A suitable nucleotide sequence encoding L-ribulose phosphate
4-epimerase may be obtained from Lactobacillus plantarum strain
NCIMB8826 (ATCC 14917) (gene described in NCBI accession code NC
004567).
D-Ribitol 4-Dehydrogenase (EC 1.1.1.56)
[0292] D-ribitol 4-dehydrogenase has the EC nomenclature number
1.1.1.56. This enzyme may also be referred to as ribitol
2-dehydrogenase, adonitol dehydrogenase or ribitol
dehydrogenase.
[0293] The term D-ribitol 4-dehydrogenase refers to an enzyme which
is capable of converting ribitol to D-ribulose and vice versa.
[0294] A D-ribitol 4-dehydrogenase mentioned herein is capable of
acting on D-ribitol.
[0295] A suitable D-ribitol 4-dehydrogenase and the corresponding
gene is described by Dothie et al, 1985.
D-Lyxose Isomerase (EC 5.3.1.15)
[0296] D-lyxose isomerase has the EC nomenclature number 5.3.1.15.
This enzyme may also be referred to as D-lyxose
ketol-isomerase.
[0297] The term D-lyxose isomerase refers to an enzyme which is
capable of converting D-lyxose to D-xylulose and vice versa.
[0298] A D-lyxose isomerase mentioned herein is capable of acting
on D-lyxose.
[0299] A nucleotide sequence encoding a D-lyxose/L-ribose isomerase
may be cloned from the organism Acinetobacter sp. strain DL-28
(Shimonishi and Izumori, 1996) or from Aerobacter aerogenes
(Anderson and Allison, 1965).
Ribose Isomerase (EC 5.3.1.20)
[0300] Ribose isomerase has the EC nomenclature number 5.3.1.20.
This enzyme may also be referred to as D-ribose isomerase or
D-ribose ketol-isomerase.
[0301] The term ribose isomerase refers to an enzyme which is
capable of converting D-ribose to D-ribulose and vice versa.
[0302] A ribose isomerase mentioned herein is capable of acting on
D-ribose.
[0303] D-ribose isomerase has been found in the organism
Mycobacterium smegmatis (Izumori et al, 1975), from where it may be
cloned.
Ribulose-5-Phosphate 3-Epimerase (EC 5.1.3.1)
[0304] Ribulose-5-phosphate 3-epimerase has the EC nomenclature
number 5.1.3.1. This enzyme may also be referred to as:
pentose-5-phosphate 3-epimerase, phosphoketopentose 3-epimerase,
phosphoketopentose epimerase, phosphoribulose epimerase,
ribulose-phosphate 3-epimerase; D-ribulose 5-phosphate epimerase;
D-ribulose phosphate-3-epimerase; D-ribulose-5-P 3-epimerase;
D-xululose-5-phosphate 3-epimerase; erythrose-4-phosphate
isomerase; ribulose 5-phosphate 3-epimerase; and xylulose phosphate
3-epimerase.
[0305] The term ribulose-5-phosphate 3-epimerase refers to an
enzyme which is capable of converting D-ribulose 5-phosphate to
D-xylulose 5-phosphate and vice versa.
[0306] Examples of ribulose-5-phosphate 3-epimerase suitable for
use as described herein include ribulose-5-phosphate 3-epimerase
encoded by: the nucleotide sequence of the S. cerevisiae RPE1 gene;
the nucleotide sequence of the S. cerevisiae strain D0002 RPE1
gene; the nucleotide sequence of NCBI accession code
NP.sub.--012414; and the ribulose-5-phosphate isomerase from P.
stiptitis that can be found in NCBI accession number
NP.sub.--012414.
Ribose-5-Phosphate Isomerase (EC 5.3.1.6)
[0307] Ribose-5-phosphate isomerase has the EC nomenclature number
5.3.1.6. This enzyme may also be referred to as
phosphopentoisomerase, phosphopentoseisomerase,
phosphopentosisomerase, phosphoriboisomerase, ribose 5-phosphate
epimerase, ribose phosphate isomerase, 5-phosphoribose isomerase,
or D-ribose-5-phosphate ketol-isomerase.
[0308] The term ribose-5-phosphate isomerase refers to an enzyme
which is capable of converting D-ribose 5-phosphate to D-ribulose
5-phosphate and vice versa.
[0309] Examples of ribose-5-phosphate isomerase suitable for use as
described herein include ribose-5-phosphate ketol-isomerase encoded
by: the nucleotide sequence of the S. cerevisiae RKI1 gene; the
nucleotide sequence of the S. cerevisiae strain D0002 RKI1 gene;
the nucleotide sequence of NCBI accession code X94335; the
nucleotide sequence of NCBI accession code NP.sub.--014738, and the
ribose-5-phosphate isomerase from P. stiptitis that can be found in
accession NC.sub.--009043.
Transketolase (EC 2.2.1.1)
[0310] Transketolase has the EC nomenclature number 2.2.1.1. This
enzyme may also be referred to as glycolaldehydetransferase.
[0311] The term transketolase refers to an enzyme which is capable
of converting sedoheptulose 7-phosphate+D-glyceraldehyde
3-phopshate to D-ribose 5-phospate+D-xylulose 5-phosphate and vice
versa.
[0312] Examples of transketolases suitable for use as described
herein include transketolase encoded by: the nucleotide sequence of
the Saccharomyces cerevisiae TKL1 gene; the nucleotide sequence of
the Saccharomyces cerevisiae strain D0002 TKL1 gene; the nucleotide
sequence of NCBI accession code X73224; the nucleotide sequence of
NCBI accession code NP.sub.--015399, and the transketolase from P.
stiptitis that can be found in accession CP000496.
Transaldolase (EC 2.2.1.2)
[0313] Transaldolase has the EC nomenclature number 2.2.1.2. This
enzyme may also be referred to as dihydroxyacetonetransferase,
dihydroxyacetone synthase, formaldehyde transketolase or glycerone
transferase.
[0314] The term transaldolase refers to an enzyme which is capable
of converting sedoheptulose 7-phosphate+D-glyceraldehyde
3-phopshate to D-erythrose 4-phospate+D-fructose 6-phosphate and
vice versa.
[0315] Examples of transaldolases suitable for use as described
herein include transaldolase encoded by: the nucleotide sequence of
the Saccharomyces cerevisiae TAL1 gene; the nucleotide sequence of
the Saccharomyces cerevisiae strain D0002 TAL1 gene; the nucleotide
sequence of NCBI accession code X15953; the nucleotide sequence of
NCBI accession code NP.sub.--013458, and the transaldolase from P.
stiptitis that can be found in accession CP000502.
Exogenous Xylose Isomerase
[0316] In one aspect, the xylose isomerase mentioned herein is an
exogenous xylose isomerase.
[0317] In one aspect, the nucleotide sequence encoding the xylose
isomerase mentioned herein encodes an exogenous xylose
isomerase.
[0318] Preferably, the exogenous xylose isomerase is derived from a
mesophilic microorganism. More preferably the exogenous xylose
isomerase is derived from a mesophilic bacterium.
[0319] As used herein the term "mesophilic microorganism" refers to
a microorganism which grows and/or thrives best at a temperature
between 15.degree. C. and 40.degree. C. Examples of mesophilic
microorganisms include Lactococcus species, Saccharomyces species
(such as S. cerevisiae), Escherichia species (such as E. coli) and
Bacillus species (such as B. subtilis).
[0320] In one embodiment, the exogenous xylose isomerase is derived
from a Lactococcus species. Preferably, the exogenous xylose
isomerase is derived from a Lactococcus species capable of growing
on xylose. Preferably, the exogenous xylose isomerase is derived
from a Lactococcus lactis. More preferably, the exogenous xylose
isomerase is derived from a Lactococcus lactis subsp. lactis. In a
highly preferred embodiment, the exogenous xylose isomerase is
derived from Lactococcus lactis strain NRRL B-4449, strain DSM
20175, strain KF147 or strain IO-1.
[0321] The microorganism Lactococcus lactis subsp. lactis may be
referred to as Lactobacillus xylosus.
Aldopentose Assay
[0322] The amount of an aldopentose (such as xylose) in a solution
(such as a culture medium) may be determined colorimetrically by
the phloroglucinol method, as described by Ebert et al (A
Simplified, Colorimetric Micromethod for Xylose in Serum or Urine,
with Phloroglucinol, 1979, Clin. Chem. 25, no. 8, pp.
1440-1443).
[0323] The colour reagent consists of 0.5 g phloroglucinol (1,3,5
trihydroxybenzene), 100 ml glacial acetic acid and 10 ml conc. HCl.
50 .mu.l of sample is added 950 .mu.l of the colour reagent. The
mixture is heated to 100.degree. C. for 4 minutes, and the
absorbance of the mixture is read at 554 nm. The amount of an
aldopentose (such as xylose) in the sample is determined according
to a standard curve made with the same aldopentose as standard.
This method may also be used to determine the amount of arabinose,
lyxose and ribose in a culture medium.
Ketopentose Assay
[0324] The amount of a ketopentose (such as xylulose) in a solution
(such as a culture medium) may be determined colorimetrically by
the cysteine-carbazole method as described by Zacharias Dische and
Ellen Borenfreund (1951; J. Biol. Chem. 192 (2): 583).
Xylose Isomerase Assay
[0325] The amount of xylose isomerase activity in a solution may be
determined as follows.
[0326] The conversion of xylose into xylulose is determined in a
solution containing an amount of the xylose isomerase, 2% xylose
and 2 mM MgCl.sub.2 in 20 mM TRIS buffer, pH 7.4, and incubated at
the temperature of choice. The reaction is started by the addition
of the xylose and stopped by placing the reaction or sample of the
reaction on ice. The reaction time is typically 30 or 60 minutes,
or samples for following the reaction may be taken at different
time points. The generated xylulose is quantified using the
cysteine/carbazole method (Dische and Borenfreund, 1951). One unit
of xylose isomerase activity is defined as the amount of activity
that catalyses the conversion of 1 nanomole of xylose into xylulose
per minute.
[0327] In a highly preferred aspect, the transformed microorganism
has a xylose isomerase activity of at least 0.2 xylose isomerase
units per mg of microorganism protein. The amount of xylose
isomerase activity is assayed as mentioned herein and the protein
content is determined as described below. The microorganisms having
been cultured beforehand for 96 hours or less in a culture medium
comprising xylose.
Protein Content
[0328] The amount of protein (i.e. the protein content) in a
solution was determined by the use of the commercially available
BCA assay kit (Pierce Biotechnology inc., USA) based on the
bicinchoninic acid assay (Smith et al, 1985), using bovine serum
albumin as standard.
Ethanol Assay
[0329] The amount of the biofuel as ethanol, in a solution (such as
a culture medium) may be determined by the use of a commercially
available ethanol assay, the K-ETOH Kit, manufactured and sold by
Megazyme International, Bray Business Park, Bray, Co. Wicklow,
Ireland; or it may be determined by, for example, the use of gas
chromatography.
Pentose Phosphate Pathway
[0330] Ketopentoses (such as xylulose) are converted into ethanol
via the pentose phosphate pathway. An example of this is shown in
FIG. 4.
[0331] Examples of enzymes involved in the pentose phosphate
pathway include: transketolase, transaldolase, ribose-5-phosphate
ketol-isomerase and ribulose-5-phosphate 3-epimerase.
[0332] In one embodiment the microorganism according to the present
invention has also been transformed to express or overexpress one
or more enzymes involved in the pentose phosphate pathway.
[0333] In one embodiment the microorganism according to the present
invention has also been transformed with one or more nucleotide
sequences that cause the microorganism to overexpress one or more
enzymes involved in the pentose phosphate pathway. For example, a
promoter is inserted into the genome of a microorganism which
enables the microorganism to overexpress an endogenous nucleotide
sequence encoding an enzyme involved in the pentose phosphate
pathway.
[0334] In another embodiment the microorganism has been transformed
with one or more nucleotide sequences encoding one or more enzymes
involved in the pentose phosphate pathway. For example, the
microorganism is transformed with an expression vector comprising a
nucleotide sequence encoding one or more enzymes involved in the
pentose phosphate pathway.
[0335] Preferably the expression vector mentioned herein comprises
one or more promoters capable of overexpressing one or more
nucleotide sequences encoding one or more enzymes involved in the
pentose phosphate pathway. Examples of such promoters include the
GPD promoter, the TEF promoter and the ADP promoter. Preferred
promoters which may be used to overexpress one or more nucleotide
sequences encoding one or more enzymes involved in the pentose
phosphate pathway can be any of the regulatory elements controlling
the expression of nucleotide sequences encoding proteins involved
in glycolysis and glucose fermentation.
[0336] As used herein, the term "overexpress" in the phrase "one or
more nucleotide sequences that cause the microorganism to
overexpress one or more enzymes involved in the pentose phosphate
pathway" and "one or more promoters capable of overexpressing one
or more nucleotide sequences encoding one or more enzymes involved
in the pentose phosphate pathway" refers to an increase in
expression from zero to a level of expression or going from a lower
level of expression to a higher level of expression (e.g.
upregulation) when the transformed microorganism is compared to the
equivalent microorganism prior to transformation. Microorganisms
overexpressing one or more enzymes involved in the pentose
phosphate pathway have an increased ability to catalyse the
conversion of a ketopentose (such as xylulose 5-phosphate) to a
biofuel (such as ethanol).
[0337] Preferably said transformed microorganism which
overexpresses one or more enzymes involved in the pentose phosphate
pathway is able to catalyse the conversion of a ketopentose (such
as xylulose 5-phosphate) to a biofuel (such as ethanol) by a rate
which is at least 10%, 15%, 20% or 25% higher than an untransformed
microorganism.
[0338] Examples of microorganisms overexpressing one or more
enzymes involved in the pentose phosphate pathway include: (i)
microorganisms transformed with one or more expression vectors
encoding one or more of transketolase, transaldolase,
ribose-5-phosphate ketol-isomerase and ribulose-5-phosphate
3-epimerase; and (ii) microorganisms transformed to upregulate the
expression of one or more endogenous nucleotide sequences encoding
one or more of transketolase, transaldolase, ribose-5-phosphate
ketol-isomerase and ribulose-5-phosphate 3-epimerase (prior to
transformation said microorganism was capable of expressing one or
more of these enzymes for a given set of culture conditions during
exponential growth but after transformation said microorganism is
capable of expression one or more of these enzymes at a higher
level, in the same culture conditions, during exponential
growth).
Variants/Homologues/Derivatives
[0339] The present invention encompasses the use of variants,
homologues and derivatives of any amino acid sequence of a protein
or of any nucleotide sequence encoding such a protein.
[0340] In particular, the present invention encompasses the use of
variants, homologues and derivatives of the amino acid sequence
encoded by SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID No 13,
SEQ ID No 19 or SEQ ID No 20.
[0341] Further, the present invention encompasses the use of
variants, homologues and derivatives of the nucleotide sequence
encoding the amino acid sequence shown as SEQ ID No 14, SEQ ID No
11, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No 20; and
the nucleotide sequence shown as SEQ ID No 1, SEQ ID No 10, SEQ ID
No 17, SEQ ID No 12, SEQ ID No 27 or SEQ ID No 28.
[0342] For example, a variant, homologue or derivative of a
nucleotide sequence (such as SEQ ID No 1, SEQ ID No 10, SEQ ID No
17, SEQ ID No 12, SEQ ID No 27 or SEQ ID No 28; or the nucleotide
sequence encoding the amino acid sequence shown as SEQ ID No 14,
SEQ ID No 11, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No
20) may comprise up to 30, 21, 15, 9, 6 or 3 nucleic acid
substitutions.
[0343] For example, a variant, homologue or derivative of an amino
acid sequence (such as SEQ ID No 14, SEQ ID No 11, SEQ ID No 18,
SEQ ID No 13, SEQ ID No 19 or SEQ ID No 20) may comprise up to 10,
7, 5, 3, 2 or 1 amino acid substitutions.
[0344] Without wishing to be bound by theory, two amino acid
residues (namely D296 and D339) in xylose isomerase may be involved
in the binding of divalent metal ions in the active site (Meng, M.
et al, 1993). In one embodiment, D296 and D339 are important for
the binding of divalent metal ions in the active site. Thus, in one
embodiment, the variant, homologue or derivative of an amino acid
sequence (such as SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID
No 13, SEQ ID No 19 or SEQ ID No 20) comprises the amino acid
aspartic acid (D) at residue 296 and/or the amino acid aspartic
acid (D) at residue 339 of SEQ ID No 14, SEQ ID No 11, SEQ ID No
18, SEQ ID No 13, SEQ ID No 19 and SEQ ID No 20 or equivalent
substitution(s) in other suitable amino acid sequences.
[0345] Further, without wishing to be bound by theory, the amino
acid residue (histidine 101) in xylose isomerase may be involved in
the active site (Lee et al 1989). In one embodiment, H101 is
important for the active site. Thus, in one embodiment, the
variant, homologue or derivative of an amino acid sequence (such as
SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID No 13, SEQ ID No
19 or SEQ ID No 20) comprises the amino acid histidine (H) at
residue 101 of SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ ID No
13, SEQ ID No 19 and SEQ ID No 20 or equivalent substitution in
other suitable amino acid sequences.
[0346] In one embodiment, the variant, homologue or derivative of
an amino acid sequence (such as SEQ ID No 14, SEQ ID No 11, SEQ ID
No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No 20) comprises the
amino acid arginine (R) at residue 391 and/or the amino acid lysine
(K) at residue 407 of SEQ ID No 14, SEQ ID No 11, SEQ ID No 18, SEQ
ID No 13, SEQ ID No 19 and SEQ ID No 20 or equivalent substitutions
in other suitable amino acid sequences.
[0347] In one embodiment, a variant, homologue or derivative of a
nucleotide sequence (such as SEQ ID No 1, SEQ ID No 10, SEQ ID No
17, SEQ ID No 12, SEQ ID No 27 or SEQ ID No 28; or the nucleotide
sequence encoding the amino acid sequence shown as SEQ ID No 14,
SEQ ID No 11, SEQ ID No 18, SEQ ID No 13, SEQ ID No 19 or SEQ ID No
20) comprises silent codon sequence changes in order that the
sequence is optimised for the codon preferences of a particular
host cell in which the sequence is expressed. For example, codon
usage in a sequence may be optimised based on the yeast codon usage
table from the Kazusa codon usage database (Nakamura et al.,
2000).
[0348] Here, the term "homologue" means an entity having a certain
homology with the subject amino acid sequences and the subject
nucleotide sequences. Here, the term "homology" can be equated with
"identity".
[0349] In the present context, a homologous sequence is taken to
include an amino acid sequence which may be at least 75, 80, 85 or
90% identical; preferably at least 95, 96, 97, 98 or 99% identical
to the subject sequence; and in a highly preferred embodiment at
least 98, 99 or 99.5% identical to the subject sequence. Typically,
the homologues will comprise the same active sites etc. as the
subject amino acid sequence. Although homology can also be
considered in terms of similarity (i.e. amino acid residues having
similar chemical properties/functions), in the context of the
present invention it is preferred to express homology in terms of
sequence identity.
[0350] In the present context, an homologous sequence is taken to
include a nucleotide sequence which may be at least 75, 80, 85 or
90% identical; preferably at least 95, 96, 97, 98 or 99% identical;
and in a highly preferred embodiment at least 98, 99 or 99.5%
identical to a nucleotide sequence encoding an enzyme of the
present invention (the subject sequence). Typically, the homologues
will comprise the same sequences that code for the active sites
etc. as the subject sequence. Although homology can also be
considered in terms of similarity (i.e. amino acid residues having
similar chemical properties/functions), in the context of the
present invention it is preferred to express homology in terms of
sequence identity.
[0351] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0352] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0353] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0354] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons. For example when using the GCG Wisconsin
Bestfit package the default gap penalty for amino acid sequences is
-12 for a gap and -4 for each extension.
[0355] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984
Nuc. Acids Research 12 p 387). Examples of other software that can
perform sequence comparisons include, but are not limited to, the
BLAST package (see Ausubel et al., 1999 Short Protocols in
Molecular Biology, 4.sup.th Ed--Chapter 18), FASTA (Altschul et
al., 1990 J. Mol. Biol. 403-410) and the GENE WORKS suite of
comparison tools. Both BLAST and FASTA are available for offline
and online searching (see Ausubel et al., 1999, Short Protocols in
Molecular Biology, pages 7-58 to 7-60). However, for some
applications, it is preferred to use the GCG Bestfit program. A new
tool, called BLAST 2 Sequences is also available for comparing
protein and nucleotide sequence (see FEMS Microbiol Lett 1999
174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and
tatiana@ncbi.nlm.nih.gov).
[0356] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. GCG
Wisconsin programs generally use either the public default values
or a custom symbol comparison table if supplied (see user manual
for further details). For some applications, it is preferred to use
the public default values for the GCG package, or in the case of
other software, the default matrix, such as BLOSUM62.
[0357] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in DNASIS.TM. (Hitachi Software),
based on an algorithm, analogous to CLUSTAL (Higgins D G &
Sharp P M (1988), Gene 73(1), 237-244).
[0358] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0359] The sequences may also have deletions, insertions or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent substance. Deliberate amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
secondary binding activity of the substance is retained. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine, valine,
glycine, alanine, asparagine, glutamine, serine, threonine,
phenylalanine, and tyrosine.
[0360] Conservative substitutions may be made, for example
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
TABLE-US-00001 ALIPHATIC Non-polar G A P I L V Polar-uncharged C S
T M N Q Polar-charged D E K R AROMATIC H F W Y
[0361] The present invention also encompasses homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue, with an
alternative residue) that may occur i.e. like-for-like substitution
such as basic for basic, acidic for acidic, polar for polar etc.
Non-homologous substitution may also occur i.e. from one class of
residue to another or alternatively involving the inclusion of
unnatural amino acids such as ornithine (hereinafter referred to as
Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine and phenylglycine.
[0362] Replacements may also be made by unnatural amino acids
include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino
acids*, lactic acid*, halide derivatives of natural amino acids
such as trifluorotyrosine*, p-Cl-phenylalanine*,
p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*,
.beta.-alanine*, L-.alpha.-amino butyric acid*, L-.gamma.-amino
butyric acid*, L-.alpha.-amino isobutyric acid*, L-.epsilon.-amino
caproic acid.sup.#, 7-amino heptanoic acid*, L-methionine
sulfone.sup.#*, L-norleucine*, L-norvaline*,
p-nitro-L-phenylalanine*, L-hydroxyproline.sup.#, L-thioproline*,
methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*,
pentamethyl-Phe*, L-Phe (4-amino).sup.#, L-Tyr (methyl)*, L-Phe
(4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl
acid)*, L-diaminopropionic acid and L-Phe (4-benzyl)*. The notation
* has been utilised for the purpose of the discussion above
(relating to homologous or non-homologous substitution), to
indicate the hydrophobic nature of the derivative whereas # has
been utilised to indicate the hydrophilic nature of the derivative,
#* indicates amphipathic characteristics.
[0363] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups such as methyl, ethyl or propyl
groups in addition to amino acid spacers such as glycine or R-|5
alanine residues. A further form of variation, involves the
presence of one or more amino acid residues in peptoid form, will
be well understood by those skilled in the art. For the avoidance
of doubt, "the peptoid form" is used to refer to variant amino acid
residues wherein the .alpha.-carbon substituent group is on the
residue's nitrogen atom rather than the .alpha.-carbon. Processes
for preparing peptides in the peptoid form are known in the art,
for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and
Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
[0364] The nucleotide sequences for use in the present invention
may include within them synthetic or modified nucleotides. A number
of different types of modification to oligonucleotides are known in
the art. These include methylphosphonate and phosphorothioate
backbones and/or the addition of acridine or polylysine chains at
the 3' and/or 5' ends of the molecule. For the purposes of the
present invention, it is to be understood that the nucleotide
sequences described herein may be modified by any method available
in the art. Such modifications may be carried out in order to
enhance the in vivo activity or life span of nucleotide sequences
of the present invention.
[0365] The present invention also encompasses the use of nucleotide
sequences that are complementary to the sequences presented herein,
or any derivative, fragment or derivative thereof. If the sequence
is complementary to a fragment thereof then that sequence can be
used as a probe to identify similar coding sequences in other
organisms etc.
[0366] Polynucleotides which are not 100% homologous to the
sequences mentioned herein can be obtained in a number of ways.
Other variants of the sequences described herein may be obtained
for example by probing DNA libraries. In addition, other homologues
may be obtained and such homologues and fragments thereof in
general will be capable of selectively hybridising to the sequences
shown in the sequence listing herein. Such sequences may be
obtained by probing cDNA libraries with probes comprising all or
part of any one of the sequences in the attached sequence listings
under conditions of medium to high stringency.
[0367] Variants and strain/species homologues may also be obtained
using degenerate PCR which will use primers designed to target
sequences within the variants and homologues encoding conserved
amino acid sequences within the sequences mentioned herein.
Conserved sequences can be predicted, for example, by aligning the
amino acid sequences from several variants/homologues. Sequence
alignments can be performed using computer software known in the
art. For example the GCG Wisconsin PileUp program is widely
used.
[0368] The primers used in degenerate PCR will contain one or more
degenerate positions and will be used at stringency conditions
lower than those used for cloning sequences with single sequence
primers against known sequences.
[0369] Alternatively, such polynucleotides may be obtained by site
directed mutagenesis of characterised sequences. This may be useful
where for example silent codon sequence changes are required to
optimise codon preferences for a particular host cell in which the
polynucleotide sequences are being expressed. Other sequence
changes may be desired in order to introduce restriction enzyme
recognition sites, or to alter the property or function of the
polypeptides encoded by the polynucleotides.
[0370] Polynucleotides (nucleotide sequences) mentioned herein may
be used to produce a primer, e.g. a PCR primer, a primer for an
alternative amplification reaction, a probe e.g. labelled with a
revealing label by conventional means using radioactive or
non-radioactive labels, or the polynucleotides may be cloned into
vectors. Such primers, probes and other fragments will be at least
15, preferably at least 20, for example at least 25, 30 or 40
nucleotides in length, and are also encompassed by the term
polynucleotides as used herein.
[0371] Polynucleotides such as DNA polynucleotides and probes
mentioned herein may be produced recombinantly, synthetically, or
by any means available to those of skill in the art. They may also
be cloned by standard techniques.
[0372] In general, primers will be produced by synthetic means,
involving a stepwise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0373] Longer polynucleotides will generally be produced using
recombinant means, for example using a PCR (polymerase chain
reaction) cloning techniques. The primers may be designed to
contain suitable restriction enzyme recognition sites so that the
amplified DNA can be cloned into a suitable cloning vector.
[0374] The invention will now be further described by way of
Examples, which are meant to serve to assist one of ordinary skill
in the art in carrying out the invention and are not intended in
any way to limit the scope of the invention.
EXAMPLES
General Recombinant DNA Methodology Techniques
[0375] The present invention employs, unless otherwise indicated,
conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA and immunology, which are within the
capabilities of a person of ordinary skill in the art. Such
techniques are explained in the literature. See, for example, J. B.
Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; M. J. Gait (Editor),
1984, Oligonucleotide Synthesis: A Practical Approach, In Press; D.
M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA
Structure Part A: Synthesis and Physical Analysis of DNA Methods in
Enzymology, Academic Press; Sambrook J., et al (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3. Cold Spring Harbor
Laboratory Press; and Ausubel F. M., et al (1995 (and periodic
supplements)) Current Protocols in Molecular Biology, Vol. ch. 9,
13 and 16. John Wiley & Sons, New York, N.Y. Each of these
general texts is herein incorporated by reference.
Organisms:
[0376] Lactococcus is family of Gram positive bacteria, commonly
found throughout many parts of the world. Strains of this family
have a long history of safe use for fermentation of dairy products
for production of e.g. cheese. Such a strain is used in these
examples, Lactococcus lactis strain DSM 20175, which can be
obtained from the deposit institute DSMZ--Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (German collection of
Microorganisms and Cell Cultures).
[0377] Saccharomyces is a family of yeast strains commonly found
throughout many parts of the world. They have a long history of
safe use for baking, brewing and winemaking purposes, and are at
the present time the most well characterized eucaryotic organism.
It is therefore a very popular organism for experimental use in
molecular biology and microbiology laboratories. The strain S.
cerevisiae BY4741 is used in the examples here. The strain can be
obtained from Euroscarf (EUROpean Saccharomyces Cerevisiae ARchive
for Functional Analysis--Germany).
[0378] The S. cerevisiae strain T0040, S. cerevisiae strain T0028,
S. cerevisiae strain T0049 and S. cerevisiae strain T0004 used in
the following examples are all isogenic variants, constructed from
BY4741 obtained from Euroscarf.
[0379] Escherichia coli is a Gram negative bacterium that is
commonly found in the lower intestine of higher animals and human
beings. Many strains of the organism have a long history of safe
use in microbiological and molecular biology laboratories. The
strain used in the following examples is the E. coli DH5-alpha
strain, commonly used in molecular biology laboratories for cloning
and propagation of genetic material. The strain may be obtained
from the company "InVitrogen" (InVitrogen Corporation, California,
USA).
[0380] Thermoanaerobacter is a family of thermophilic,
anaerobically growing, Gram positive bacterial strains. The two
strains from which enzymes are used in the following examples,
Thermoanaerobacter thermohydrosulfuricus (DSM no. 15750) and
Thermoanaerobacterium thermosulfurigenes (ATCC 33743) are both
originally isolated from thermal springs, DSM no. 15750 in Ayas,
Turkey and ATCC 33743 in Yellowstone National Park, USA. They can
be obtained from DSMZ--Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (German collection of Microorganisms and Cell
Cultures). The genetic material encoding the enzymes from these
strains and used in the examples is however made synthetically,
based on a backtranslation of the amino acid sequences as specified
in the sequence listing as SEQ ID No 15 and SEQ ID No 16.
[0381] Pichia stipitis is a yeast species that has been isolated
from the guts of insect larvae and thermites. The Pichia stipitis
strain from which genes have been used in the following examples is
the strain Pichia stipitis DSM no. 3651, which can be obtained from
DSMZ--Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(German collection of Microorganisms and Cell Cultures).
[0382] Pseudomonas is a diverse family of rodshaped, aerobic Gram
negative bacteria commonly found in water and in plant seeds and
plant tissue. The strain used in these examples is the Pseudomonas
syringae strain DSM 50315, originally isolated from wounded plant
tissue. The strain can be obtained from the deposit institute
DSMZ--Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(German collection of Microorganisms and Cell Cultures).
Example 1
Construction of an S. cerevisiae Strain Expressing the
Lactobacillus xylosus XI and the Pichia stipitis Xylulose
Kinase
[0383] Example 1a
TOPO Cloning of the D-Xylose Isomerase Gene from Lactobacillus
xylosus (Lactococcus lactis, Strain DSM 20175), Using Primer
Sequences that are Based on NCBI Accession Code AF092042 (SEQ ID No
1)
[0384] The entire L. xylosus D-xylose isomerase gene (Lx XI) was
PCR amplified from DNA obtained from the strain DSM 20175 using the
primers identified by SEQ. ID. NO. 2 and SEQ. ID. NO. 3. The coding
region of xylose isomerase gene cloned by PCR of Lactococcus lactis
strain DSM 20175, using primers shown in SEQ ID No 2 and SEQ ID No
3, is shown in SEQ ID No 17. A restriction-site for NheI, proximal
to the ATG-start codon and a restriction-site for XhoI, distal to
the stop-codon was introduced flanking the Lx XI gene. As template,
DNA from the L. xylosus strain was used in a concentration of 0.2
ng/.mu.l PCR-reaction. PCR was performed at 30 cycles of 30 seconds
at 96.degree. C., 30 seconds at 50.degree. C., and 150 seconds at
72.degree. C., followed by a final incubation of 10 minutes at
72.degree. C. using Phusion High Fidelity DNA polymerase (Finnzymes
Oy, Finland). The PCR product was electrophoretically separated on
a 1% low melt agarose gel and a 1339 bp fragment was isolated. The
DNA fragment was TOPO cloned into the pCR-Blunt II-TOPO vector
(Invitrogen, USA) according to the manufacturer's instructions and
the resulting plasmid was used for the transformation of E. coli
TOP10. The plasmid was named pCR-Blunt 2 LxXI.
Example 1b
Construction of Plasmid LxXI-2a Containing the L. xylosus Xylose
Isomerase (LxXI) Gene Under Control of the GPD Promoter and the
CYC1 Terminator from S. cerevisiae
[0385] The E. coli/S. cerevisiae high-copy shuttle vector P426-GPD
(Mumberg et al., 1995) was digested with SpeI and XhoI and the
resulting termini were dephosphorylated with alkaline phosphatase.
Similarly, the DNA fragment encoding the L. xylosus xylose
isomerase gene was released from the vector pCR-Blunt 2 LxXI
(described in Example 1a) by digestion with NheI and XhoI. The
resulting linearized plasmid P426-GPD and the DNA fragment encoding
ThXI were electrophoretically separated on a 1% low melt agarose
gel and isolated. The two DNA fragments were ligated together
resulting in the plasmid named LxXI-2a.
Example 1c
TOPO Cloning of the D-Xylulokinase Gene from the Pichia stipitis
Strain DSM3651 Based on NCBI Accession Code AF127802
[0386] The entire P. stipitis D-xylulokinase gene (PsXKS) was PCR
amplified from DNA obtained from the strain DSM3651 using the
primers identified by SEQ. ID. NO. 4 and SEQ. ID. NO. 5. A
restriction-site for NheI, proximal to the ATG-start codon and a
restriction-site for XhoI, distal to the stop-codon was introduced
flanking the PsXKS gene. As template, DNA from the P. stipitis
strain was used in a concentration of 0.2 ng/.mu.l PCR-reaction.
PCR was performed at 30 cycles of 30 seconds at 96.degree. C., 30
seconds at 50.degree. C., and 150 seconds at 72.degree. C.,
followed by a final incubation of 10 minutes at 72.degree. C. using
Phusion High Fidelity DNA polymerase (Finnzymes Oy, Finland). The
PCR product was electrophoretically separated on a 1% low melt
agarose gel and a 1891 bp fragment was isolated. The DNA fragment
was TOPO cloned into the pCR-Blunt II-TOPO vector (Invitrogen, USA)
according to the manufacturer's instructions and the resulting
plasmid was used for the transformation of E. coli TOP10. The
plasmid was named pCR-Blunt 2 P. stip XKS.
[0387] Example 1d
Construction of Plasmid PsXKS-14a Containing the P. stipitis
D-xylulokinase (PsXKS) Gene Under Control of the GPD Promoter and
the CYC1 Terminator from S. cerevisiae
[0388] The E. coli/S. cerevisiae high-copy shuttle vector P425-GPD
(Mumberg et al., 1995) was digested with SpeI and XhoI and the
resulting termini were dephosphorylated with alkaline phosphatase.
Similarly, the DNA fragment encoding the P. stipitis xylulose
kinase gene was released from the vector pCR-Blunt 2 P. stip XKS
(described in Example 1c), by digestion with NheI and XhoI. The
resulting linearized plasmid P425-GPD and the DNA fragment encoding
PsXKS were electrophoretically separated on a 1% low melt agarose
gel and isolated. The two DNA fragments were ligated together
resulting in the plasmid named PsXKS-14a.
Example 1e
Construction of S. cerevisiae Strains Containing the LxXI-2a and
the PsXKS-14a
[0389] 200 ng each of the plasmids were combined and used for the
transformation of S. cerevisiae yeast strain BY4741 (Euroscarf,
Germany) by means of electroporation using the Biorad Gene Pulser
II system (Biorad, USA) according to the manufacturer's
instruction. Yeast cells were made competent according to a
standard protocol (Becker and Guarente, 1991). Selection for clones
transformed with both plasmids was done on solid synthetic complete
dropout media omitting uracil and leucine and supplemented with 2%
D-glucose (SC-Ura, Leu) (Rose et al., 1990). Medium-size primary
clones were restreaked on SC-Ura, Leu and one colony of strain
T0004 transformed with the plasmids LxXI-1a and PsXKS-14a was
obtained.
Example 2
Construction of an S. cerevisiae Strain Expressing the
Thermoanaerobacter thermohydrosulfuricus XI and the Pichia stipitis
Xylulose Kinase
Example 2a
Construction of Synthetic Xylose Isomerase Gene Encoding
Thermoanaerobacter thermohydrosulfuricus Xylose Isomerase Based on
NCBI Accession Code D00756
[0390] The entire T. thermohydrosulfuricus xylose isomerase gene
(ThXI) was synthesized and assembled by Geneart AG (Regensburg,
Germany). Codon usage in the sequence was optimised based on the
yeast codon usage table from the Kazusa codon usage database
(Nakamura et al., 2000). Flanking the open reading frame, a
restriction-site for NheI, proximal to the ATG-start codon and a
restriction-site for XhoI, distal to the stop-codon, were included
in the synthetic construct. The integrity of the ThXI synthetic
gene was determined by sequencing of both strands. The nucleotide
sequence of ThXI including flanking restriction-sites is identified
as SEQ. ID. NO. 6, showing the synthesized DNA sequence with the
amino acid translation of the coding region shown above the
nucleotide sequence. The harbouring plasmid was named
0717046pGA14.
Example 2b
Construction of Plasmid ThXI-5a Containing the T.
thermohydrosulfuricus Xylose Isomerase (ThXI) Gene Under Control of
the GPD Promoter and the CYC1 Terminator from S. cerevisiae
[0391] The E. coli/S. cerevisiae high-copy shuttle vector P426-GPD
(Mumberg et al., 1995) was digested with SpeI and XhoI and the
resulting termini were dephosphorylated with alkaline phosphatase.
Similarly, the DNA fragment encoding the T. thermohydrosulfuricus
xylose isomerase gene was released from the vector 0717046pGA14
(described in Example 2a) by digestion with NheI and XhoI. The
resulting linearized plasmid P426-GPD and the DNA fragment encoding
ThXI were electrophoretically separated on a 1% low melt agarose
gel and isolated. The two DNA fragments were ligated together
resulting in the plasmid named ThXI-5a.
Example 2c
Construction of S. cerevisiae Strains Containing the ThXI-5a and
the PsXKS-14a 200 ng each of the plasmids were combined and used
for the transformation of S. cerevisiae yeast strain BY4741
(Euroscarf, Germany) by means of electroporation using the Biorad
Gene Pulser II system (Biorad, USA) according to the manufacturer's
instruction. Yeast cells were made competent according to a
standard protocol (Becker and Guarente, 1991). Selection for clones
transformed with both plasmids was done on solid synthetic complete
dropout media omitting uracil and leucine and supplemented with 2%
D-glucose (SC-Ura, Leu) (Rose et al., 1990). Medium-size primary
clones were restreaked on SC-Ura, Leu and one colony of strain
T0049 transformed with the plasmids ThXI-5a and PsXKS-14a was
obtained.
Example 3
Construction of an S. cerevisiae Strain Expressing the
Thermoanaerobacter thermosulphurigenes Xylose Isomerase and the
Pichia stipitis Xylulose Kinase
Example 3a
Construction of Synthetic Xylose Isomerase Gene Encoding
Thermoanaerobacter thermosulphurigenes Xylose Isomerase Based on
NCBI Accession Code J05650
[0392] The entire T. thermosulphurigenes xylose isomerase gene
(TsXI) was synthesized and assembled by Geneart AG (Regensburg,
Germany). Codon usage in the sequence was optimised based on the
yeast codon usage table from the Kazusa codon usage database
(Nakamura et al., 2000). Flanking the open reading frame, a
restriction-site for NheI, proximal to the ATG-start codon and a
restriction-site for XhoI, distal to the stop-codon, were included
in the synthetic construct. The integrity of the TsXI synthetic
gene was determined by sequencing of both strands. The nucleotide
sequence of TsXI including flanking restriction-sites is identified
as SEQ. ID. NO. 7, showing the synthesized DNA sequence with the
amino acid translation of the coding region shown above the
nucleotide sequence. The harbouring plasmid was named
0717047pGA18.
Example 3b
Construction of Plasmid TsXI-28a Containing the T.
thermosulphurigenes Xylose Isomerase (TsXI) Gene Under Control of
the GPD Promoter and the CYC1 Terminator from S. cerevisiae
[0393] The E. coli/S. cerevisiae high-copy shuttle vector P426-GPD
(Mumberg et al., 1995) was digested with SpeI and XhoI and the
resulting termini were dephosphorylated with alkaline phosphatase.
Similarly, the DNA fragment encoding the T. thermosulphurigenes
xylose isomerase gene was released from the vector 0717047pGA18
(described in Example 3a) by digestion with NheI and XhoI. The
resulting linearized plasmid P426-GPD and the DNA fragment encoding
TsXI were electrophoretically separated on a 1% low melt agarose
gel and isolated. The two DNA fragments were ligated together
resulting in the plasmid named TsXI-28a.
Example 3c
Construction of S. cerevisiae Strains Containing the TsXI-28a and
the PsXKS-14a 200 ng each of the plasmids were combined and used
for the transformation of S. cerevisiae yeast strain BY4741
(Euroscarf, Germany) by means of electroporation using the Biorad
Gene Pulser II system (Biorad, USA) according to the manufacturer's
instruction. Yeast cells were made competent according to a
standard protocol (Becker & Guarente, 1991). Selection for
clones transformed with both plasmids was done on solid synthetic
complete dropout media omitting uracil and leucine and supplemented
with 2% D-glucose (SC-Ura, Leu) (Rose et al., 1990). Medium-size
primary clones were restreaked on SC-Ura, Leu and one colony of
strain T0028 transformed with the plasmids TsXI-28a and PsXKS-14a
was obtained.
Example 4
Construction of a S. cerevisiae Strain Expressing the Pseudomonas
syringae XylA (PsXI) and the Pichia stipitis Xylulose Kinase
[0394] Example 4a
TOPO Cloning of the D-Xylose Isomerase Gene (PsXI) from Pseudomonas
syringae pv Tomato
[0395] The entire P. syringae D-xylose isomerase gene (Ps XI) was
PCR amplified from DNA obtained from the strain DSM 50315 using the
primers identified by SEQ. ID. NO. 8 and SEQ. ID. NO. 9. A
restriction-site for NheI, proximal to the ATG-start codon and a
restriction-site for XhoI, distal to the stop-codon was introduced
flanking the PsXI gene. As template, DNA from the P. syringae
strain was used in a concentration of 0.2 ng/.mu.l PCR-reaction.
PCR was performed at 30 cycles of 30 seconds at 96.degree. C., 30
seconds at 50.degree. C., and 150 seconds at 72.degree. C.,
followed by a final incubation of 10 minutes at 72.degree. C. using
Phusion High Fidelity DNA polymerase (Finnzymes Oy, Finland). The
PCR product was electrophoretically separated on a 1% low melt
agarose gel and a 1323 bp fragment was isolated. The DNA fragment
was TOPO cloned into the pCR-Blunt II-TOPO vector (Invitrogen, USA)
according to the manufacturer's instructions and the resulting
plasmid was used for the transformation of E. coli TOP10. The
plasmid was named pCR-Blunt 2 PsXI.
Example 4b
Construction of Plasmid PsXI-34a Containing the P. syringae Xylose
Isomerase (PsXI) Gene Under Control of the GPD Promoter and the
CYC1 Terminator from S. cerevisiae
[0396] The E. coli/S. cerevisiae high-copy shuttle vector P426-GPD
(Mumberg et al., 1995) was digested with SpeI and XhoI and the
resulting termini were dephosphorylated with alkaline phosphatase.
Similarly, the DNA fragment encoding the P. syringae xylose
isomerase gene was released from the vector pCR-Blunt 2 PsXI
(described in Example 4a) by digestion with NheI and XhoI. The
resulting linearized plasmid P426-GPD and the DNA fragment encoding
PsXI were electrophoretically separated on a 1% low melt agarose
gel and isolated. The two DNA fragments were ligated together
resulting in the plasmid named PsXI-34a.
Example 4c
Construction of S. cerevisiae Strains Containing the PsXI-34a and
the PsXKS-14a 200 ng each of the plasmids were combined and used
for the transformation of S. cerevisiae yeast strain BY4741
(Euroscarf, Germany) by means of electroporation using the Biorad
Gene Pulser II system (Biorad, USA) according to the manufacturer's
instruction. Yeast cells were made competent according to a
standard protocol (Becker and Guarente, 1991). Selection for clones
transformed with both plasmids was done on solid synthetic complete
dropout media omitting uracil and leucine and supplemented with 2%
D-glucose (SC-Ura, Leu) (Rose et al., 1990). Medium-size primary
clones were restreaked on SC-Ura, Leu and one colony of strain
T0040 transformed with the plasmids PsXI-34a and PsXKS-14a was
obtained.
Example 5
Determination and Comparing of Specific Xylose Isomerase Activities
Obtained by Expression of Four Different Bacterial Xylose
Isomerases in Saccharomyces cerevisiae
[0397] The four different strains: [0398] S. cerevisiae strain
T0040 containing the PsXI-34a and the PsXKS-14a; [0399] S.
cerevisiae strains T0028 containing the TsXI-28a and the POCKS-14a;
[0400] S. cerevisiae strains T0049 containing the ThXI-5a and the
PsXKS-14a; and [0401] S. cerevisiae strains T0004 containing the
LxXI-2a and the PsXKS-14a; were streaked on plates and grown
overnight on solid synthetic complete dropout media omitting uracil
and leucine and supplemented with 2% D-glucose (SC-Ura, Leu) (Rose
et al., 1990).
[0402] 50 ml tubes containing 10 ml synthetic complete dropout
media omitting uracil and leucine and supplemented with 2%
D-glucose (SC-Ura, Leu) (Rose et al, 1990), were then inoculated
with colonies from the streaked plates to an adjusted OD600 of 0.1
and grown for 18 hours under shaking, 200 rpm, at 30.degree. C. The
cultures were harvested by centrifugation, 4000 rpm for 15 minutes,
washed by resuspension in 2 ml of water and spun down again. Cells
were lysed by addition of 300 .mu.l of CellLytic Y Plus Kit
(Sigma-Aldrich) followed by incubation at room temperature for 30
minutes under gentle shaking and centrifugation at 16000.times.g
for 10 minutes to remove the cell debris. The supernatant was
isolated and used for determination of xylose isomerase activity
and protein content. Xylose isomerase activity was determined using
the cysteine-carbazole method as described by Dische and
Borenfreund (Dische and Borenfreund, 1951). Protein content was
determined by the use of commercially available bicinchoninic acid
assay based protein assay (BCA assay kit, Pierce Biotechnology
inc., USA).
[0403] Results are shown in FIG. 5. The highest total xylose
isomerase activity per mg total yeast protein, obtained by
expressing the L. xylosus xylose isomerase, is about 10 times
higher than the second best result, obtained by expressing the
xylose isomerase from T. thermohydrosulphuricus. It is clear that
the L. xylosus xylose isomerase is far superior to the other
bacterial isomerases. The obtained results, xylose isomerase
units/mg yeast protein for the two thermophilic bacteria, are in
line with other obtained results using xylose isomerases from
thermophilic bacteria (Walfriedsson et al, 1996; Lorin et al,
2003), and L. xylosus isomerase specific activity equals or tops
the specific activity obtained by the use of the xylose isomerase
from the fungus Piromyces sp. EII (Kuyper et al, 2003).
Example 6
Growth on Xylose as the Carbon Source of S. cerevisiae Strains
Expressing LxXI-2a and POCKS-14a (Strain T0004)
[0404] S. cerevisiae strain T0004, described in Example 1e,
comprises an expression cassette containing SEQ ID No 15 (which
encodes xylose isomerase) and an expression cassette containing the
nucleotide sequence encoding P. stipitis xylulokinase.
[0405] Strain T0004, described in Example 1e, was gradually adapted
to growth on xylose by serial dilution of the glucose content,
followed by growth on xylose as the only carbon source. This was
carried out using the following procedure: [0406] Recipe of
Synthetic Complete growth medium without uracil and leucine
(SC-Ura&Leu medium): [0407] Bacto-yeast nitrogen base without
amino acids: 6.7 g/l; [0408] supplied with 40 mg/l of all amino
acids except Leucine; [0409] and supplied with p-aminobenzoic acid,
40 mg/l.
[0410] Inoculating a first culture tube containing 10 ml
SC-Ura&Leu medium containing 2% xylose+0.5% glucose with strain
T0004. Incubating the tube at 30.degree. C. and 150 rpm rotary
shaking until OD600 reached 1.5. This culture is herein referred to
as `1. culture`.
[0411] Inoculating a second culture tube containing 9 ml
SC-Ura&Leu medium containing 2% xylose with 1 ml of the above
described `1. culture`. Incubating the culture tube at 30.degree.
C. and 150 rpm rotary shaking until OD600 reached approximately
1.0. This culture is herein referred to as `2. culture`.
[0412] Inoculating a third culture tube containing 9 ml
SC-Ura&Leu medium containing 2% xylose with 1 ml of the above
described `2. culture`. Incubating the culture tube at 30.degree.
C. and 150 rpm rotary shaking until OD600 was between 0.5 and 1.0.
This culture is herein referred to as `3. culture`.
[0413] Inoculating a 250 ml shakeflask with baffles, containing 50
ml SC-Ura&Leu medium containing 2% xylose with an amount of the
`3. culture` to reach an OD600 value of 0.05. Incubating the
culture tube at 30.degree. C. and 150 rpm rotary shaking and
following the growth by OD600 measurements through a culture period
of 240 hours.
[0414] The growth of strain T0004 on xylose as the carbon source
can be seen in FIG. 6. After a lag phase, the maximum growth rate
reached 0.05/h without other modifications of the strain.
Example 7
Demonstration of High Xylose Isomerase Activity in S. cerevisiae
Cells by Intracellular Expression of Three Naturally Occurring
Variants and Two Artificial Variants of a Lactococcus Isomerase
[0415] The naturally occurring xylose isomerases with amino acid
sequences as shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 18
where expressed intracellular in Saccharomyces cerevisiae strain
BY4741 (Euroscarf, Germany). In addition, two related variants, not
found in any organism, were generated (as detailed below) and also
expressed. The amino acid sequences of these two variants are shown
in SEQ ID NO 19 and SEQ ID NO 20. Except for variation in amino
acid position 391 (arginine or lysine), position 407 (glutamic acid
or lysine) or 416 (tyrosine or histidine), all variants have
identical amino acid sequences. This means that when compared to
SEQ ID No 18, SEQ ID No 13 has the amino acid K at position 391,
the amino acid K at position 407 and the amino acid H at position
416. These amino acids in SEQ ID No 13 are underlined in the
sequence listing. Compared to SEQ ID No 18, SEQ ID No 14 has the
amino acid K at position 391. This amino acid in SEQ ID No 14 is
underlined in the sequence listing. Compared to SEQ ID No 18, SEQ
ID No 19 has the amino acid K at position 407 and the amino acid H
at position 416. These amino acids in SEQ ID No 19 are underlined
in the sequence listing. Compared to SEQ ID No 18, SEQ ID No 20 has
the amino acid K at position 407. This amino acid in SEQ ID No 20
is underlined in the sequence listing.
[0416] DNA sequences encoding the amino acid sequences SEQ ID NO
13, SEQ ID NO 14, SEQ ID NO 19 and SEQ ID NO 20 used in this
example were all generated by site directed mutagenesis of the DNA
sequence shown as SEQ ID NO 17, using the construct obtained as
described in Example 1a as initial template. (The amino acid
sequence SEQ ID No 18 is encoded by SEQ ID No 17.) Site directed
mutagenesis was done by using the "Quick Change" kit (Stratagene,
La Jolla, USA) to change the codons encoding amino acid residues
391, 407 and 416 respectively. The kit was used according to the
manufacturer's protocol. The sequences of the mutagenic DNA primers
used are shown in SEQ ID NO 21 and SEQ ID NO 22 (R391K substitution
and removal of a Psp1406I restriction site), SEQ ID NO 23 and SEQ
ID NO 24 (E407K substitution and addition of a HindIII restriction
site) and SEQ ID NO 25 and SEQ ID NO 26 (combined E407K and Y416H
substitution and addition of a restriction site). The addition or
removal of restriction sites allow for easy identification of
mutated vectors. The correct overall DNA sequences of the generated
DNA constructs were subsequently confirmed by DNA sequencing.
[0417] A modified version of the E. coli/S. cerevisiae high-copy
shuttle vector pRS426-gpd (Mumberg et al., 1995) was used for
intracellular expression of the xylose isomerase variants. The
vector was modified by exchanging the URA3 marker in pRS426-gpd
with the nourseothricin resistance marker from vector pAG35
(Euroscarf, Germany), using standard molecular biology subcloning
techniques. The resultant shuttle vector confers nourseothricin
resistance to S. cerevisiae.
[0418] The five different xylose isomerase genes were isolated and
inserted into the modified expression vector according to the
description in Example 1b. The resulting plasmid constructs were
named pRS426gpd_nat::XI13, pRS426gpd_nat::XI14,
pRS426gpd_nat::XI18, pRS426gpd_nat::XI19 and pRS426gpd_nat::XI20,
respectively.
[0419] Each variant plasmid construct was transformed into a strain
of S. cerevisiae BY4741 (Euroscarf, Germany) by means of
electroporation using the Biorad Gene Pulser II system (Biorad,
USA) according to the manufacturer's instruction. Yeast cells were
made competent according to a standard protocol (Becker and
Guarente, 1991). Selection for transformed clones was done on YPD
plates (20 g/l tryptone, 10 g/l yeast extract and 20 g/l glucose
with 15 g/l bactoagar added for solidification) containing 100 mg/l
nourseothricin.
[0420] Five cell lines, each expressing one of the variant
Lactococcus xylose isomerases were obtained.
[0421] The specific xylose isomerase activity of each cell line was
determined in duplicate and compared as described in Example 5,
except that the medium used for yeast growth was YPD (20 g/l
tryptone, 10 g/l yeast extract and 20 g/l glucose) supplied with
100 mg/l nourseothricin
[0422] The obtained average specific xylose isomerase activity of
the duplicate determinations, calculated as xylose isomerase
units/mg of total extracted yeast protein, of the five strains is
shown in FIG. 7.
[0423] All tested Lactococcus xylose isomerase variants are highly
active when expressed in S. cerevisiae. This can be therefore be
regarded to be general for lactococcus xylose isomerase genes that
encodes active xylose isomerases in Lactococcus. It appears that,
comparing the activity of SEQ ID 19 and SEQ ID 20, the Y416H
substitution makes very little difference in activity. In contrast,
the K391R substitution and the E407K substitution both increase the
specific activity in yeast by approximately 15-20%, an effect that
appears to be additive if those two substitutions are combined as
in SEQ ID NO 19 and SEQ ID NO 20.
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[0452] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in biochemistry and molecular
biology or related fields are intended to be within the scope of
the following claims.
TABLE-US-00002 SEQUENCE LISTING SEQ ID No. 1: Coding region of
Lactococcus lactis subsp. lactis (Lactobacillus xylosus) strain
NRRL- 4449 xylose isomerase gene as described in accession number
AF092042. The amino acid sequence encoded by this nucleotide
sequence is shown as SEQ ID No 14. M A Y F N D I A P I K Y E G T K
1 ATG GCT TAC TTT AAC GAC ATC GCA CCT ATC AAA TAC GAA GGT ACA AAA T
K N M F A F R H Y N P E E V V 49 ACT AAA AAT ATG TTT GCC TTT CGC
CAT TAT AAT CCA GAA GAA GTA GTT A G K T M E E Q L H F A L A F W 97
GCT GGT AAA ACA ATG GAA GAA CAA CTT CAT TTT GCC CTT GCA TTT TGG H T
I T M D G S D P F G G A T M 145 CAT ACA ATT ACA ATG GAT GGG TCA GAT
CCC TTT GGG GGA GCA ACA ATG E R P W D L E G G S E L D R A H 193 GAA
CGC CCT TGG GAT TTG GAA GGT GGT TCT GAA CTT GAC CGT GCT CAC R R V D
A F F E I A E K L G V K 241 CGT CGA GTA GAT GCT TTC TTT GAA ATT GCT
GAA AAA TTA GGT GTT AAA Y Y C F H D I D I A P T G N S L 289 TAT TAT
TGT TTC CAT GAT ATT GAT ATT GCA CCT ACT GGA AAT TCT TTG K E F Y A N
L D E I T D H L L E 337 AAA GAA TTT TAT GCT AAT TTG GAC GAA ATT ACT
GAC CAC CTT CTT GAA K Q K A T G I K L L W N T A N M 385 AAA CAA AAA
GCA ACA GGG ATT AAA TTA CTT TGG AAT ACA GCA AAC ATG F S N P R Y M N
G V S T S N R A 433 TTT TCA AAT CCC CGC TAT ATG AAT GGT GTT TCA ACT
TCT AAT CGT GCT E V F A Y G A A Q V K K G L E L 481 GAA GTC TTT GCT
TAT GGT GCT GCA CAA GTT AAA AAA GGT CTT GAA CTT S K K L G G E N Y V
F W G G R E 529 TCT AAA AAA CTC GGT GGT GAA AAT TAC GTC TTC TGG GGT
GGT CGT GAA G Y E S L L N T D M G L E M D H 577 GGT TAT GAA TCA CTT
TTG AAT ACA GAT ATG GGT CTT GAA ATG GAT CAT M A K F F H L A I D Y A
K S I N 625 ATG GCA AAA TTC TTC CAT TTG GCA ATT GAT TAT GCA AAA TCA
ATC AAC H L P I F L I E P K P K E P M T 673 CAC TTG CCC ATT TTC TTG
ATT GAA CCA AAA CCA AAA GAA CCA ATG ACT H Q Y D F D S A T A L A F L
Q K 721 CAC CAA TAT GAT TTT GAC TCA GCA ACA GCT CTT GCT TTC TTG CAA
AAA Y D L D K Y F K L N L E T N H A 769 TAT GAT TTG GAC AAA TAT TTC
AAA CTC AAT CTT GAA ACA AAT CAT GCT W L A G H T F E H E L N T A R T
817 TGG TTG GCT GGA CAC ACT TTT GAA CAC GAA TTA AAT ACT GCT CGT ACT
F N A L G S I D A N Q G N Y L L 865 TTC AAT GCT TTG GGT TCT ATT GAT
GCC AAT CAA GGA AAT TAC TTG CTT G W D T D E F P T L V I D I T L 913
GGT TGG GAT ACA GAT GAA TTC CCA ACA CTT GTT ATT GAT ATC ACA CTT A M
H Q I L L N G G L G K G G I 961 GCG ATG CAC CAA ATT CTT TTG AAC GGT
GGA CTT GGC AAA GGT GGA ATT N F D A K V R R T S F K A E D L 1009
AAC TTT GAT GCG AAA GTA CGT CGT ACA AGT TTC AAA GCA GAA GAT TTA I L
A H I A G M D T Y A R A L K 1057 ATT CTT GCT CAT ATT GCA GGG ATG
GAT ACT TAT GCG CGT GCT TTG AAA G A A A I I E D K F L S D I V D
1105 GGT GCA GCA GCA ATC ATT GAA GAT AAA TTC TTG TCT GAT ATT GTT
GAC E R Y S S Y K N T E V G Q S I E 1153 GAA CGT TAT AGT TCA TAC
AAA AAT ACA GAA GTT GGT CAA TCC ATT GAA N G T A T F E S L A A F A L
E Y 1201 AAT GGA ACA GCA ACT TTT GAA AGT CTT GCC GCA TTT GCA CTT
GAA TAT G D D I E L D S N H L E Y I K S 1249 GGT GAT GAT ATT GAA
CTT GAT TCT AAT CAC TTG GAA TAC ATC AAA TCA V E N D Y L V * 1297
GTA TTG AAT GAC TAT CTT GTT TAA SEQ ID No. 2:
GCTAGCCATGGCTTACTTTAACGACATCGCACCTATC SEQ ID No. 3:
CTCGAGCCTAGGCTAAACAAGATAGTCATTCAATACTGATTTG SEQ ID No. 4:
GCTAGCCATGGCCACTACCCCATTTGATGCTCCAGATAAG SEQ ID No. 5:
CTCGAGCCTAGGCTAGTGTTTCAATTCACTTTCCATCTTGGCC SEQ ID No. 6:
Artificial Thermoanaerobacter thermohydrosulfuricus xylose
isomerase gene, with N- terminal and C-terminal flanking regions.
The coding region is based on the amino acid sequence from GenBank
Accession code D00756. The amino acid sequence encoded by the
coding nucleotide sequence is shown as SEQ ID No 15. 1
GAATTCCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGGAGCTAGCC M E Y F K N V P Q
I K Y E G P K 49 ATG GAA TAT TTC AAA AAC GTG CCA CAG ATC AAG TAT
GAA GGT CCT AAA S N N P Y A F K F Y N P D E I I 97 AGC AAT AAC CCT
TAT GCA TTT AAG TTC TAT AAC CCA GAT GAA ATT ATA D G K P L K E H L R
F S V A Y W 145 GAT GGA AAA CCA TTA AAA GAA CAC TTA AGA TTT AGC GTA
GCC TAC TGG H T F T A N G T D P F G A P T M 193 CAT ACA TTT ACC GCT
AAC GGA ACG GAT CCA TTT GGT GCA CCG ACT ATG Q R P W D H F T D P M D
I A K A 241 CAG CGT CCT TGG GAT CAT TTT ACC GAC CCT ATG GAC ATA GCA
AAA GCA R V E A A F E L F E K L D V P F 289 CGT GTG GAA GCC GCA TTC
GAG CTT TTT GAA AAA TTG GAT GTT CCA TTC F C E H D R D I A P E G E T
L R 337 TTC TGT TTT CAT GAC AGA GAT ATA GCT CCG GAA GGT GAA ACA TTG
AGA E T N K N L D T I V A M I K D Y 385 GAA ACC AAC AAA AAC TTA GAT
ACT ATC GTT GCT ATG ATT AAA GAC TAC L K T S K T K V L W G T A N L F
433 TTA AAA ACG TCA AAG ACT AAA GTT CTT TGG GGC ACT GCT AAT TTG TTT
S N P R F V H G A A T S C N A D 481 TCT AAT CCA CGT TTC GTG CAT GGC
GCT GCC ACA TCA TGT AAT GCA GAC V F A Y A A A Q V K K A L E I T 529
GTA TTT GCT TAT GCA GCC GCT CAA GTT AAA AAG GCC TTA GAG ATT ACC K E
L G G Q N Y V F W G G R E G 577 AAA GAG TTA GGA GGC CAG AAT TAT GTT
TTC TGG GGT GGT CGT GAG GGA Y E T L L N T D M E L E L D N L 625 TAT
GAG ACA CTT TTA AAT ACT GAT ATG GAG TTG GAA TTA GAT AAT TTA A R F L
H M A V E Y A Q E I G F 673 GCA AGA TTC TTA CAC ATG GCA GTA GAA TAT
GCT CAG GAA ATT GGT TTT E G Q F L I E P K P K E P T K H 721 GAA GGA
CAG TTC TTG ATC GAG CCT AAA CCA AAG GAA CCA ACA AAG CAT Q Y D F D A
A S V H A F L K K Y 769 CAG TAT GAT TTC GAC GCT GCT TCT GTA CAC GCC
TTT TTG AAG AAG TAT D L D K Y F K L N I E A N H A T 817 GAT TTG GAT
AAA TAC TTT AAG TTG AAC ATA GAG GCT AAT CAC GCA ACG L A G H D F Q H
E L R Y A R I N 865 TTG GCA GGT CAC GAT TTT CAA CAC GAA TTG AGA TAC
GCC CGT ATT AAT N M L G S I D A N M G D M L L G 913 AAC ATG TTA GGT
TCC ATA GAT GCC AAC ATG GGT GAC ATG TTG CTG GGT W D T D Q Y P T D I
R M T T L A 961 TGG GAT ACT GAT CAA TAC CCA ACG GAT ATT AGA ATG ACA
ACT TTA GCA M Y E V I K M G G F N K G G L N 1009 ATG TAC GAG GTC
ATT AAA ATG GGA GGT TTT AAC AAA GGA GGT TTG AAT F D A K V R R A S F
E P E D L F 1057 TTC GAT GCT AAA GTG CGT CGT GCC TCT TTT GAA CCT
GAA GAC CTT TTT L G H I A G M D A F A K G F K V 1105 CTT GGA CAT
ATT GCC GGA ATG GAT GCA TTT GCA AAA GGT TTC AAG GTC A Y K L V K D G
V F D R F I E E 1153 GCT TAT AAG CTT GTT AAG GAT GGT GTA TTT GAT
AGA TTC ATT GAA GAG R Y K S Y R E G I G A E I V S G 1201 AGA TAC
AAA TCC TAT CGT GAA GGT ATA GGT GCT GAA ATC GTT TCA GGT K A N F K T
L E E Y A L N N P K 1249 AAG GCC AAT TTT AAG ACT TTA GAG GAA TAT
GCA TTG AAT AAC CCA AAA I E N K S G K Q E L L E S I L N 1297 ATC
GAA AAC AAA AGC GGT AAA CAG GAA CTG CTG GAA TCT ATT TTG AAT Q Y L F
S E * 1345 CAA TAT TTG TTC TCT GAA TAG CCTAGGCTCGAGGAATTC SEQ ID
No. 7: Artificial Thermoanaerobacter thermosulphurigenes xylose
isomerase gene, with N- terminal and C-terminal flanking regions.
The coding region is based on amino acid sequence from GenBank
Accession code J05650, except that the second amino acid has been
changed from N into A, in order to enable the use of the "Kozak
consensus sequence" for eucaryotic expression. C.
therrnosulfurogenes is an alternative name for Thermoanaerobacter
thermosulphurigenes (Lee et al 1993). The amino acid sequence
encoded by the coding region of this nucleotide sequence is shown
as SEQ ID No 16. 1 GAATTCCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGGAGCTAGCC
M A K Y F E N V S K I K Y E G P 49 ATG GCC AAG TAT TTT GAG AAT GTT
TCC AAG ATT AAG TAT GAA GGT CCT K S N N P Y S F K F Y N P E E V 97
AAG TCA AAC AAC CCT TAC TCC TTC AAA TTC TAT AAT CCA GAA GAA GTT I D
G K T M E E H L R F S I A Y 145 ATA GAC GGT AAA ACG ATG GAG GAA CAC
TTA AGA TTT TCT ATT GCT TAC W H T F T A D G T D Q F G K A T 193 TGG
CAC ACA TTT ACT GCC GAC GGC ACA GAC CAA TTC GGA AAG GCT ACT M Q R P
W N H Y T D P M D I A K 241 ATG CAA AGA CCG TGG AAC CAT TAC ACT GAT
CCA ATG GAC ATA GCC AAA A R V E A A F E F F D K I N A P 289 GCC AGA
GTG GAG GCT GCA TTC GAG TTC TTC GAT AAG ATA AAC GCC CCT Y F C F H D
R D I A P E G D T L 337 TAC TTC TGC TTC CAT GAT CGT GAC ATT GCT CCG
GAA GGC GAT ACC TTG R E T N K N L D T I V A M I K D 385 AGA GAA ACC
AAC AAA AAT CTT GAC ACC ATT GTC GCA ATG ATA AAA GAT Y L K T S K T K
V L W G T A N L 433 TAT TTG AAG ACG TCT AAA ACC AAA GTT TTG TGG GGT
ACG GCA AAC TTA F S N P R F V H G A S T S C N A 481 TTT TCT AAT CCG
AGA TTT GTT CAT GGT GCT TCC ACA TCC TGC AAC GCA D V F A Y S A A Q V
K K A L E I 529 GAT GTC TTT GCT TAT TCC GCT GCT CAA GTC AAA AAG GCT
CTT GAG ATA
T K E L G G E N Y V F W G G R E 577 ACC AAA GAA TTA GGT GGT GAA AAC
TAC GTT TTC TGG GGT GGC AGA GAA G Y E T L L N T D M E F E L D N 625
GGT TAT GAG ACT CTT TTA AAT ACA GAT ATG GAA TTT GAA CTT GAC AAT F A
R F L H M A V D Y A K E I G 673 TTC GCA AGA TTC TTG CAC ATG GCT GTC
GAT TAT GCC AAG GAG ATA GGT F E G Q F L I E P K P K E P T K 721 TTT
GAA GGC CAG TTC TTG ATT GAA CCG AAA CCA AAG GAA CCT ACC AAA H Q Y D
F D V A N V L A F L R K 769 CAT CAG TAT GAC TTT GAT GTC GCA AAT GTT
TTG GCT TTC TTG AGA AAA Y D L D K Y F K V N I E A N H A 817 TAC GAT
TTA GAT AAG TAT TTC AAA GTA AAT ATT GAA GCT AAT CAT GCA T L A F H D
F Q H E L R Y A R I 865 ACG TTG GCC TTC CAT GAT TTC CAG CAC GAG TTG
AGA TAC GCT AGA ATC N G V L G S I D A N T G D M L L 913 AAT GGA GTC
TTA GGA TCT ATC GAT GCT AAT ACC GGT GAT ATG CTG CTG G W D T D Q F P
T D I R M T T L 961 GGA TGG GAT ACT GAT CAA TTT CCG ACC GAT ATT CGT
ATG ACT ACC CTG A M Y E V I K M G G F D K G G L 1009 GCA ATG TAT
GAA GTT ATT AAG ATG GGT GGA TTT GAT AAA GGC GGT CTG N F D A K V R R
A S F E P E D L 1057 AAC TTC GAT GCA AAA GTA AGA AGA GCT TCT TTT
GAA CCT GAA GAT TTG F L G H I A G M D A F A K G F K 1105 TTT TTA
GGA CAC ATC GCT GGC ATG GAC GCA TTT GCT AAA GGT TTT AAG V A Y K L V
K D R V F D K F I E 1153 GTA GCC TAT AAG CTT GTA AAA GAT AGA GTG
TTC GAC AAA TTC ATC GAA E R Y A S Y K D G I G A D I V S 1201 GAG
AGA TAT GCT TCT TAT AAG GAC GGA ATA GGA GCC GAT ATA GTT TCC G K A D
F R S L E K Y A L E R S 1249 GGT AAG GCC GAT TTC AGA TCT CTT GAG
AAA TAC GCC TTG GAA AGA TCA Q I V N K S G R Q E L L E S I L 1297
CAA ATC GTG AAC AAA TCA GGC CGT CAA GAA TTG TTA GAA TCA ATT CTT N Q
Y L F A E * 1345 AAT CAA TAC CTG TTC GCT GAA TAG CCTAGGCTCGAGGAATTC
SEQ ID No. 8: TAGCTAGCATGTCGTACTTCCCCACTGTCGAC SEQ ID No. 9:
ATAAGCTTTCAGGTGTAGATAAAGCGGTTGACC SEQ ID No. 10: Lactococcus lactis
subsp. lactis, strain KF147 xylose isomerase gene. Based on GenBank
accession number EU255918. The amino acid sequence encoded by this
nucleotide sequence is shown as SEQ ID No 11. 1 atg gct tac ttt aac
gac atc gca cct atc aaa tac gaa ggt aca aaa 49 act aaa aat atg ttt
gcc ttt cgt cat tat aat cca gaa gaa gta gtt 97 gct ggt aaa aca atg
gaa gaa caa ctt cat ttt gcc ctt gca ttt tgg 145 cat aca att acg atg
gat ggg tca gat ccc ttt ggg gga gca aca atg 193 gaa cgt cct tgg gat
ttg gaa ggt ggt tct gaa ctt gac cgt gct cac 241 cgt cga gta gat gct
ttc ttt gaa att gct gaa aaa tta ggt gtt aaa 289 tat tat tgt ttc cat
gat att gat att gca cct act gga aat tct ttg 337 aaa gaa ttt tat gct
aat ttg gac gaa att act gac cac ctt ctt gaa 385 aaa caa aaa gca aca
ggc att aaa tta ctt tgg aat aca gca aac atg 433 ttt tca aat ccc cgc
tat atg aat ggt gtt tca act tct aat cgt gct 481 gaa gtc ttt gct tat
ggt gct gca caa gtt aaa aaa ggt ctt gaa ctt 529 tct aaa aaa ctc ggt
ggt gaa aat tat gtc ttc tgg ggt ggt cgt gaa 577 ggt tat gaa tca ctt
ttg aat aca gat atg ggt ctt gaa atg gat cat 625 atg gca aaa ttc ttc
cat ttg gca att gat tat gca aaa tca atc aac 673 cac ttg cct att ttc
ttg att gaa cca aaa cca aaa gaa cca atg act 721 cac caa tat gat ttt
gac tca gca aca gct ctt gct ttc ttg caa aaa 769 tat gac ttg gac aaa
tac ttc aaa ctc aat ctt gaa aca aat cat gct 817 tgg ttg gct ggg cac
act ttt gaa cac gaa tta aat act gca cgt act 865 ttc aat gct ttg ggt
tct att gat gcc aat caa gga aat tac ttg ctt 913 ggt tgg gat aca gat
gaa ttc cca aca ctt gtt att gat atc aca ctt 961 gcg atg cac caa att
ctt ttg aac ggt gga ctt ggc aaa ggt gga att 1009 aac ttt gat gcg
aaa gta cgt cgt aca agt ttc aaa gca gaa gat tta 1057 att ctt gct
cat att gca ggg atg gat act tat gcg cgt gct ttg aaa 1105 ggt gca
gca gca atc att gaa gat aaa ttc ttg tct gat att gtt gac 1153 gaa
cgt tat agt tca tac aaa aat aca gaa gtt ggt caa tcc att gaa 1201
aat gga aca gca act ttt gaa agt ctt gct gca ttt gca ctt gaa cat
1249 ggt gac gat att gaa ctt gat tct aat cac ttg gaa tac atc aaa
tca 1297 gta ttg aat gac tat ctt gtt taa SEQ ID No. 11: Lactococcus
lactis subsp. lactis Strain KF147 xylose isomerase gene. Based on
GenBank accession number ABX75758. The amino acid sequence encoded
by the nucleotide sequence of SEQ ID No 10.
MAYFNDIAPIKYEGTKTKNMFAFRHYNPEEVVAGKTMEEQLHFALAFWHTITMDGSDPFG
GATMERPWDLEGGSELDRAHRRVDAFFEIAEKLGVKYYCFHDIDIAPTGNSLKEFYANLD
EITDHLLEKQKATGIKLLWNTANMFSNPRYMNGVSTSNRAEVFAYGAAQVKKGLELSKKL
GGENYVFWGGREGYESLLNTDMGLEMDHMAKFFHLAIDYAKSINHLPIFLIEPKPKEPMT
HQYDFDSATALAFLQKYDLDKYFKLNLETNHAWLAGHTFEHELNTARTFNALGSIDANQG
NYLLGWDTDEFPTLVIDITLAMHQILLNGGLGKGGINFDAKVRRTSFKAEDLILAHIAGM
DTYARALKGAAAIIEDKFLSDIVDERYSSYKNTEVGQSIENGTATFESLAAFALEHGDDI
ELDSNHLEYIKSVLNDYLV SEQ ID No 12: Lactococcus lactis subsp. lactis,
strain IO-1 xylose isomerase gene. Based on GenBank accession
number AF092041. The amino acid sequence encoded by this nucleotide
sequence is shown as SEQ ID No 13. 1 atg gct tac ttt aac gac atc
gca cct atc aaa tac gaa ggt aca aaa 49 act aaa aat atg ttt gcc ttt
cgc cat tat aat cca gaa gaa gta gtt 97 gct ggt aaa aca atg gaa gaa
caa ctt cat ttt gcc ctt gca ttt tgg 145 cat aca att aca atg gat ggg
tca gac ccc ttt ggg gga gca aca atg 193 gaa cgt cct tgg gat ttg gaa
ggt ggt tct gaa ctt gac cgt gct cac 241 cgt cga gta gat gct ttc ttt
gaa att gct gaa aaa tta ggt gtt aaa 289 tat tat tgt ttc cat gat att
gat att gca cct act gga aat tct ttg 337 aaa gaa ttt tat gct aat ttg
gac gaa att act gac cac ctt ctt gaa 385 aaa caa aaa gca aca ggg att
aaa tta ctt tgg aat aca gca aac atg 433 ttt tca aat ccc cgc tat atg
aat ggt gtt tca act tct aac cgt gct 481 gaa gtc ttt gct tat ggt gct
gca caa gtt aaa aaa ggt ctt gaa ctt 529 tct aaa aaa ctc ggt ggt gaa
aat tac gtc ttc tgg ggt ggt cgt gaa 577 ggt tat gaa tca ctt ttg aat
aca gat atg ggt ctt gaa atg gat cat 625 atg gca aaa ttc ttc cat ttg
gca att gat tat gca aaa tca atc aac 673 cac ttg ccc att ttc ttg atc
gaa cca aaa cca aaa gaa cca atg act 721 cac caa tat gat ttt gac tca
gca aca gct ctt gct ttc ttg caa aaa 769 tat gat ttg gac aaa tat ttc
aaa ctc aat ctt gaa aca aat cat gct 817 tgg ttg gct gga cac act ttt
gaa cac gaa tta aat act gct cgt act 865 ttc aat gct ttg ggt tct att
gat gcc aat caa gga aat tac ttg ctt 913 ggt tgg gat aca gat gaa ttc
cca aca ctt gtt att gat atc aca ctt 961 gcg atg cac caa att ctt ttg
aac ggt gga ctt ggc aaa ggt gga att 1009 aac ttt gat gcg aaa gta
cgt cgt aca agt ttc aaa gca gaa gat tta 1057 att ctt gct cat att
gca ggg atg gat act tat gcg cgt gct ttg aaa 1105 ggt gca gca gca
atc att gaa gat aaa ttc ttg tct gat att gtt gac 1153 gaa cgt tat
agt tca tac aaa aat aca gaa gtt gga caa tcc att gaa 1201 aat gga
aca gca act ttt aaa agt ctt gcc gca ttt gca ctt gaa cat 1249 ggt
gac gat att gaa ctt gat tct aat cac ttg gaa tac atc aaa tca 1297
gta ttg aat gac tat ctt gtt taa SEQ ID No 13: Lactococcus lactis
subsp. lactis, Strain IO-1 xylose isomerase. Based on GenBank
accession number AAD20249. The amino acid sequence encoded by the
nucleotide sequence of SEQ ID No 12. The amino acids which differ
to SEQ ID No 18 are underlined.
MAYFNDIAPIKYEGTKTKNMFAFRHYNPEEVVAGKTMEEQLHFALAFWHTITMDGSDPFG
GATMERPWDLEGGSELDRAHRRVDAFFEIAEKLGVKYYCFHDIDIAPTGNSLKEFYANLD
EITDHLLEKQKATGIKLLWNTANMFSNPRYMNGVSTSNRAEVFAYGAAQVKKGLELSKKL
GGENYVFWGGREGYESLLNTDMGLEMDHMAKFFHLAIDYAKSINHLPIFLIEPKPKEPMT
HQYDFDSATALAFLQKYDLDKYFKLNLETNHAWLAGHTFEHELNTARTFNALGSIDANQG
NYLLGWDTDEFPTLVIDITLAMHQILLNGGLGKGGINFDAKVRRTSFKAEDLILAHIAGM
DTYARALKGAAAIIEDKFLSDIVDERYSSYKNTEVGQSIENGTATFKSLAAFALEHGDDI
ELDSNHLEYIKSVLNDYLV SEQ ID No 14: The amino acid sequence encoded
by the nucleotide sequence of SEQ ID No 1. The amino acid which
differs to SEQ ID No 18 is underlined. M A Y F N D I A P I K Y E G
T K T K N M F A F R H Y N P E E V V A G K T M E E Q L H F A L A F W
H T I T M D G S D P F G G A T M E R P W D L E C G S E L D R A H R R
V D A F F E I A E K L G V K Y Y C F H D I D I A P T G N S L K E F Y
A N L D E I T D H L L E K Q K A T G I K L L W N T A N M F S N P R Y
M N G V S T S N R A E V F A Y G A A Q V K K G L E L S K K L G G E N
Y V F W G G R E G Y E S L L N T D M G L E M D H M A K F F H L A I D
Y A K S I N H L P I F L I E P K P K E P M T H Q Y D F D S A T A L A
F L Q K Y D L D K Y F K L N L E T N H A W L A G H T F E H E L N T A
R T F N A L G S I D A N Q G N Y L L G W D T D E F P T L V I D I T L
A M H Q I L L N G G L G K G G I N F D A K V R R T S F K A E D L I L
A H I A G M D T Y A R A L K G A A A I I E D K F L S D I V D E R Y S
S Y K N T E V G Q S I E N G T A T F E S L A A F A L E Y G D D I E L
D S N H L E Y I K S V L N D Y L V SEQ ID No 15: GenBank accession
number BAA00652. The amino acid sequence encoded by the coding
region of nucleotide sequence of SEQ ID No 6. M E Y F K N V P Q I K
Y E G P K S N N P Y A F K E Y N P D E I I D G K P L K E H L R F S V
A Y W H T F T A N G T D P F G A P T M Q R P W D H F T D P M D I A K
A R V E A A F E L F E K L D V P F F C F H D R D I A P E G E T L R E
T N K N L D T I V A M I K D Y L K T S K T K V L W G T A N L F S N P
R F V H G A A T S C N A D V F A Y A A A Q V K K A L E I T K E L G G
Q N Y V F W G G R E G Y E T L L N T D M E L E L D N L A R F L H M A
V E Y A Q E I G F E G Q F L I E P K P K E P T K H Q Y D F D A A S V
H A F L K K Y D L D K Y F K L N I E A N H A T L A G H D F Q H E L R
Y A R I N N M L G S I D A N M G D M L L G W D T D Q Y P T D I R M I
T L A M Y E V I K M G G F N K G G L N F D A K V R R A S F E P E D L
F L G H I A G M D A F A K G F K V A Y K L V K D G V F D R F I E E R
Y K S Y R E G I G A E I V S G K A N F K T L E E Y A L N N P K I E N
K S G K Q E L L E S I L N Q Y L F S E SEQ ID No 16: GenBank
accession number AAA23285. The amino acid sequence encoded by the
coding region of nucleotide sequence of SEQ ID No 7. M A K Y F E N
V S K I K Y E G P K S N N P Y S F K F Y N P E E V I D G K T M E E H
L R F S I A Y W H T F T A D G T D Q F G K A T M Q R P W N H Y T D P
M D I A K A R V E A A F E F F D K I N A P Y F C F H D R D I A P E G
D T L R E T N K N L D T I V A M I K D Y L K T S K T K V L W G T A N
L F S N P R F V H G A S T S C N A D V F A Y S A A Q V K K A L E I T
K E L G G E N Y V F W G G R E G Y E T L L N T D M E F E L D N F A R
F L H M A V D Y A K E I G F E G Q F L I E P K P K E P T K H Q Y D F
D V A N V L A F L R K Y D L D K Y F K V N I E A N H A T L A F H D F
Q H E L R Y A R I N G V L G S I D A N T G D M L L G W D T D Q F P T
D I R M T T L A M Y E V I K M G G F D K G G L N F D A K V R R A S F
E P E D L F L G H I A G M D A F A K G F K V A Y K L V K D R V F D K
F I E E R Y A S Y K D G I G A D I V S G K A D F R S L E K Y A L E R
S
Q I V N K S G R Q E L L E S I L N Q Y L F A E SEQ ID No 17: Coding
region of Xylose isomerase gene cloned by PCR out of Lactococcus
lactis strain designated DSM 20175, using primers shown in SEQ ID
No 2 and SEQ ID No 3. 1 atg gct tac ttt aac gac atc gca cct atc aaa
tac gaa ggt aca aaa 49 act aaa aat atg ttt gcc ttt cgc cat tat aat
cca gaa gaa gta gtt 97 gct ggt aaa aca atg gaa gaa caa ctt cat ttt
gcc ctt gca ttt tgg 145 cat aca att aca atg gat ggg tca gat ccc ttt
ggg gga gca aca atg 193 gaa cgc cct tgg gat ttg gaa ggt ggt tct gaa
ctt gac cgt gct cac 241 cgt cga gta gat gct ttc ttt gaa att gct gaa
aaa tta ggt gtt aaa 289 tat tat tgt ttc cat gat att gat att gca cct
act gga aat tct ttg 337 aaa gaa ttt tat gct aat ttg gac gaa att act
gac cac ctt ctt gaa 385 aaa caa aaa gca aca ggg att aaa tta ctt tgg
aat aca gca aac atg 433 ttt tca aat ccc cgc tat atg aat ggt gtt tca
act tct aat cgt gct 481 gaa gtc ttt gct tat ggt gct gca caa gtt aaa
aaa ggt ctt gaa ctt 529 tct aaa aaa ctc ggt ggt gaa aat tac gtc ttc
tgg ggt ggt cgt gaa 577 ggt tat gaa tca ctt ttg aat aca gat atg ggt
ctt gaa atg gat cat 625 atg gca aaa ttc ttc cat ttg gca att gat tat
gca aaa tca atc aac 673 cac ttg ccc att ttc ttg att gaa cca aaa cca
aaa gaa cca atg act 721 cac caa tat gat ttt gac tca gca aca gct ctt
gct ttc ttg caa aaa 769 tat gat ttg gac aaa tat ttc aaa ctc aat ctt
gaa aca aat cat gct 817 tgg ttg gct gga cac act ttt gaa cac gaa tta
aat act gct cgt act 865 ttc aat gct ttg ggt tct att gat gcc aat caa
gga aat tac ttg ctt 913 ggt tgg gat aca gat gaa ttc cca aca ctt gtt
att gat atc aca ctt 961 gcg atg cac caa att ctt ttg aac ggt gga ctt
ggc aaa ggt gga att 1009 aac ttt gat gcg aaa gta cgt cgt aca agt
ttc aaa gca gaa gat tta 1057 att ctt gct cat att gca ggg atg gat
act tat gcg cgt gct ttg aaa 1105 ggt gca gca gca atc att gaa gat
aaa ttc ttg tct gat att gtt gac 1153 gaa cgt tat agt tca tac aga
aat aca gaa gtt ggt caa tcc att gaa 1201 aat gga aca gca act ttt
gaa agt ctt goo gca ttt gca ctt gaa tat 1249 ggt gat gat att gaa
ctt gat tct aat cac ttg gaa tac atc aaa tca 1297 gta ttg aat gac
tat ctt gtt taa SEQ ID No 18: Amino acid sequence encoded by SEQ ID
17. MAYFNDIAPIKYEGTKTKNMFAFRHYNPEEVVAGKTMEEQLHFALAFWHTITM
DGSDPFGGATMERPWDLEGGSELDRAHRRVDAFFEIAEKLGVKYYCFHDIDIAP
TGNSLKEFYANLDEITDHLLEKQKATGIKLLWNTANMFSNPRYMNGVSTSNRAE
VFAYGAAQVKKGLELSKKLGGENYVFWGGREGYESLLNTDMGLEMDHMAKFF
HLAIDYAKSINHLPIFLIEPKPKEPMTHQYDFDSATALAFLQKYDLDKYFKLNLET
NHAWLAGHTFEHELNTARTFNALGSIDANQGNYLLGWDTDEFPTLVIDITLAMH
QILLNGGLGKGGINFDAKVRRTSFKAEDLILAHIAGMDTYARALKGAAAIIEDKF
LSDIVDERYSSYRNTEVGQSIENGTATFESLAAFALEYGDDIELDSNHLEYIKSVL NDYLV SEQ
ID No 19: Artificial variant of Lactococcus xylose isomerase, amino
acid sequence (SEQ ID No 18). The amino acids which differ to SEQ
ID No 18 are underlined. The nucleotide sequence encoding this
amino acid sequence is shown as SEQ ID No. 27
MAYFNDIAPIKYEGTKTKNMFAFRHYNPEEVVAGKTMEEQLHFALAFWHTITMDGSDPFG
GATMERPWDLEGGSELDRAHRRVDAFFEIAEKLGVKYYCFHDIDIAPTGNSLKEFYANLD
EITDHLLEKQKATGIKLLWNTANMFSNPRYMNGVSTSNRAEVFAYGAAQVKKGLELSKKL
GGENYVFWGGREGYESLLNTDMGLEMDHMAKFFHLAIDYAKSINHLPIFLIEPKPKEPMT
HQYDFDSATALAFLQKYDLDKYFKLNLETNHAWLAGHTFEHELNTARTFNALGSIDANQG
NYLLGWDTDEFPTLVIDITLAMHQILLNGGLGKGGINFDAKVRRTSFKAEDLILAHIAGM
DTYARALKGAAAIIEDKFLSDIVDERYSSYRNTEVGQSIENGTATFKSLAAFALEHGDDI
ELDSNHLEYIKSVLNDYLV SEQ ID No 20: Artificial variant of Lactococcus
xylose isomerase, amino acid sequence (SEQ ID No 18). The amino
acid which differs to SEQ ID No 18 is underlined. The nucleotide
sequence encoding this amino acid sequence is shown as SEQ ID No.
28 MAYFNDIAPIKYEGTKTKNMFAFRHYNPEEVVAGKTMEEQLHFALAFWHTITMDGSDPFG
GATMERPWDLEGGSELDRAHRRVDAFFEIAEKLGVKYYCFHDIDIAPTGNSLKEFYANLD
EITDHLLEKQKATGIKLLWNTANMFSNPRYMNGVSTSNRAEVFAYGAAQVKKGLELSKKL
GGENYVFWGGREGYESLLNTDMGLEMDHMAKFFHLAIDYAKSINHLPIFLIEPKPKEPMT
HQYDFDSATALAFLQKYDLDKYFKLNLETNHAWLAGHTFEHELNTARTFNALGSIDANQG
NYLLGWDTDEEPTLVIDITLAMHQILLNGGLGKGGINFDAKVRRTSFKAEDLILAHIAGM
DTYARALKGAAAIIEDKELSDIVDERYSSYRNTEVGQSIENGTATEKSLAAFALEYGDDI
ELDSNHLEYIKSVLNDYLV SEQ ID No 21: R391K Sense primer:
GTTGACGAACGATATAGTTCATACAAAAATACAGAAGTTGG SEQ ID No 22: R391K
antisense primer: CCAACTTCTGTATTTTTGTATGAACTATATCGTTCGTCAAC SEQ ID
No 23: E407K sense primer:
ACAGCAACTTTTAAAAGCTTAGCCGCATTTGCACTTGAATATGGTGATGATATTG SEQ ID No
24: E407K antisense primer:
CAATATCATCACCATATTCAAGTGCAAATGCGGCTAAGCTTTTAAAAGTTGCTGT SEQ ID No
25: E407K-Y416H sense primer:
ACAGCAACTTTTAAAAGCTTAGCCGCATTTGCACTTGAACATGGTGATGATATTG SEQ ID No
26: E407K-Y416H antisense primer:
CAATATCATCACCATGTTCAAGTGCAAATGCGGCTAAGCTTTTAAAAGTTGCTGT SEQ ID No
27: The nucleotide sequence encoding the amino acid sequence shown
as SEQ ID No. 19 1 atg gct tac ttt aac gac atc gca cct atc aaa tac
gaa ggt aca aaa 49 act aaa aat atg ttt gcc ttt cgc cat tat aat cca
gaa gaa gta gtt 97 gct ggt aaa aca atg gaa gaa caa ctt cat ttt gcc
ctt gca ttt tgg 145 cat aca att aca atg gat ggg tca gat ccc ttt ggg
gga gca aca atg 193 gaa cgc cct tgg gat ttg gaa ggt ggt tct gaa ctt
gac cgt gct cac 241 cgt cga gta gat gct ttc ttt gaa att gct gaa aaa
tta ggt gtt aaa 289 tat tat tgt ttc cat gat att gat att gca cct act
gga aat tct ttg 337 aaa gaa ttt tat gct aat ttg gac gaa att act gac
cac ctt ctt gaa 385 aaa caa aaa gca aca ggg att aaa tta ctt tgg aat
aca gca aac atg 433 ttt tca aat ccc cgc tat atg aat ggt gtt tca act
tct aat cgt gct 481 gaa gtc ttt gct tat ggt gct gca caa gtt aaa aaa
ggt ctt gaa ctt 529 tct aaa aaa ctc ggt ggt gaa aat tac gtc ttc tgg
ggt ggt cgt gaa 577 ggt tat gaa tca ctt ttg aat aca gat atg ggt ctt
gaa atg gat cat 625 atg gca aaa ttc ttc cat ttg gca att gat tat gca
aaa tca atc aac 673 cac ttg ccc att ttc ttg att gaa cca aaa cca aaa
gaa cca atg act 721 cac caa tat gat ttt gac tca gca aca gct ctt gct
ttc ttg caa aaa 769 tat gat ttg gac aaa tat ttc aaa ctc aat ctt gaa
aca aat cat gct 817 tgg ttg gct gga cac act ttt gaa cac gaa tta aat
act gct cgt act 865 ttc aat gct ttg ggt tct att gat gcc aat caa gga
aat tac ttg ctt 913 ggt tgg gat aca gat gaa ttc cca aca ctt gtt att
gat atc aca ctt 961 gcg atg cac caa att ctt ttg aac ggt gga ctt ggc
aaa ggt gga att 1009 aac ttt gat gcg aaa gta cgt cgt aca agt ttc
aaa gca gaa gat tta 1057 att ctt gct cat att gca ggg atg gat act
tat gcg cgt gct ttg aaa 1105 ggt gca gca gca atc att gaa gat aaa
ttc ttg tct gat att gtt gac 1153 gaa cgt tat agt tca tac aga aat
aca gaa gtt ggt caa tcc att gaa 1201 aat gga aca gca act ttt aaa
agc tta gcc gca ttt gca ctt gaa cat 1249 ggt gat gat att gaa ctt
gat tct aat cac ttg gaa tac atc aaa tca 1297 gta ttg aat gac tat
ctt gtt taa SEQ ID No 28: The nucleotide sequence encoding the
amino acid sequence shown as SEQ ID No. 20 1 atg gct tac ttt aac
gac atc gca cct atc aaa tac gaa ggt aca aaa 49 act aaa aat atg ttt
gcc ttt cgc cat tat aat cca gaa gaa gta gtt 97 gct ggt aaa aca atg
gaa gaa caa ctt cat ttt gcc ctt gca ttt tgg 145 cat aca att aca atg
gat ggg tca gat ccc ttt ggg gga gca aca atg 193 gaa cgc cct tgg gat
ttg gaa ggt ggt tct gaa oft gac cgt gct cac 241 cgt cga gta gat gct
ttc ttt gaa att gct gaa aaa tta ggt gtt aaa 289 tat tat tgt ttc cat
gat att gat att gca cct act gga aat tct ttg 337 aaa gaa ttt tat gct
aat ttg gac gaa att act gac cac ctt ctt gaa 385 aaa caa aaa gca aca
ggg att aaa tta ctt tgg aat aca gca aac atg 433 ttt tca aat ccc cgc
tat atg aat ggt gtt tca act tct aat cgt gct 481 gaa gtc ttt gct tat
ggt gct gca caa gtt aaa aaa ggt ctt gaa ctt 529 tct aaa aaa ctc ggt
ggt gaa aat tac gtc ttc tgg ggt ggt cgt gaa 577 ggt tat gaa tca ctt
ttg aat aca gat atg ggt ctt gaa atg gat cat 625 atg gca aaa ttc ttc
cat ttg gca att gat tat gca aaa tca atc aac 673 cac ttg ccc att ttc
ttg att gaa cca aaa cca aaa gaa cca atg act 721 cac caa tat gat ttt
gac tca gca aca gct ctt gct ttc ttg caa aaa 769 tat gat ttg gac aaa
tat ttc aaa etc aat ctt gaa aca aat cat gct 817 tgg ttg gct gga cac
act ttt gaa cac gaa tta aat act gct cgt act 865 ttc aat gct ttg ggt
tct att gat gcc aat caa gga aat tac ttg ctt 913 ggt tgg gat aca gat
gaa ttc cca aca ctt gtt att gat atc aca ctt 961 gcg atg cac caa att
ctt ttg aac ggt gga ctt ggc aaa ggt gga att 1009 aac ttt gat gcg
aaa gta cgt cgt aca agt ttc aaa gca gaa gat tta 1057 att ott gct
cat att gca ggg atg gat act tat gcg cgt gct ttg aaa 1105 ggt gca
gca gca atc att gaa gat aaa ttc ttg tct gat att gtt gac 1153 gaa
cgt tat agt tca tac aga aat aca gaa gtt ggt caa tcc att gaa 1201
aat gga aca gca act ttt aaa agc tta gcc gca ttt gca ctt gaa tat
1249 ggt gat gat att gaa ctt gat tct aat cac ttg gaa tac atc aaa
tca 1297 gta ttg aat gac tat ctt gtt taa
Sequence CWU 1
1
2811320DNALactococcus lactis subsp. lactisCDS(1)..(1320) 1atg gct
tac ttt aac gac atc gca cct atc aaa tac gaa ggt aca aaa 48Met Ala
Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu Gly Thr Lys1 5 10 15act
aaa aat atg ttt gcc ttt cgc cat tat aat cca gaa gaa gta gtt 96Thr
Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro Glu Glu Val Val 20 25
30gct ggt aaa aca atg gaa gaa caa ctt cat ttt gcc ctt gca ttt tgg
144Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe Ala Leu Ala Phe Trp
35 40 45cat aca att aca atg gat ggg tca gat ccc ttt ggg gga gca aca
atg 192His Thr Ile Thr Met Asp Gly Ser Asp Pro Phe Gly Gly Ala Thr
Met 50 55 60gaa cgc cct tgg gat ttg gaa ggt ggt tct gaa ctt gac cgt
gct cac 240Glu Arg Pro Trp Asp Leu Glu Gly Gly Ser Glu Leu Asp Arg
Ala His65 70 75 80cgt cga gta gat gct ttc ttt gaa att gct gaa aaa
tta ggt gtt aaa 288Arg Arg Val Asp Ala Phe Phe Glu Ile Ala Glu Lys
Leu Gly Val Lys 85 90 95tat tat tgt ttc cat gat att gat att gca cct
act gga aat tct ttg 336Tyr Tyr Cys Phe His Asp Ile Asp Ile Ala Pro
Thr Gly Asn Ser Leu 100 105 110aaa gaa ttt tat gct aat ttg gac gaa
att act gac cac ctt ctt gaa 384Lys Glu Phe Tyr Ala Asn Leu Asp Glu
Ile Thr Asp His Leu Leu Glu 115 120 125aaa caa aaa gca aca ggg att
aaa tta ctt tgg aat aca gca aac atg 432Lys Gln Lys Ala Thr Gly Ile
Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135 140ttt tca aat ccc cgc
tat atg aat ggt gtt tca act tct aat cgt gct 480Phe Ser Asn Pro Arg
Tyr Met Asn Gly Val Ser Thr Ser Asn Arg Ala145 150 155 160gaa gtc
ttt gct tat ggt gct gca caa gtt aaa aaa ggt ctt gaa ctt 528Glu Val
Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys Gly Leu Glu Leu 165 170
175tct aaa aaa ctc ggt ggt gaa aat tac gtc ttc tgg ggt ggt cgt gaa
576Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190ggt tat gaa tca ctt ttg aat aca gat atg ggt ctt gaa atg
gat cat 624Gly Tyr Glu Ser Leu Leu Asn Thr Asp Met Gly Leu Glu Met
Asp His 195 200 205atg gca aaa ttc ttc cat ttg gca att gat tat gca
aaa tca atc aac 672Met Ala Lys Phe Phe His Leu Ala Ile Asp Tyr Ala
Lys Ser Ile Asn 210 215 220cac ttg ccc att ttc ttg att gaa cca aaa
cca aaa gaa cca atg act 720His Leu Pro Ile Phe Leu Ile Glu Pro Lys
Pro Lys Glu Pro Met Thr225 230 235 240cac caa tat gat ttt gac tca
gca aca gct ctt gct ttc ttg caa aaa 768His Gln Tyr Asp Phe Asp Ser
Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250 255tat gat ttg gac aaa
tat ttc aaa ctc aat ctt gaa aca aat cat gct 816Tyr Asp Leu Asp Lys
Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala 260 265 270tgg ttg gct
gga cac act ttt gaa cac gaa tta aat act gct cgt act 864Trp Leu Ala
Gly His Thr Phe Glu His Glu Leu Asn Thr Ala Arg Thr 275 280 285ttc
aat gct ttg ggt tct att gat gcc aat caa gga aat tac ttg ctt 912Phe
Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asn Tyr Leu Leu 290 295
300ggt tgg gat aca gat gaa ttc cca aca ctt gtt att gat atc aca ctt
960Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu Val Ile Asp Ile Thr
Leu305 310 315 320gcg atg cac caa att ctt ttg aac ggt gga ctt ggc
aaa ggt gga att 1008Ala Met His Gln Ile Leu Leu Asn Gly Gly Leu Gly
Lys Gly Gly Ile 325 330 335aac ttt gat gcg aaa gta cgt cgt aca agt
ttc aaa gca gaa gat tta 1056Asn Phe Asp Ala Lys Val Arg Arg Thr Ser
Phe Lys Ala Glu Asp Leu 340 345 350att ctt gct cat att gca ggg atg
gat act tat gcg cgt gct ttg aaa 1104Ile Leu Ala His Ile Ala Gly Met
Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365ggt gca gca gca atc att
gaa gat aaa ttc ttg tct gat att gtt gac 1152Gly Ala Ala Ala Ile Ile
Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375 380gaa cgt tat agt
tca tac aaa aat aca gaa gtt ggt caa tcc att gaa 1200Glu Arg Tyr Ser
Ser Tyr Lys Asn Thr Glu Val Gly Gln Ser Ile Glu385 390 395 400aat
gga aca gca act ttt gaa agt ctt gcc gca ttt gca ctt gaa tat 1248Asn
Gly Thr Ala Thr Phe Glu Ser Leu Ala Ala Phe Ala Leu Glu Tyr 405 410
415ggt gat gat att gaa ctt gat tct aat cac ttg gaa tac atc aaa tca
1296Gly Asp Asp Ile Glu Leu Asp Ser Asn His Leu Glu Tyr Ile Lys Ser
420 425 430gta ttg aat gac tat ctt gtt taa 1320Val Leu Asn Asp Tyr
Leu Val 435237DNAArtificial SequencePrimer 2gctagccatg gcttacttta
acgacatcgc acctatc 37343DNAArtificial SequencePrimer 3ctcgagccta
ggctaaacaa gatagtcatt caatactgat ttg 43440DNAArtificial
SequencePrimer 4gctagccatg gccactaccc catttgatgc tccagataag
40543DNAArtificial SequencePrimer 5ctcgagccta ggctagtgtt tcaattcact
ttccatcttg gcc 4361383DNAArtificial SequenceArtificial
Thermoanaerobacter thermohydrosulfuricus xylose isomerase gene
6gaattcctag aaataatttt gtttaacttt aagaaggagg agctagcc atg gaa tat
57 Met Glu Tyr 1ttc aaa aac gtg cca cag atc aag tat gaa ggt cct aaa
agc aat aac 105Phe Lys Asn Val Pro Gln Ile Lys Tyr Glu Gly Pro Lys
Ser Asn Asn 5 10 15cct tat gca ttt aag ttc tat aac cca gat gaa att
ata gat gga aaa 153Pro Tyr Ala Phe Lys Phe Tyr Asn Pro Asp Glu Ile
Ile Asp Gly Lys20 25 30 35cca tta aaa gaa cac tta aga ttt agc gta
gcc tac tgg cat aca ttt 201Pro Leu Lys Glu His Leu Arg Phe Ser Val
Ala Tyr Trp His Thr Phe 40 45 50acc gct aac gga acg gat cca ttt ggt
gca ccg act atg cag cgt cct 249Thr Ala Asn Gly Thr Asp Pro Phe Gly
Ala Pro Thr Met Gln Arg Pro 55 60 65tgg gat cat ttt acc gac cct atg
gac ata gca aaa gca cgt gtg gaa 297Trp Asp His Phe Thr Asp Pro Met
Asp Ile Ala Lys Ala Arg Val Glu 70 75 80gcc gca ttc gag ctt ttt gaa
aaa ttg gat gtt cca ttc ttc tgt ttt 345Ala Ala Phe Glu Leu Phe Glu
Lys Leu Asp Val Pro Phe Phe Cys Phe 85 90 95cat gac aga gat ata gct
ccg gaa ggt gaa aca ttg aga gaa acc aac 393His Asp Arg Asp Ile Ala
Pro Glu Gly Glu Thr Leu Arg Glu Thr Asn100 105 110 115aaa aac tta
gat act atc gtt gct atg att aaa gac tac tta aaa acg 441Lys Asn Leu
Asp Thr Ile Val Ala Met Ile Lys Asp Tyr Leu Lys Thr 120 125 130tca
aag act aaa gtt ctt tgg ggc act gct aat ttg ttt tct aat cca 489Ser
Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu Phe Ser Asn Pro 135 140
145cgt ttc gtg cat ggc gct gcc aca tca tgt aat gca gac gta ttt gct
537Arg Phe Val His Gly Ala Ala Thr Ser Cys Asn Ala Asp Val Phe Ala
150 155 160tat gca gcc gct caa gtt aaa aag gcc tta gag att acc aaa
gag tta 585Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr Lys
Glu Leu 165 170 175gga ggc cag aat tat gtt ttc tgg ggt ggt cgt gag
gga tat gag aca 633Gly Gly Gln Asn Tyr Val Phe Trp Gly Gly Arg Glu
Gly Tyr Glu Thr180 185 190 195ctt tta aat act gat atg gag ttg gaa
tta gat aat tta gca aga ttc 681Leu Leu Asn Thr Asp Met Glu Leu Glu
Leu Asp Asn Leu Ala Arg Phe 200 205 210tta cac atg gca gta gaa tat
gct cag gaa att ggt ttt gaa gga cag 729Leu His Met Ala Val Glu Tyr
Ala Gln Glu Ile Gly Phe Glu Gly Gln 215 220 225ttc ttg atc gag cct
aaa cca aag gaa cca aca aag cat cag tat gat 777Phe Leu Ile Glu Pro
Lys Pro Lys Glu Pro Thr Lys His Gln Tyr Asp 230 235 240ttc gac gct
gct tct gta cac gcc ttt ttg aag aag tat gat ttg gat 825Phe Asp Ala
Ala Ser Val His Ala Phe Leu Lys Lys Tyr Asp Leu Asp 245 250 255aaa
tac ttt aag ttg aac ata gag gct aat cac gca acg ttg gca ggt 873Lys
Tyr Phe Lys Leu Asn Ile Glu Ala Asn His Ala Thr Leu Ala Gly260 265
270 275cac gat ttt caa cac gaa ttg aga tac gcc cgt att aat aac atg
tta 921His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile Asn Asn Met
Leu 280 285 290ggt tcc ata gat gcc aac atg ggt gac atg ttg ctg ggt
tgg gat act 969Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu Gly
Trp Asp Thr 295 300 305gat caa tac cca acg gat att aga atg aca act
tta gca atg tac gag 1017Asp Gln Tyr Pro Thr Asp Ile Arg Met Thr Thr
Leu Ala Met Tyr Glu 310 315 320gtc att aaa atg gga ggt ttt aac aaa
gga ggt ttg aat ttc gat gct 1065Val Ile Lys Met Gly Gly Phe Asn Lys
Gly Gly Leu Asn Phe Asp Ala 325 330 335aaa gtg cgt cgt gcc tct ttt
gaa cct gaa gac ctt ttt ctt gga cat 1113Lys Val Arg Arg Ala Ser Phe
Glu Pro Glu Asp Leu Phe Leu Gly His340 345 350 355att gcc gga atg
gat gca ttt gca aaa ggt ttc aag gtc gct tat aag 1161Ile Ala Gly Met
Asp Ala Phe Ala Lys Gly Phe Lys Val Ala Tyr Lys 360 365 370ctt gtt
aag gat ggt gta ttt gat aga ttc att gaa gag aga tac aaa 1209Leu Val
Lys Asp Gly Val Phe Asp Arg Phe Ile Glu Glu Arg Tyr Lys 375 380
385tcc tat cgt gaa ggt ata ggt gct gaa atc gtt tca ggt aag gcc aat
1257Ser Tyr Arg Glu Gly Ile Gly Ala Glu Ile Val Ser Gly Lys Ala Asn
390 395 400ttt aag act tta gag gaa tat gca ttg aat aac cca aaa atc
gaa aac 1305Phe Lys Thr Leu Glu Glu Tyr Ala Leu Asn Asn Pro Lys Ile
Glu Asn 405 410 415aaa agc ggt aaa cag gaa ctg ctg gaa tct att ttg
aat caa tat ttg 1353Lys Ser Gly Lys Gln Glu Leu Leu Glu Ser Ile Leu
Asn Gln Tyr Leu420 425 430 435ttc tct gaa tag cctaggctcg aggaattc
1383Phe Ser Glu71386DNAArtificial SequenceArtificial
Thermoanaerobacter thermosulphurigenes xylose isomerase gene
7gaattcctag aaataatttt gtttaacttt aagaaggagg agctagcc atg gcc aag
57 Met Ala Lys 1tat ttt gag aat gtt tcc aag att aag tat gaa ggt cct
aag tca aac 105Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
Lys Ser Asn 5 10 15aac cct tac tcc ttc aaa ttc tat aat cca gaa gaa
gtt ata gac ggt 153Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu
Val Ile Asp Gly20 25 30 35aaa acg atg gag gaa cac tta aga ttt tct
att gct tac tgg cac aca 201Lys Thr Met Glu Glu His Leu Arg Phe Ser
Ile Ala Tyr Trp His Thr 40 45 50ttt act gcc gac ggc aca gac caa ttc
gga aag gct act atg caa aga 249Phe Thr Ala Asp Gly Thr Asp Gln Phe
Gly Lys Ala Thr Met Gln Arg 55 60 65ccg tgg aac cat tac act gat cca
atg gac ata gcc aaa gcc aga gtg 297Pro Trp Asn His Tyr Thr Asp Pro
Met Asp Ile Ala Lys Ala Arg Val 70 75 80gag gct gca ttc gag ttc ttc
gat aag ata aac gcc cct tac ttc tgc 345Glu Ala Ala Phe Glu Phe Phe
Asp Lys Ile Asn Ala Pro Tyr Phe Cys 85 90 95ttc cat gat cgt gac att
gct ccg gaa ggc gat acc ttg aga gaa acc 393Phe His Asp Arg Asp Ile
Ala Pro Glu Gly Asp Thr Leu Arg Glu Thr100 105 110 115aac aaa aat
ctt gac acc att gtc gca atg ata aaa gat tat ttg aag 441Asn Lys Asn
Leu Asp Thr Ile Val Ala Met Ile Lys Asp Tyr Leu Lys 120 125 130acg
tct aaa acc aaa gtt ttg tgg ggt acg gca aac tta ttt tct aat 489Thr
Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu Phe Ser Asn 135 140
145ccg aga ttt gtt cat ggt gct tcc aca tcc tgc aac gca gat gtc ttt
537Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala Asp Val Phe
150 155 160gct tat tcc gct gct caa gtc aaa aag gct ctt gag ata acc
aaa gaa 585Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr
Lys Glu 165 170 175tta ggt ggt gaa aac tac gtt ttc tgg ggt ggc aga
gaa ggt tat gag 633Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg
Glu Gly Tyr Glu180 185 190 195act ctt tta aat aca gat atg gaa ttt
gaa ctt gac aat ttc gca aga 681Thr Leu Leu Asn Thr Asp Met Glu Phe
Glu Leu Asp Asn Phe Ala Arg 200 205 210ttc ttg cac atg gct gtc gat
tat gcc aag gag ata ggt ttt gaa ggc 729Phe Leu His Met Ala Val Asp
Tyr Ala Lys Glu Ile Gly Phe Glu Gly 215 220 225cag ttc ttg att gaa
ccg aaa cca aag gaa cct acc aaa cat cag tat 777Gln Phe Leu Ile Glu
Pro Lys Pro Lys Glu Pro Thr Lys His Gln Tyr 230 235 240gac ttt gat
gtc gca aat gtt ttg gct ttc ttg aga aaa tac gat tta 825Asp Phe Asp
Val Ala Asn Val Leu Ala Phe Leu Arg Lys Tyr Asp Leu 245 250 255gat
aag tat ttc aaa gta aat att gaa gct aat cat gca acg ttg gcc 873Asp
Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala Thr Leu Ala260 265
270 275ttc cat gat ttc cag cac gag ttg aga tac gct aga atc aat gga
gtc 921Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile Asn Gly
Val 280 285 290tta gga tct atc gat gct aat acc ggt gat atg ctg ctg
gga tgg gat 969Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
Gly Trp Asp 295 300 305act gat caa ttt ccg acc gat att cgt atg act
acc ctg gca atg tat 1017Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr
Thr Leu Ala Met Tyr 310 315 320gaa gtt att aag atg ggt gga ttt gat
aaa ggc ggt ctg aac ttc gat 1065Glu Val Ile Lys Met Gly Gly Phe Asp
Lys Gly Gly Leu Asn Phe Asp 325 330 335gca aaa gta aga aga gct tct
ttt gaa cct gaa gat ttg ttt tta gga 1113Ala Lys Val Arg Arg Ala Ser
Phe Glu Pro Glu Asp Leu Phe Leu Gly340 345 350 355cac atc gct ggc
atg gac gca ttt gct aaa ggt ttt aag gta gcc tat 1161His Ile Ala Gly
Met Asp Ala Phe Ala Lys Gly Phe Lys Val Ala Tyr 360 365 370aag ctt
gta aaa gat aga gtg ttc gac aaa ttc atc gaa gag aga tat 1209Lys Leu
Val Lys Asp Arg Val Phe Asp Lys Phe Ile Glu Glu Arg Tyr 375 380
385gct tct tat aag gac gga ata gga gcc gat ata gtt tcc ggt aag gcc
1257Ala Ser Tyr Lys Asp Gly Ile Gly Ala Asp Ile Val Ser Gly Lys Ala
390 395 400gat ttc aga tct ctt gag aaa tac gcc ttg gaa aga tca caa
atc gtg 1305Asp Phe Arg Ser Leu Glu Lys Tyr Ala Leu Glu Arg Ser Gln
Ile Val 405 410 415aac aaa tca ggc cgt caa gaa ttg tta gaa tca att
ctt aat caa tac 1353Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile
Leu Asn Gln Tyr420 425 430 435ctg ttc gct gaa tag cctaggctcg
aggaattc 1386Leu Phe Ala Glu832DNAArtificial SequencePrimer
8tagctagcat gtcgtacttc cccactgtcg ac 32933DNAArtificial
SequencePrimer 9ataagctttc aggtgtagat aaagcggttg acc
33101320DNALactococcus lactis subsp. lactis 10atggcttact ttaacgacat
cgcacctatc aaatacgaag gtacaaaaac taaaaatatg 60tttgcctttc gtcattataa
tccagaagaa gtagttgctg gtaaaacaat ggaagaacaa 120cttcattttg
cccttgcatt ttggcataca attacgatgg atgggtcaga tccctttggg
180ggagcaacaa tggaacgtcc ttgggatttg gaaggtggtt ctgaacttga
ccgtgctcac 240cgtcgagtag atgctttctt tgaaattgct gaaaaattag
gtgttaaata ttattgtttc 300catgatattg atattgcacc tactggaaat
tctttgaaag aattttatgc taatttggac 360gaaattactg accaccttct
tgaaaaacaa aaagcaacag gcattaaatt actttggaat 420acagcaaaca
tgttttcaaa tccccgctat atgaatggtg tttcaacttc taatcgtgct
480gaagtctttg cttatggtgc tgcacaagtt aaaaaaggtc ttgaactttc
taaaaaactc 540ggtggtgaaa attatgtctt ctggggtggt cgtgaaggtt
atgaatcact tttgaataca 600gatatgggtc ttgaaatgga tcatatggca
aaattcttcc atttggcaat tgattatgca 660aaatcaatca accacttgcc
tattttcttg attgaaccaa aaccaaaaga accaatgact 720caccaatatg
attttgactc agcaacagct cttgctttct tgcaaaaata tgacttggac
780aaatacttca aactcaatct tgaaacaaat catgcttggt tggctgggca
cacttttgaa 840cacgaattaa atactgcacg tactttcaat gctttgggtt
ctattgatgc caatcaagga 900aattacttgc ttggttggga tacagatgaa
ttcccaacac ttgttattga tatcacactt 960gcgatgcacc aaattctttt
gaacggtgga cttggcaaag gtggaattaa ctttgatgcg 1020aaagtacgtc
gtacaagttt
caaagcagaa gatttaattc ttgctcatat tgcagggatg 1080gatacttatg
cgcgtgcttt gaaaggtgca gcagcaatca ttgaagataa attcttgtct
1140gatattgttg acgaacgtta tagttcatac aaaaatacag aagttggtca
atccattgaa 1200aatggaacag caacttttga aagtcttgct gcatttgcac
ttgaacatgg tgacgatatt 1260gaacttgatt ctaatcactt ggaatacatc
aaatcagtat tgaatgacta tcttgtttaa 132011439PRTLactococcus lactis
subsp. lactis 11Met Ala Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu
Gly Thr Lys1 5 10 15Thr Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro
Glu Glu Val Val 20 25 30Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe
Ala Leu Ala Phe Trp 35 40 45His Thr Ile Thr Met Asp Gly Ser Asp Pro
Phe Gly Gly Ala Thr Met 50 55 60Glu Arg Pro Trp Asp Leu Glu Gly Gly
Ser Glu Leu Asp Arg Ala His65 70 75 80Arg Arg Val Asp Ala Phe Phe
Glu Ile Ala Glu Lys Leu Gly Val Lys 85 90 95Tyr Tyr Cys Phe His Asp
Ile Asp Ile Ala Pro Thr Gly Asn Ser Leu 100 105 110Lys Glu Phe Tyr
Ala Asn Leu Asp Glu Ile Thr Asp His Leu Leu Glu 115 120 125Lys Gln
Lys Ala Thr Gly Ile Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135
140Phe Ser Asn Pro Arg Tyr Met Asn Gly Val Ser Thr Ser Asn Arg
Ala145 150 155 160Glu Val Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys
Gly Leu Glu Leu 165 170 175Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val
Phe Trp Gly Gly Arg Glu 180 185 190Gly Tyr Glu Ser Leu Leu Asn Thr
Asp Met Gly Leu Glu Met Asp His 195 200 205Met Ala Lys Phe Phe His
Leu Ala Ile Asp Tyr Ala Lys Ser Ile Asn 210 215 220His Leu Pro Ile
Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Met Thr225 230 235 240His
Gln Tyr Asp Phe Asp Ser Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250
255Tyr Asp Leu Asp Lys Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala
260 265 270Trp Leu Ala Gly His Thr Phe Glu His Glu Leu Asn Thr Ala
Arg Thr 275 280 285Phe Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly
Asn Tyr Leu Leu 290 295 300Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu
Val Ile Asp Ile Thr Leu305 310 315 320Ala Met His Gln Ile Leu Leu
Asn Gly Gly Leu Gly Lys Gly Gly Ile 325 330 335Asn Phe Asp Ala Lys
Val Arg Arg Thr Ser Phe Lys Ala Glu Asp Leu 340 345 350Ile Leu Ala
His Ile Ala Gly Met Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365Gly
Ala Ala Ala Ile Ile Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375
380Glu Arg Tyr Ser Ser Tyr Lys Asn Thr Glu Val Gly Gln Ser Ile
Glu385 390 395 400Asn Gly Thr Ala Thr Phe Glu Ser Leu Ala Ala Phe
Ala Leu Glu His 405 410 415Gly Asp Asp Ile Glu Leu Asp Ser Asn His
Leu Glu Tyr Ile Lys Ser 420 425 430Val Leu Asn Asp Tyr Leu Val
435121320DNALactococcus lactis subsp. lactis 12atggcttact
ttaacgacat cgcacctatc aaatacgaag gtacaaaaac taaaaatatg 60tttgcctttc
gccattataa tccagaagaa gtagttgctg gtaaaacaat ggaagaacaa
120cttcattttg cccttgcatt ttggcataca attacaatgg atgggtcaga
cccctttggg 180ggagcaacaa tggaacgtcc ttgggatttg gaaggtggtt
ctgaacttga ccgtgctcac 240cgtcgagtag atgctttctt tgaaattgct
gaaaaattag gtgttaaata ttattgtttc 300catgatattg atattgcacc
tactggaaat tctttgaaag aattttatgc taatttggac 360gaaattactg
accaccttct tgaaaaacaa aaagcaacag ggattaaatt actttggaat
420acagcaaaca tgttttcaaa tccccgctat atgaatggtg tttcaacttc
taaccgtgct 480gaagtctttg cttatggtgc tgcacaagtt aaaaaaggtc
ttgaactttc taaaaaactc 540ggtggtgaaa attacgtctt ctggggtggt
cgtgaaggtt atgaatcact tttgaataca 600gatatgggtc ttgaaatgga
tcatatggca aaattcttcc atttggcaat tgattatgca 660aaatcaatca
accacttgcc cattttcttg atcgaaccaa aaccaaaaga accaatgact
720caccaatatg attttgactc agcaacagct cttgctttct tgcaaaaata
tgatttggac 780aaatatttca aactcaatct tgaaacaaat catgcttggt
tggctggaca cacttttgaa 840cacgaattaa atactgctcg tactttcaat
gctttgggtt ctattgatgc caatcaagga 900aattacttgc ttggttggga
tacagatgaa ttcccaacac ttgttattga tatcacactt 960gcgatgcacc
aaattctttt gaacggtgga cttggcaaag gtggaattaa ctttgatgcg
1020aaagtacgtc gtacaagttt caaagcagaa gatttaattc ttgctcatat
tgcagggatg 1080gatacttatg cgcgtgcttt gaaaggtgca gcagcaatca
ttgaagataa attcttgtct 1140gatattgttg acgaacgtta tagttcatac
aaaaatacag aagttggaca atccattgaa 1200aatggaacag caacttttaa
aagtcttgcc gcatttgcac ttgaacatgg tgacgatatt 1260gaacttgatt
ctaatcactt ggaatacatc aaatcagtat tgaatgacta tcttgtttaa
132013439PRTLactococcus lactis subsp. lactis 13Met Ala Tyr Phe Asn
Asp Ile Ala Pro Ile Lys Tyr Glu Gly Thr Lys1 5 10 15Thr Lys Asn Met
Phe Ala Phe Arg His Tyr Asn Pro Glu Glu Val Val 20 25 30Ala Gly Lys
Thr Met Glu Glu Gln Leu His Phe Ala Leu Ala Phe Trp 35 40 45His Thr
Ile Thr Met Asp Gly Ser Asp Pro Phe Gly Gly Ala Thr Met 50 55 60Glu
Arg Pro Trp Asp Leu Glu Gly Gly Ser Glu Leu Asp Arg Ala His65 70 75
80Arg Arg Val Asp Ala Phe Phe Glu Ile Ala Glu Lys Leu Gly Val Lys
85 90 95Tyr Tyr Cys Phe His Asp Ile Asp Ile Ala Pro Thr Gly Asn Ser
Leu 100 105 110Lys Glu Phe Tyr Ala Asn Leu Asp Glu Ile Thr Asp His
Leu Leu Glu 115 120 125Lys Gln Lys Ala Thr Gly Ile Lys Leu Leu Trp
Asn Thr Ala Asn Met 130 135 140Phe Ser Asn Pro Arg Tyr Met Asn Gly
Val Ser Thr Ser Asn Arg Ala145 150 155 160Glu Val Phe Ala Tyr Gly
Ala Ala Gln Val Lys Lys Gly Leu Glu Leu 165 170 175Ser Lys Lys Leu
Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu 180 185 190Gly Tyr
Glu Ser Leu Leu Asn Thr Asp Met Gly Leu Glu Met Asp His 195 200
205Met Ala Lys Phe Phe His Leu Ala Ile Asp Tyr Ala Lys Ser Ile Asn
210 215 220His Leu Pro Ile Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro
Met Thr225 230 235 240His Gln Tyr Asp Phe Asp Ser Ala Thr Ala Leu
Ala Phe Leu Gln Lys 245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Leu
Asn Leu Glu Thr Asn His Ala 260 265 270Trp Leu Ala Gly His Thr Phe
Glu His Glu Leu Asn Thr Ala Arg Thr 275 280 285Phe Asn Ala Leu Gly
Ser Ile Asp Ala Asn Gln Gly Asn Tyr Leu Leu 290 295 300Gly Trp Asp
Thr Asp Glu Phe Pro Thr Leu Val Ile Asp Ile Thr Leu305 310 315
320Ala Met His Gln Ile Leu Leu Asn Gly Gly Leu Gly Lys Gly Gly Ile
325 330 335Asn Phe Asp Ala Lys Val Arg Arg Thr Ser Phe Lys Ala Glu
Asp Leu 340 345 350Ile Leu Ala His Ile Ala Gly Met Asp Thr Tyr Ala
Arg Ala Leu Lys 355 360 365Gly Ala Ala Ala Ile Ile Glu Asp Lys Phe
Leu Ser Asp Ile Val Asp 370 375 380Glu Arg Tyr Ser Ser Tyr Lys Asn
Thr Glu Val Gly Gln Ser Ile Glu385 390 395 400Asn Gly Thr Ala Thr
Phe Lys Ser Leu Ala Ala Phe Ala Leu Glu His 405 410 415Gly Asp Asp
Ile Glu Leu Asp Ser Asn His Leu Glu Tyr Ile Lys Ser 420 425 430Val
Leu Asn Asp Tyr Leu Val 43514439PRTLactococcus lactis subsp. lactis
14Met Ala Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu Gly Thr Lys1
5 10 15Thr Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro Glu Glu Val
Val 20 25 30Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe Ala Leu Ala
Phe Trp 35 40 45His Thr Ile Thr Met Asp Gly Ser Asp Pro Phe Gly Gly
Ala Thr Met 50 55 60Glu Arg Pro Trp Asp Leu Glu Gly Gly Ser Glu Leu
Asp Arg Ala His65 70 75 80Arg Arg Val Asp Ala Phe Phe Glu Ile Ala
Glu Lys Leu Gly Val Lys 85 90 95Tyr Tyr Cys Phe His Asp Ile Asp Ile
Ala Pro Thr Gly Asn Ser Leu 100 105 110Lys Glu Phe Tyr Ala Asn Leu
Asp Glu Ile Thr Asp His Leu Leu Glu 115 120 125Lys Gln Lys Ala Thr
Gly Ile Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135 140Phe Ser Asn
Pro Arg Tyr Met Asn Gly Val Ser Thr Ser Asn Arg Ala145 150 155
160Glu Val Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys Gly Leu Glu Leu
165 170 175Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly
Arg Glu 180 185 190Gly Tyr Glu Ser Leu Leu Asn Thr Asp Met Gly Leu
Glu Met Asp His 195 200 205Met Ala Lys Phe Phe His Leu Ala Ile Asp
Tyr Ala Lys Ser Ile Asn 210 215 220His Leu Pro Ile Phe Leu Ile Glu
Pro Lys Pro Lys Glu Pro Met Thr225 230 235 240His Gln Tyr Asp Phe
Asp Ser Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250 255Tyr Asp Leu
Asp Lys Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala 260 265 270Trp
Leu Ala Gly His Thr Phe Glu His Glu Leu Asn Thr Ala Arg Thr 275 280
285Phe Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asn Tyr Leu Leu
290 295 300Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu Val Ile Asp Ile
Thr Leu305 310 315 320Ala Met His Gln Ile Leu Leu Asn Gly Gly Leu
Gly Lys Gly Gly Ile 325 330 335Asn Phe Asp Ala Lys Val Arg Arg Thr
Ser Phe Lys Ala Glu Asp Leu 340 345 350Ile Leu Ala His Ile Ala Gly
Met Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365Gly Ala Ala Ala Ile
Ile Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375 380Glu Arg Tyr
Ser Ser Tyr Lys Asn Thr Glu Val Gly Gln Ser Ile Glu385 390 395
400Asn Gly Thr Ala Thr Phe Glu Ser Leu Ala Ala Phe Ala Leu Glu Tyr
405 410 415Gly Asp Asp Ile Glu Leu Asp Ser Asn His Leu Glu Tyr Ile
Lys Ser 420 425 430Val Leu Asn Asp Tyr Leu Val
43515438PRTArtificial SequenceSynthetic Construct 15Met Glu Tyr Phe
Lys Asn Val Pro Gln Ile Lys Tyr Glu Gly Pro Lys1 5 10 15Ser Asn Asn
Pro Tyr Ala Phe Lys Phe Tyr Asn Pro Asp Glu Ile Ile 20 25 30Asp Gly
Lys Pro Leu Lys Glu His Leu Arg Phe Ser Val Ala Tyr Trp 35 40 45His
Thr Phe Thr Ala Asn Gly Thr Asp Pro Phe Gly Ala Pro Thr Met 50 55
60 Gln Arg Pro Trp Asp His Phe Thr Asp Pro Met Asp Ile Ala Lys
Ala65 70 75 80Arg Val Glu Ala Ala Phe Glu Leu Phe Glu Lys Leu Asp
Val Pro Phe 85 90 95Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly
Glu Thr Leu Arg 100 105 110Glu Thr Asn Lys Asn Leu Asp Thr Ile Val
Ala Met Ile Lys Asp Tyr 115 120 125Leu Lys Thr Ser Lys Thr Lys Val
Leu Trp Gly Thr Ala Asn Leu Phe 130 135 140Ser Asn Pro Arg Phe Val
His Gly Ala Ala Thr Ser Cys Asn Ala Asp145 150 155 160Val Phe Ala
Tyr Ala Ala Ala Gln Val Lys Lys Ala Leu Glu Ile Thr 165 170 175Lys
Glu Leu Gly Gly Gln Asn Tyr Val Phe Trp Gly Gly Arg Glu Gly 180 185
190Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn Leu
195 200 205Ala Arg Phe Leu His Met Ala Val Glu Tyr Ala Gln Glu Ile
Gly Phe 210 215 220Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu
Pro Thr Lys His225 230 235 240Gln Tyr Asp Phe Asp Ala Ala Ser Val
His Ala Phe Leu Lys Lys Tyr 245 250 255Asp Leu Asp Lys Tyr Phe Lys
Leu Asn Ile Glu Ala Asn His Ala Thr 260 265 270Leu Ala Gly His Asp
Phe Gln His Glu Leu Arg Tyr Ala Arg Ile Asn 275 280 285Asn Met Leu
Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu Gly 290 295 300Trp
Asp Thr Asp Gln Tyr Pro Thr Asp Ile Arg Met Thr Thr Leu Ala305 310
315 320Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asn Lys Gly Gly Leu
Asn 325 330 335Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu
Asp Leu Phe 340 345 350Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala
Lys Gly Phe Lys Val 355 360 365Ala Tyr Lys Leu Val Lys Asp Gly Val
Phe Asp Arg Phe Ile Glu Glu 370 375 380 Arg Tyr Lys Ser Tyr Arg Glu
Gly Ile Gly Ala Glu Ile Val Ser Gly385 390 395 400Lys Ala Asn Phe
Lys Thr Leu Glu Glu Tyr Ala Leu Asn Asn Pro Lys 405 410 415Ile Glu
Asn Lys Ser Gly Lys Gln Glu Leu Leu Glu Ser Ile Leu Asn 420 425
430Gln Tyr Leu Phe Ser Glu 43516439PRTArtificial SequenceSynthetic
Construct 16Met Ala Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu
Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro
Glu Glu Val 20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe
Ser Ile Ala Tyr 35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln
Phe Gly Lys Ala Thr 50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp
Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu
Phe Phe Asp Lys Ile Asn Ala Pro 85 90 95Tyr Phe Cys Phe His Asp Arg
Asp Ile Ala Pro Glu Gly Asp Thr Leu 100 105 110Arg Glu Thr Asn Lys
Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp 115 120 125Tyr Leu Lys
Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu 130 135 140Phe
Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150
155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu
Ile 165 170 175Thr Lys Glu Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly
Gly Arg Glu 180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu
Phe Glu Leu Asp Asn 195 200 205Phe Ala Arg Phe Leu His Met Ala Val
Asp Tyr Ala Lys Glu Ile Gly 210 215 220Phe Glu Gly Gln Phe Leu Ile
Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp
Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys 245 250 255Tyr Asp
Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala 260 265
270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met
Leu Leu 290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg
Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly
Gly Phe Asp Lys Gly Gly Leu 325 330 335Asn Phe Asp Ala Lys Val Arg
Arg Ala Ser Phe Glu Pro Glu Asp Leu 340 345 350Phe Leu Gly His Ile
Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys 355 360 365Val Ala Tyr
Lys Leu Val Lys Asp Arg Val Phe Asp Lys Phe Ile Glu 370 375 380Glu
Arg Tyr Ala Ser Tyr Lys Asp Gly Ile Gly Ala Asp Ile Val Ser385 390
395 400Gly Lys Ala Asp Phe Arg Ser Leu Glu Lys Tyr Ala Leu Glu Arg
Ser 405 410 415Gln Ile Val Asn Lys Ser Gly
Arg Gln Glu Leu Leu Glu Ser Ile Leu 420 425 430Asn Gln Tyr Leu Phe
Ala Glu 435171320DNALactococcus lactis 17atggcttact ttaacgacat
cgcacctatc aaatacgaag gtacaaaaac taaaaatatg 60tttgcctttc gccattataa
tccagaagaa gtagttgctg gtaaaacaat ggaagaacaa 120cttcattttg
cccttgcatt ttggcataca attacaatgg atgggtcaga tccctttggg
180ggagcaacaa tggaacgccc ttgggatttg gaaggtggtt ctgaacttga
ccgtgctcac 240cgtcgagtag atgctttctt tgaaattgct gaaaaattag
gtgttaaata ttattgtttc 300catgatattg atattgcacc tactggaaat
tctttgaaag aattttatgc taatttggac 360gaaattactg accaccttct
tgaaaaacaa aaagcaacag ggattaaatt actttggaat 420acagcaaaca
tgttttcaaa tccccgctat atgaatggtg tttcaacttc taatcgtgct
480gaagtctttg cttatggtgc tgcacaagtt aaaaaaggtc ttgaactttc
taaaaaactc 540ggtggtgaaa attacgtctt ctggggtggt cgtgaaggtt
atgaatcact tttgaataca 600gatatgggtc ttgaaatgga tcatatggca
aaattcttcc atttggcaat tgattatgca 660aaatcaatca accacttgcc
cattttcttg attgaaccaa aaccaaaaga accaatgact 720caccaatatg
attttgactc agcaacagct cttgctttct tgcaaaaata tgatttggac
780aaatatttca aactcaatct tgaaacaaat catgcttggt tggctggaca
cacttttgaa 840cacgaattaa atactgctcg tactttcaat gctttgggtt
ctattgatgc caatcaagga 900aattacttgc ttggttggga tacagatgaa
ttcccaacac ttgttattga tatcacactt 960gcgatgcacc aaattctttt
gaacggtgga cttggcaaag gtggaattaa ctttgatgcg 1020aaagtacgtc
gtacaagttt caaagcagaa gatttaattc ttgctcatat tgcagggatg
1080gatacttatg cgcgtgcttt gaaaggtgca gcagcaatca ttgaagataa
attcttgtct 1140gatattgttg acgaacgtta tagttcatac agaaatacag
aagttggtca atccattgaa 1200aatggaacag caacttttga aagtcttgcc
gcatttgcac ttgaatatgg tgatgatatt 1260gaacttgatt ctaatcactt
ggaatacatc aaatcagtat tgaatgacta tcttgtttaa 132018439PRTLactococcus
lactis 18Met Ala Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu Gly
Thr Lys1 5 10 15Thr Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro Glu
Glu Val Val 20 25 30Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe Ala
Leu Ala Phe Trp 35 40 45His Thr Ile Thr Met Asp Gly Ser Asp Pro Phe
Gly Gly Ala Thr Met 50 55 60Glu Arg Pro Trp Asp Leu Glu Gly Gly Ser
Glu Leu Asp Arg Ala His65 70 75 80Arg Arg Val Asp Ala Phe Phe Glu
Ile Ala Glu Lys Leu Gly Val Lys 85 90 95Tyr Tyr Cys Phe His Asp Ile
Asp Ile Ala Pro Thr Gly Asn Ser Leu 100 105 110Lys Glu Phe Tyr Ala
Asn Leu Asp Glu Ile Thr Asp His Leu Leu Glu 115 120 125Lys Gln Lys
Ala Thr Gly Ile Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135 140Phe
Ser Asn Pro Arg Tyr Met Asn Gly Val Ser Thr Ser Asn Arg Ala145 150
155 160Glu Val Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys Gly Leu Glu
Leu 165 170 175Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly
Gly Arg Glu 180 185 190Gly Tyr Glu Ser Leu Leu Asn Thr Asp Met Gly
Leu Glu Met Asp His 195 200 205Met Ala Lys Phe Phe His Leu Ala Ile
Asp Tyr Ala Lys Ser Ile Asn 210 215 220His Leu Pro Ile Phe Leu Ile
Glu Pro Lys Pro Lys Glu Pro Met Thr225 230 235 240His Gln Tyr Asp
Phe Asp Ser Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250 255Tyr Asp
Leu Asp Lys Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala 260 265
270Trp Leu Ala Gly His Thr Phe Glu His Glu Leu Asn Thr Ala Arg Thr
275 280 285Phe Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asn Tyr
Leu Leu 290 295 300Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu Val Ile
Asp Ile Thr Leu305 310 315 320Ala Met His Gln Ile Leu Leu Asn Gly
Gly Leu Gly Lys Gly Gly Ile 325 330 335Asn Phe Asp Ala Lys Val Arg
Arg Thr Ser Phe Lys Ala Glu Asp Leu 340 345 350Ile Leu Ala His Ile
Ala Gly Met Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365Gly Ala Ala
Ala Ile Ile Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375 380Glu
Arg Tyr Ser Ser Tyr Arg Asn Thr Glu Val Gly Gln Ser Ile Glu385 390
395 400Asn Gly Thr Ala Thr Phe Glu Ser Leu Ala Ala Phe Ala Leu Glu
Tyr 405 410 415Gly Asp Asp Ile Glu Leu Asp Ser Asn His Leu Glu Tyr
Ile Lys Ser 420 425 430Val Leu Asn Asp Tyr Leu Val
43519439PRTArtificial SequenceVariant of Lactococcus xylose
isomerase 19Met Ala Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu Gly
Thr Lys1 5 10 15Thr Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro Glu
Glu Val Val 20 25 30Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe Ala
Leu Ala Phe Trp 35 40 45His Thr Ile Thr Met Asp Gly Ser Asp Pro Phe
Gly Gly Ala Thr Met 50 55 60Glu Arg Pro Trp Asp Leu Glu Gly Gly Ser
Glu Leu Asp Arg Ala His65 70 75 80Arg Arg Val Asp Ala Phe Phe Glu
Ile Ala Glu Lys Leu Gly Val Lys 85 90 95Tyr Tyr Cys Phe His Asp Ile
Asp Ile Ala Pro Thr Gly Asn Ser Leu 100 105 110Lys Glu Phe Tyr Ala
Asn Leu Asp Glu Ile Thr Asp His Leu Leu Glu 115 120 125Lys Gln Lys
Ala Thr Gly Ile Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135 140Phe
Ser Asn Pro Arg Tyr Met Asn Gly Val Ser Thr Ser Asn Arg Ala145 150
155 160Glu Val Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys Gly Leu Glu
Leu 165 170 175Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly
Gly Arg Glu 180 185 190Gly Tyr Glu Ser Leu Leu Asn Thr Asp Met Gly
Leu Glu Met Asp His 195 200 205Met Ala Lys Phe Phe His Leu Ala Ile
Asp Tyr Ala Lys Ser Ile Asn 210 215 220His Leu Pro Ile Phe Leu Ile
Glu Pro Lys Pro Lys Glu Pro Met Thr225 230 235 240His Gln Tyr Asp
Phe Asp Ser Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250 255Tyr Asp
Leu Asp Lys Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala 260 265
270Trp Leu Ala Gly His Thr Phe Glu His Glu Leu Asn Thr Ala Arg Thr
275 280 285Phe Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asn Tyr
Leu Leu 290 295 300Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu Val Ile
Asp Ile Thr Leu305 310 315 320Ala Met His Gln Ile Leu Leu Asn Gly
Gly Leu Gly Lys Gly Gly Ile 325 330 335Asn Phe Asp Ala Lys Val Arg
Arg Thr Ser Phe Lys Ala Glu Asp Leu 340 345 350Ile Leu Ala His Ile
Ala Gly Met Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365Gly Ala Ala
Ala Ile Ile Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375 380Glu
Arg Tyr Ser Ser Tyr Arg Asn Thr Glu Val Gly Gln Ser Ile Glu385 390
395 400Asn Gly Thr Ala Thr Phe Lys Ser Leu Ala Ala Phe Ala Leu Glu
His 405 410 415Gly Asp Asp Ile Glu Leu Asp Ser Asn His Leu Glu Tyr
Ile Lys Ser 420 425 430Val Leu Asn Asp Tyr Leu Val
43520439PRTArtificial SequenceVariant of Lactococcus xylose
isomerase 20Met Ala Tyr Phe Asn Asp Ile Ala Pro Ile Lys Tyr Glu Gly
Thr Lys1 5 10 15Thr Lys Asn Met Phe Ala Phe Arg His Tyr Asn Pro Glu
Glu Val Val 20 25 30Ala Gly Lys Thr Met Glu Glu Gln Leu His Phe Ala
Leu Ala Phe Trp 35 40 45His Thr Ile Thr Met Asp Gly Ser Asp Pro Phe
Gly Gly Ala Thr Met 50 55 60Glu Arg Pro Trp Asp Leu Glu Gly Gly Ser
Glu Leu Asp Arg Ala His65 70 75 80Arg Arg Val Asp Ala Phe Phe Glu
Ile Ala Glu Lys Leu Gly Val Lys 85 90 95Tyr Tyr Cys Phe His Asp Ile
Asp Ile Ala Pro Thr Gly Asn Ser Leu 100 105 110Lys Glu Phe Tyr Ala
Asn Leu Asp Glu Ile Thr Asp His Leu Leu Glu 115 120 125Lys Gln Lys
Ala Thr Gly Ile Lys Leu Leu Trp Asn Thr Ala Asn Met 130 135 140Phe
Ser Asn Pro Arg Tyr Met Asn Gly Val Ser Thr Ser Asn Arg Ala145 150
155 160Glu Val Phe Ala Tyr Gly Ala Ala Gln Val Lys Lys Gly Leu Glu
Leu 165 170 175Ser Lys Lys Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly
Gly Arg Glu 180 185 190Gly Tyr Glu Ser Leu Leu Asn Thr Asp Met Gly
Leu Glu Met Asp His 195 200 205Met Ala Lys Phe Phe His Leu Ala Ile
Asp Tyr Ala Lys Ser Ile Asn 210 215 220His Leu Pro Ile Phe Leu Ile
Glu Pro Lys Pro Lys Glu Pro Met Thr225 230 235 240His Gln Tyr Asp
Phe Asp Ser Ala Thr Ala Leu Ala Phe Leu Gln Lys 245 250 255Tyr Asp
Leu Asp Lys Tyr Phe Lys Leu Asn Leu Glu Thr Asn His Ala 260 265
270Trp Leu Ala Gly His Thr Phe Glu His Glu Leu Asn Thr Ala Arg Thr
275 280 285Phe Asn Ala Leu Gly Ser Ile Asp Ala Asn Gln Gly Asn Tyr
Leu Leu 290 295 300Gly Trp Asp Thr Asp Glu Phe Pro Thr Leu Val Ile
Asp Ile Thr Leu305 310 315 320Ala Met His Gln Ile Leu Leu Asn Gly
Gly Leu Gly Lys Gly Gly Ile 325 330 335Asn Phe Asp Ala Lys Val Arg
Arg Thr Ser Phe Lys Ala Glu Asp Leu 340 345 350Ile Leu Ala His Ile
Ala Gly Met Asp Thr Tyr Ala Arg Ala Leu Lys 355 360 365Gly Ala Ala
Ala Ile Ile Glu Asp Lys Phe Leu Ser Asp Ile Val Asp 370 375 380Glu
Arg Tyr Ser Ser Tyr Arg Asn Thr Glu Val Gly Gln Ser Ile Glu385 390
395 400Asn Gly Thr Ala Thr Phe Lys Ser Leu Ala Ala Phe Ala Leu Glu
Tyr 405 410 415Gly Asp Asp Ile Glu Leu Asp Ser Asn His Leu Glu Tyr
Ile Lys Ser 420 425 430Val Leu Asn Asp Tyr Leu Val
4352141DNAArtificial SequencePrimer 21gttgacgaac gatatagttc
atacaaaaat acagaagttg g 412241DNAArtificial SequencePrimer
22ccaacttctg tatttttgta tgaactatat cgttcgtcaa c 412355DNAArtificial
SequencePrimer 23acagcaactt ttaaaagctt agccgcattt gcacttgaat
atggtgatga tattg 552455DNAArtificial SequencePrimer 24caatatcatc
accatattca agtgcaaatg cggctaagct tttaaaagtt gctgt
552555DNAArtificial SequencePrimer 25acagcaactt ttaaaagctt
agccgcattt gcacttgaac atggtgatga tattg 552655DNAArtificial
SequencePrimer 26caatatcatc accatgttca agtgcaaatg cggctaagct
tttaaaagtt gctgt 55271320DNAArtificial SequenceVariant of
Lactococcus xylose isomerase 27atggcttact ttaacgacat cgcacctatc
aaatacgaag gtacaaaaac taaaaatatg 60tttgcctttc gccattataa tccagaagaa
gtagttgctg gtaaaacaat ggaagaacaa 120cttcattttg cccttgcatt
ttggcataca attacaatgg atgggtcaga tccctttggg 180ggagcaacaa
tggaacgccc ttgggatttg gaaggtggtt ctgaacttga ccgtgctcac
240cgtcgagtag atgctttctt tgaaattgct gaaaaattag gtgttaaata
ttattgtttc 300catgatattg atattgcacc tactggaaat tctttgaaag
aattttatgc taatttggac 360gaaattactg accaccttct tgaaaaacaa
aaagcaacag ggattaaatt actttggaat 420acagcaaaca tgttttcaaa
tccccgctat atgaatggtg tttcaacttc taatcgtgct 480gaagtctttg
cttatggtgc tgcacaagtt aaaaaaggtc ttgaactttc taaaaaactc
540ggtggtgaaa attacgtctt ctggggtggt cgtgaaggtt atgaatcact
tttgaataca 600gatatgggtc ttgaaatgga tcatatggca aaattcttcc
atttggcaat tgattatgca 660aaatcaatca accacttgcc cattttcttg
attgaaccaa aaccaaaaga accaatgact 720caccaatatg attttgactc
agcaacagct cttgctttct tgcaaaaata tgatttggac 780aaatatttca
aactcaatct tgaaacaaat catgcttggt tggctggaca cacttttgaa
840cacgaattaa atactgctcg tactttcaat gctttgggtt ctattgatgc
caatcaagga 900aattacttgc ttggttggga tacagatgaa ttcccaacac
ttgttattga tatcacactt 960gcgatgcacc aaattctttt gaacggtgga
cttggcaaag gtggaattaa ctttgatgcg 1020aaagtacgtc gtacaagttt
caaagcagaa gatttaattc ttgctcatat tgcagggatg 1080gatacttatg
cgcgtgcttt gaaaggtgca gcagcaatca ttgaagataa attcttgtct
1140gatattgttg acgaacgtta tagttcatac agaaatacag aagttggtca
atccattgaa 1200aatggaacag caacttttaa aagcttagcc gcatttgcac
ttgaacatgg tgatgatatt 1260gaacttgatt ctaatcactt ggaatacatc
aaatcagtat tgaatgacta tcttgtttaa 1320281320DNAArtificial
SequenceVariant of Lactococcus xylose isomerase 28atggcttact
ttaacgacat cgcacctatc aaatacgaag gtacaaaaac taaaaatatg 60tttgcctttc
gccattataa tccagaagaa gtagttgctg gtaaaacaat ggaagaacaa
120cttcattttg cccttgcatt ttggcataca attacaatgg atgggtcaga
tccctttggg 180ggagcaacaa tggaacgccc ttgggatttg gaaggtggtt
ctgaacttga ccgtgctcac 240cgtcgagtag atgctttctt tgaaattgct
gaaaaattag gtgttaaata ttattgtttc 300catgatattg atattgcacc
tactggaaat tctttgaaag aattttatgc taatttggac 360gaaattactg
accaccttct tgaaaaacaa aaagcaacag ggattaaatt actttggaat
420acagcaaaca tgttttcaaa tccccgctat atgaatggtg tttcaacttc
taatcgtgct 480gaagtctttg cttatggtgc tgcacaagtt aaaaaaggtc
ttgaactttc taaaaaactc 540ggtggtgaaa attacgtctt ctggggtggt
cgtgaaggtt atgaatcact tttgaataca 600gatatgggtc ttgaaatgga
tcatatggca aaattcttcc atttggcaat tgattatgca 660aaatcaatca
accacttgcc cattttcttg attgaaccaa aaccaaaaga accaatgact
720caccaatatg attttgactc agcaacagct cttgctttct tgcaaaaata
tgatttggac 780aaatatttca aactcaatct tgaaacaaat catgcttggt
tggctggaca cacttttgaa 840cacgaattaa atactgctcg tactttcaat
gctttgggtt ctattgatgc caatcaagga 900aattacttgc ttggttggga
tacagatgaa ttcccaacac ttgttattga tatcacactt 960gcgatgcacc
aaattctttt gaacggtgga cttggcaaag gtggaattaa ctttgatgcg
1020aaagtacgtc gtacaagttt caaagcagaa gatttaattc ttgctcatat
tgcagggatg 1080gatacttatg cgcgtgcttt gaaaggtgca gcagcaatca
ttgaagataa attcttgtct 1140gatattgttg acgaacgtta tagttcatac
agaaatacag aagttggtca atccattgaa 1200aatggaacag caacttttaa
aagcttagcc gcatttgcac ttgaatatgg tgatgatatt 1260gaacttgatt
ctaatcactt ggaatacatc aaatcagtat tgaatgacta tcttgtttaa 1320
* * * * *