U.S. patent application number 14/120176 was filed with the patent office on 2014-08-28 for method for production of isoprenoid compounds.
The applicant listed for this patent is Richard Burlingame, Bryan Julien. Invention is credited to Richard Burlingame, Bryan Julien.
Application Number | 20140242658 14/120176 |
Document ID | / |
Family ID | 41669654 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140242658 |
Kind Code |
A1 |
Julien; Bryan ; et
al. |
August 28, 2014 |
Method for production of isoprenoid compounds
Abstract
The present invention is directed to variant squalene synthase
enzymes, including Saccharomyces cerevisiae squalene synthase
enzymes, and to nucleic acid molecules encoding these variant
enzymes. These variant enzymes produce squalene at a lower rate
than the wild-type enzyme, allowing more farnesyl pyrophosphate to
be utilized for production of isoprenoid compounds, while still
producing sufficient squalene to allow the S. cerevisiae cells to
grow without the requirement for supplementation by sterols such as
ergosterol. These variant enzymes, therefore, are highly suitable
for the efficient production of isoprenoids.
Inventors: |
Julien; Bryan; (Lexington,
KY) ; Burlingame; Richard; (Nicholasville,
KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Julien; Bryan
Burlingame; Richard |
Lexington
Nicholasville |
KY
KY |
US
US |
|
|
Family ID: |
41669654 |
Appl. No.: |
14/120176 |
Filed: |
May 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13986436 |
May 1, 2013 |
8753842 |
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14120176 |
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12540094 |
Aug 12, 2009 |
8481286 |
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13986436 |
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12540050 |
Aug 12, 2009 |
8609371 |
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13986436 |
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61088288 |
Aug 12, 2008 |
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61088288 |
Aug 12, 2008 |
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Current U.S.
Class: |
435/166 ;
435/193; 435/252.33; 435/254.21; 435/320.1; 435/471; 506/10;
536/23.2 |
Current CPC
Class: |
C12N 15/1079 20130101;
C12P 5/002 20130101; C12P 5/007 20130101; C12N 9/1085 20130101 |
Class at
Publication: |
435/166 ;
536/23.2; 435/320.1; 435/254.21; 435/252.33; 435/193; 506/10;
435/471 |
International
Class: |
C12N 9/10 20060101
C12N009/10; C12N 15/10 20060101 C12N015/10; C12P 5/00 20060101
C12P005/00 |
Claims
1. A nucleic acid molecule that encodes a variant squalene synthase
enzyme, wherein the variant enzyme comprises a sequence of amino
acid residues that is at least 95% identical to the squalene
synthase enzyme whose sequence is set forth in SEQ ID NO: 4,
whereby: the variant enzyme exhibits reduced cellular squalene
synthase activity compared to the enzyme whose sequence is set
forth in SEQ ID NO: 4; the variant enzyme, when present and
expressed in vivo in a eukaryotic microbial host as the only
squalene synthase species, catalyzes the synthesis of squalene at a
sufficiently high rate such that supplementation of the eukaryotic
microbial host with a sterol is not required for growth; and the
eukaryotic microbial host produces a terpene product in medium
lacking a sterol.
2. The nucleic acid molecule of claim 1, wherein the eukaryotic
microbial host is a fungal microbial host.
3. The nucleic acid molecule of claim 2, wherein the fungal
microbial host is a yeast microbial host.
4. The nucleic acid molecule of claim 1, wherein the microbial host
is selected from among Saccharomyces, Zygosaccharomyces,
Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and
Torulopsis.
5. The nucleic acid molecule of claim 4, wherein the yeast
microbial host is Saccharomyces cerevisiae.
6. The isolated nucleic acid molecule of claim 1, wherein the
encoded variant squalene synthase enzyme comprises a sequence that
has at least 97.5% sequence identity with the sequence of amino
acids set forth in SEQ ID NO: 4.
7. The isolated nucleic acid molecule of claim 1, wherein the
encoded variant squalene synthase enzyme comprises a sequence that
has at least 99% sequence identity with the sequence of amino acids
set forth in SEQ ID NO: 4.
8. A vector, comprising the nucleic acid molecule of claim 1.
9. A host cell, comprising the nucleic acid molecule of claim 1,
wherein the host cell is a prokaryotic cell.
10. An isolated host cell, comprising the nucleic acid molecule of
claim 1, wherein the host cell is a eukaryotic cell.
11. A host cell, comprising the nucleic acid molecule of claim 1,
wherein the host cell is a yeast host cell.
12. The host cell of claim 10 that is selected from among
Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida,
Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis.
13. The host cell of claim 12 that is a Saccharomyces cerevisiae
cell.
14. A variant squalene synthase enzyme, comprising a sequence of
amino acid residues that is at least 95% identical to the squalene
synthase enzyme whose sequence is set forth in SEQ ID NO: 4,
whereby: the variant enzyme exhibits reduced cellular squalene
synthase activity compared to the wild-type enzyme, but when
present and expressed in vivo in a eukaryotic microbial host as the
only squalene synthase species, catalyzes the synthesis of squalene
at a sufficiently high rate that supplementation of the eukaryotic
microbial host with a sterol is not required for growth; the host
produces a terpene product in medium lacking a sterol; and the
eukaryotic microbial host is a yeast microbial host.
15. The variant squalene synthase enzyme of claim 14, wherein the
yeast microbial host is Saccharomyces cerevisiae.
16. A method for producing a terpene, comprising culturing a cell
of claim 9 under conditions for production of a terpene
product.
17. A method of isolating a defective ERG9 gene that permits growth
of a host in sterol-free medium and production of terpenes in
sterol-free medium, comprising: (a) isolating a wild-type ERG9 gene
to produce an isolated wild-type ERG9 gene; (b) subjecting the
isolated wild-type ERG9 gene to mutagenesis to generate a pool of
erg9 mutants; (c) transforming mutants from the pool of erg9
mutants generated in step (b) into a strain of a eukaryotic
microbial host that contains an expressed terpene synthase gene
that produces a detectable and measurable terpene product, the
strain of the eukaryotic microbial host being transformed in such a
manner that replacement of the preexisting ERG9 allele with an erg9
mutation allows the strain to grow in a sterol-free medium; (d)
growing the transformants in sterol-free medium; and (e) isolating
a transformant from step (d) that grows in sterol-free medium and
produces a terpene product in the sterol-free medium.
18. The method of claim 17, wherein the ERG9 gene is a
Saccharomyces ERG9 gene.
19. The method of claim 18, wherein the Saccharomyces ERG9 gene is
a Saccharomyces cerevisiae ERG9 gene.
20. The method of claim 17, wherein the eukaryotic microbial host
is a fungal microbial host.
21. The method of claim 20, wherein the fungal microbial host is a
yeast microbial host.
22. The method of claim 21, wherein the yeast microbial host is a
yeast of the genus Saccharomyces.
23. The method of claim 22, wherein the yeast of the genus
Saccharomyces is Saccharomyces cerevisiae.
24. The method of claim 17, wherein the terpene synthase gene that
produces a detectable and measurable terpene product is a
Hyoscyamus muticus premnaspirodiene synthase (HPS) gene.
25. The method of claim 23, wherein the strain of S. cerevisiae is
ALX7-95 (his3, trp1, erg9::HIS3, HMGcat/TRP1::rDNA, dpp1), a
leucine prototroph of strain CALI-5, that contains a plasmid
expressing the Hyoscyamus muticus premnaspirodiene synthase (HPS)
gene.
26. A host cell, comprising the variant squalene synthase enzyme of
claim 14, wherein the host cell is a bacterial cell or a yeast
cell.
27. An isolated host cell, comprising the variant squalene synthase
enzyme of claim 14.
28. The method of claim 16, wherein the terpene product is
isolated.
29. A method for producing a host cell that encodes a variant
squalene synthase, comprising introducing a nucleic acid molecule
of claim 1 into the cell.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 13/986,436, filed May 1, 2013, entitled
"METHOD FOR PRODUCTION OF ISOPRENOID COMPOUNDS," which is a
continuation of U.S. patent application Ser. No. 12/540,094, now
issued U.S. Pat. No. 8,481,286, filed Aug. 12, 2009, entitled
"METHOD FOR PRODUCTION OF ISOPRENOID COMPOUNDS," which claims the
benefit of priority to U.S. Provisional Application Ser. No.
61/088,288, filed Aug. 12, 2008, and is a continuation of U.S.
patent application Ser. No. 12/540,050, now issued U.S. Pat. No.
8,609,371, filed Aug. 12, 2009, entitled "ISOPRENOID COMPOUNDS,"
which also claims the benefit of priority to U.S. Provisional
Application Ser. No. 61/088,288, filed Aug. 12, 2008. The subject
matter of each of the above-referenced applications is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention is directed to yeast strains that overproduce
a precursor to terpenes, farnesyl pyrophosphate (FPP), and are
capable of growing without sterol supplementation.
BACKGROUND OF THE INVENTION
[0003] With over 50,000 identified members, terpenoids comprise the
largest known class of natural products. These compounds are
structurally diverse, although based on related carbon skeletons.
The structural diversity found among these compounds allows them to
perform a variety of essential biochemical functions. These
compounds serve as attractants for pollinators, antimicrobial and
antiherbivorial defense compounds, and may react with reactive
oxygen species to protect against oxidative damage (Dudereva et
al., Plant Physiol. 135:1893-1902 (2004)). As components of the
essential oils of aromatic plants, they are largely responsible for
the distinct flavors and fragrances associated with their host
plants. Moreover, the value of these small molecules extends beyond
their biological utility. Many terpenoids have commercial value as
antibiotics, pest control agents, fragrances, flavors, and
anti-cancer agents, among other important uses.
[0004] A specific class of these natural products,
sesquiterpenoids, is derived from a common 15-carbon building
block. This common 15-carbon building block is farnesyl
pyrophosphate (FPP). Many important products, such as the flavoring
nootkatone, the cosmetic additive bisabolol, and
amorpha-4,11-diene, a precursor to the antimalarial compound
artemisinin, are sesquiterpenoids and thus are based on the
15-carbon skeleton of FPP. Therefore, methods that can increase the
yield of FPP that can be utilized in sesquiterpenoid synthesis are
of extreme importance.
[0005] To maximize production of terpenes, mutations in squalene
synthase have been used to prevent or minimize conversion of
farnesyl pyrophosphate to squalene. In practice, this has been done
by either eliminating the corresponding gene, reducing its
expression using weak promoters, or controlling its expression with
a regulated promoter. Squalene is a precursor to sterols, which are
essential to viability of yeast and other organisms. Accordingly,
complete elimination of the gene requires feeding of sterols. The
yeast Saccharomyces cerevisiae is not normally capable of taking up
sterols under aerobic conditions, so in order to feed sterols to
mutants, secondary mutations enabling sterol uptake are
required.
[0006] Various solutions have been proposed in order to obtain high
yields of farnesyl pyrophosphate for maximum production of
terpenes. In one approach, the ERG9 gene of the yeast is completely
eliminated. The gene ERG9 encodes the enzyme squalene synthase.
However, because these mutants in which ERG9 is eliminated cannot
synthesize squalene, which is a precursor to sterols, they must be
fed sterols (Takahashi, et al., Biotech. Bioengineer. 97:170-181
(2007)). In another approach, PCT Patent Application Publication
No. WO 06/102342 by Bailey et al., describes production of high
yields of farnesyl pyrophosphate by modifying the expression or
activity of one or more polypeptides involved in generating
cytosolic acetyl-CoA and/or NADPH. In another approach, a promoter,
the MET3 promoter, is used in place of the native ERG9 promoter to
downregulate the expression of squalene synthase by repressing its
synthesis by adding methionine, which acts as a repressor with
respect to the MET3 promoter (Asadollahi et al., Biotech.
Bioengineer. 99:666-677 (2007)). In a similar approach, in addition
to repression of ERG9 production, overproduction of a soluble,
truncated form of 3-hydroxy-3-methylglutaryl-coenzyme A reductase,
and enhancement of the activity of the transcription factor UPC2
was employed (Paradise et al., Cell. Metabol. Engineer.
Bioengineer. 100:371-378 (2008); Ro et al., Nature 440: 940-943
(2006)).
[0007] However, there is a need for improved strains of
Saccharomyces cerevisiae that can overproduce FPP without the need
for sterol supplementation and without regulating expression.
Preferably, these improved strains would grow efficiently and
produce high levels of farnesyl pyrophosphate for subsequent
terpenoid synthesis.
SUMMARY OF THE INVENTION
[0008] A number of mutations of the Saccharomyces cerevisiae
squalene synthase gene have been isolated and characterized. These
mutants produce a sufficient quantity of squalene synthesis enzyme
so that the enzyme catalyzes the synthesis of squalene at a
sufficiently high rate so that sterol supplementation for the S.
cerevisiae cells is not required, while having reduced activity so
that more farnesyl pyrophosphate is available for isoprenoid
biosynthesis. The reduced activity may be the result of reduced
catalytic efficiency of the enzyme, or of reduced intracellular
concentration of the protein, or both.
[0009] Accordingly, one aspect of the present invention is an
isolated nucleic acid molecule that encodes a squalene synthase
enzyme that, when present and expressed in vivo in a eukaryotic
microbial host as the only squalene synthase species, catalyzes the
synthesis of squalene at a sufficiently high rate so that
supplementation of the eukaryotic microbial host with a sterol is
not required for growth, and also has a reduced squalene synthase
activity (referred to herein for convenience as a variant squalene
synthase enzyme). In one alternative, a host cell containing the
nucleic acid molecule, when expressed in vivo, produces a greater
concentration of an isoprenoid in grams of isoprenoid per liter of
culture than a corresponding host containing a wild-type nucleic
acid molecule.
[0010] The variant squalene synthase enzyme of the present
invention can be a squalene synthase enzyme of any suitable
species. In one aspect, variant squalene synthase enzymes are from
Saccharomyces cerevisiae.
[0011] Isolated nucleic acid molecules encoding a variant S.
cerevisiae squalene synthase enzyme according to the present
invention include, but are not limited to the following isolated
nucleic acid molecules:
[0012] (1) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 691 A.fwdarw.G, resulting in the amino acid
change E.fwdarw.G at amino acid residue 149; (b) 748 G.fwdarw.T,
resulting in the amino acid change G.fwdarw.V at amino acid residue
168; (c) 786 T.fwdarw.A, resulting in the amino acid change
Y.fwdarw.N at amino acid residue 181; (d) 1114 A.fwdarw.T,
resulting in the amino acid change Q.fwdarw.L at amino acid residue
290; (e) 1213 T.fwdarw.C, resulting in the amino acid change
I.fwdarw.T at amino acid residue 323; and (f) 1290 T.fwdarw.C,
resulting in no change of the amino acid L at amino acid residue
349 (silent mutation);
[0013] (2) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 72 C.fwdarw.A (in the non-coding region);
(b) 110 .DELTA.A (in the non-coding region); and (c) 801
G.fwdarw.A, resulting in the amino acid change V.fwdarw.1 at amino
acid residue 186;
[0014] (3) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 989 T.fwdarw.A, resulting in no change of
the amino acid P at amino acid residue 248 (silent mutation); (b)
1112 G.fwdarw.A, resulting in no change of the amino acid E at
amino acid residue 289 (silent mutation); (c) 1220 G.fwdarw.A,
resulting in no change of the amino acid K at amino acid residue
325 (silent mutation); and (d) 1233 T.fwdarw.C, resulting in the
amino acid change Y.fwdarw.H at amino acid residue 330;
[0015] (4) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 786 T.fwdarw.A, resulting in the amino acid
change Y.fwdarw.N at amino acid residue 181; (b) 1025 A.fwdarw.G,
resulting in no change of the amino acid Q at amino acid residue
260 (silent mutation); (c) 1056 T.fwdarw.A, resulting in the amino
acid change L.fwdarw.I at amino acid residue 271; (d) 1068
A.fwdarw.G, resulting in the amino acid change S.fwdarw.G at amino
acid residue 275; and (e) 1203 A.fwdarw.G, resulting in the amino
acid change N.fwdarw.D at amino acid residue 320;
[0016] (5) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 886 T.fwdarw.C, resulting in the amino acid
change M.fwdarw.T at amino acid residue 214; (b) 969 A.fwdarw.G,
resulting in the amino acid change I.fwdarw.V at amino acid residue
242; (c) 1075 T.fwdarw.C, resulting in the amino acid change
V.fwdarw.A at amino acid residue 277; and (d) 1114 A.fwdarw.T,
resulting in the amino acid change Q.fwdarw.L at amino acid residue
290;
[0017] (6) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: 84 T.fwdarw.A (in the non-coding region); (b)
283 A.fwdarw.T, resulting in the amino acid change E.fwdarw.V at
amino acid residue 13; (c) 424 T.fwdarw.C, resulting in the amino
acid change L.fwdarw.P at amino acid residue 60; (d) 440
A.fwdarw.G, resulting in no change of the amino acid R at amino
acid residue 65 (silent mutation); and (e) 1076 T.fwdarw.C,
resulting in no change of the amino acid V at amino acid residue
277 (silent mutation);
[0018] (7) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 619 A.fwdarw.T, resulting in the amino acid
change D.fwdarw.V at amino acid residue 125; (b) 634 T.fwdarw.C,
resulting in the amino acid change L.fwdarw.P at amino acid residue
130; and (c) 962 C.fwdarw.T, resulting in no change of the amino
acid P at amino acid residue 239 (silent mutation);
[0019] (8) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 150 A.fwdarw.T (in the non-coding region);
(b) 410 T.fwdarw.G, resulting in no change of the amino acid A at
amino acid residue 55 (silent mutation); (c) 411 G.fwdarw.T,
resulting in the amino acid change V.fwdarw.L at amino acid residue
56; and (d) 1248 T.fwdarw.C, resulting in the amino acid change
S.fwdarw.P at amino acid residue 335;
[0020] (9) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme, in which the following nucleic
acid changes occur: (a) 510 C.fwdarw.T, resulting in the amino acid
change H.fwdarw.Y at amino acid residue 89; (b) 573 T.fwdarw.C,
resulting in the amino acid change F.fwdarw.L at amino acid residue
110; (c) 918 A.fwdarw.G, resulting in the amino acid change
R.fwdarw.G at amino acid residue 224; and (d) 997 A.fwdarw.G,
resulting in the amino acid change K.fwdarw.G at amino acid residue
251;
[0021] (10) an isolated nucleic acid molecule identical to any of
(3), (4), (6), (7), or (8), above, except that one or more of the
silent mutations in nucleic acid molecules (3), (4), (6), (7), or
(8) are omitted;
[0022] (11) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme in which the wild-type S.
cerevisiae squalene synthase enzyme is mutated with the same amino
acid changes as in any of (1) through (10) above;
[0023] (12) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme containing any of the amino
acid changes in any of (1) through (10) above; and
[0024] (13) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme in which the squalene synthase
enzyme differs from the squalene synthase enzyme encoded by the
isolated nucleic acid molecule of any of (1) through (12) above by
one to three conservative amino acid substitutions, wherein a
conservative amino acid substitution is defined as one of the
following substitutions: A.fwdarw.G or S; R.fwdarw.K; N.fwdarw.Q or
H; D.fwdarw.E; C.fwdarw.S; Q.fwdarw.N; G.fwdarw.D; G.fwdarw.A. or
P; H.fwdarw.N or Q; I.fwdarw.L or V; L.fwdarw.I or V; K.fwdarw.R or
Q or E; M.fwdarw.L or Y or I; F.fwdarw.M or L or Y; S.fwdarw.T;
T.fwdarw.S; W.fwdarw.Y; Y.fwdarw.W or F; and V.fwdarw.I or L.
[0025] The present invention also encompasses an isolated nucleic
acid molecule that is at least 95% identical to any of the isolated
nucleic acid molecules described above that encodes a variant S.
cerevisiae squalene synthase enzyme, such that the isolated nucleic
acid molecule also encodes a variant S. cerevisiae squalene
synthase enzyme that, when present and expressed in vivo in
Saccharomyces cerevisiae, catalyzes the synthesis of squalene at a
sufficiently high rate that supplementation of the S. cerevisiae
with a sterol is not required and that has a reduced squalene
synthase activity.
[0026] Also within the scope of the present invention is an
isolated nucleic acid molecule that includes therein, as a
discrete, continuous nucleic acid segment, the isolated nucleic
acid molecule encoding the variant squalene synthase as described
above. This embodiment of the invention can include, at either the
5'-terminus, the 3'-terminus, or both, additional nucleic acid
sequences such as linkers, adaptors, restriction endonuclease
cleavage sites, regulatory sequences such as promoters, enhancers,
or operators, or coding sequences, to which the discrete,
continuous nucleic acid segment is operatively linked.
[0027] Also within the scope of the present invention are vectors
including therein nucleic acid segments according to the present
invention as described above, as well as host cells transformed or
transfected with the vectors or host cells including therein a
nucleic acid segment encoding the variant squalene synthase enzyme
according to the present invention, as described above.
[0028] The present invention further includes a variant squalene
synthase enzyme encoded by a nucleic acid sequence according to the
present invention as described above. The variant squalene synthase
enzyme can be, but is not limited to a variant S. cerevisiae
squalene synthase enzyme. Variant S. cerevisiae squalene synthase
enzymes according to the present invention include, but are not
limited to, at least one of the mutants listed in Table 2.
[0029] Another aspect of the present invention includes a host cell
containing and/or expressing a variant squalene synthase enzyme of
the present invention as described above (a variant squalene
synthase enzyme). The host cell, in this alternative, includes at
least one copy of a nucleic acid sequence encoding the enzyme. The
copy of the nucleic acid sequence encoding the enzyme can be
present in the chromosome of a prokaryotic (bacterial) cell or in
one chromosome of a eukaryotic cell. Alternatively, the copy of the
nucleic acid sequence encoding the enzyme can be present in a
vector or plasmid that is present in the cell.
[0030] Another aspect of the invention is a method of isolating a
defective ERG9 gene. In general, this method comprises the steps
of:
[0031] (1) isolating a wild-type ERG9 gene to produce an isolated
wild-type ERG9 gene;
[0032] (2) subjecting the isolated wild-type ERG9 gene to
mutagenesis to generate a pool of erg9 mutants;
[0033] (3) transforming mutants from the pool of erg9 mutants
generated in step (2) into a strain of a eukaryotic microbial host
that contains a plasmid expressing a terpene synthase gene that
produces a detectable and measurable terpene product, the strain of
the eukaryotic microbial host being transformed in such a manner
that replacement of the preexisting ERG9 allele with an erg9
mutation allows the strain to grow in a sterol-free medium; and
[0034] (4) isolating a transformant from step (3) that produces a
level of terpene product at least equivalent to the level of
terpene product produced by a strain of the eukaryotic microbial
host expressing the terpene synthase gene that requires a sterol in
the medium for growth.
[0035] Another aspect of the present invention is a method of
isolating a variant squalene synthase enzyme. The variant squalene
synthase enzyme to be isolated by the methods of the invention is
as described above.
[0036] In general, this method comprises the steps of:
[0037] (a) culturing a host cell that expresses a variant squalene
synthase gene according to the present invention or that contains a
variant squalene synthase enzyme according to the present
invention; and
[0038] (b) isolating the variant squalene synthase enzyme from the
host cell.
[0039] Yet another aspect of the present invention is a method of
producing an isoprenoid using a host cell containing a mutated ERG9
gene, which defective ERG9 gene encodes a variant squalene synthase
enzyme.
[0040] In one alternative, a host cell that includes a mutated ERG9
gene encoding a variant squalene synthase enzyme further includes
at least one isoprenoid synthase gene, so that the farnesyl
pyrophosphate produced in the host cell, which is available in
greater concentrations for isoprenoid biosynthesis can be converted
to an isoprenoid by the isoprenoid synthase encoded by the
isoprenoid synthase gene.
[0041] This alternative, in general, comprises the steps of:
[0042] (1) providing a host cell that includes a mutated ERG9 gene
that encodes a variant squalene synthase enzyme according to the
present invention and at least one isoprenoid synthase gene;
[0043] (2) allowing the host cell to produce farnesyl pyrophosphate
and to synthesize the isoprenoid from the farnesyl pyrophosphate;
and
[0044] (3) isolating the isoprenoid synthesized by the host
cell.
[0045] In another alternative method for producing an isoprenoid,
the method, in general, comprises the steps of:
[0046] (1) providing a host cell that includes a mutated ERG9 gene
that encodes a variant synthase enzyme according to the present
invention;
[0047] (2) allowing the host cell to produce farnesyl
pyrophosphate;
[0048] (3) isolating farnesyl pyrophosphate from the host cell;
[0049] (4) reacting the farnesyl pyrophosphate in vitro with one or
more isoprenoid synthases to synthesize the isoprenoid; and
[0050] (5) isolating the isoprenoid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying
drawings.
[0052] FIG. 1 depicts a graph showing the concentration of
premnaspirodiene in mg/L for each of the 25 isolates grown with and
without ergosterol supplementation.
[0053] FIGS. 2A-2D depict the sequences of the wild-type ERG9 gene
including sequences 245 base pairs upstream of the start site (SEQ
ID NO: 3). In FIGS. 2A-2D, the underlined nucleotides shown at the
5'-terminus and 3'-terminus of the ERG9 gene sequence represent the
upstream primer (7-162.1) 5'-CCATCTTCAACAACAATACCG-3' (SEQ ID NO:
1) and the downstream primer (7-162.2) 5'-GTACTTAGTTATTGTTCGG-3'
(SEQ ID NO: 2).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0054] As used herein, the term "nucleic acid," "nucleic acid
sequence," "polynucleotide," or similar terms, refers to a
deoxyribonucleotide or ribonucleotide oligonucleotide or
polynucleotide, including single- or double-stranded forms, and
coding or non-coding (e.g., "antisense") forms. The term
encompasses nucleic acids containing known analogues of natural
nucleotides. The term also encompasses nucleic acids including
modified or substituted bases as long as the modified or
substituted bases interfere neither with the Watson-Crick binding
of complementary nucleotides or with the binding of the nucleotide
sequence by proteins that bind specifically. The term also
encompasses nucleic-acid-like structures with synthetic backbones.
DNA backbone analogues provided by the invention include
phosphodiester, phosphorothioate, phosphorodithioate, methyl
phosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,
3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino
carbamate, and peptide nucleic acids (PNAs). (Oligonucleotides and
Analogues, a Practical Approach, edited by F. Eckstein, IRL Press
at Oxford University Press (1991); Antisense Strategies, Annals of
the New York Academy of Sciences, Volume 600, Eds. Baserga and
Denhardt (NYAS 1992); Milligan, J. Med. Chem. 36:1923-1937 (1993);
Antisense Research and Applications (1993, CRC Press). PNAs contain
non-ionic backbones, such as N-(2-aminoethyl) glycine units.
Phosphorothioate linkages are described in, e.g. U.S. Pat. Nos.
6,031,092; 6,001,982; 5,684,148; WO 97/03211; WO 96/39154; Mata,
Toxicol. Appl. Pharmacol. 144:189-197 (1997). Other synthetic
backbones encompassed by the term include methylphosphonate
linkages or alternating methylphosphonate and phosphodiester
linkages (U.S. Pat. No. 5,962,674; Strauss-Soukup, Biochemistry
36:8692-8698 (1997)), and benzylphosphonate linkages (U.S. Pat. No.
5,532,226; Samstag, Antisense Nucleic Acid Drug Dev 6:153-156
(1996)). Bases included in nucleic acids include any of the known
base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyl adenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N.sup.6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N.sup.6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
.beta.-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. DNA
may be in the form of cDNA, in vitro polymerized DNA, plasmid DNA,
parts of a plasmid DNA, genetic material derived from a virus,
linear DNA, vectors (e.g. P1, PAC, BAC, YAC, and artificial
chromosomes), expression cassettes, chimeric sequences, recombinant
DNA, chromosomal DNA, an oligonucleotide, anti-sense DNA, or
derivatives of these groups. RNA may be in the form of
oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear
RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), in vitro
polymerized RNA, recombinant RNA, chimeric sequences, anti-sense
RNA, siRNA (small interfering RNA), ribozymes, or derivatives of
these groups. Additionally, the terms "nucleic acid" or "nucleic
acid molecule" refer to a deoxyribonucleotide or ribonucleotide
polymer in either single- or double-stranded form, and unless
otherwise limited, would encompass known analogs of natural
nucleotides that can function in a similar manner as naturally
occurring nucleotides. A "nucleotide sequence" also refers to a
polynucleotide molecule or oligonucleotide molecule in the form of
a separate fragment or as a component of a larger nucleic acid. The
nucleotide sequence or molecule may also be referred to as a
"nucleotide probe." Some of the nucleic acid molecules of the
invention are derived from DNA or RNA isolated at least once in
substantially pure form and in a quantity or concentration enabling
identification, manipulation, and recovery of its component
nucleotide sequence by standard biochemical methods. Examples of
such methods, including methods for PCR protocols that may be used
herein, are disclosed in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
New York (1989), Ausubel, F. A., et al., eds., Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., New York (1987), and
Innis, M., et al. (Eds.) PCR Protocols: A Guide to Methods and
Applications, Academic Press, San Diego, Calif. (1990). Reference
to a nucleic acid molecule also includes its complement as
determined by the standard Watson-Crick base-pairing rules, with
uracil (U) in RNA replacing thymine (T) in DNA where necessary,
unless the complement is specifically excluded.
[0055] As described herein, the nucleic acid molecules of the
invention include DNA in both single-stranded and double-stranded
form, as well as the DNA or RNA complement thereof. DNA includes,
for example, DNA, genomic DNA, chemically synthesized DNA, DNA
amplified by PCR, and combinations thereof. Genomic DNA, including
translated, non-translated and control regions, may be isolated by
conventional techniques, e.g., using any one of the cDNAs of the
invention, or suitable fragments thereof, as a probe, to identify a
piece of genomic DNA which can then be cloned using methods
commonly known in the art.
[0056] Polypeptides encoded by the nucleic acids of the invention
are encompassed by the invention. As used herein, reference to a
nucleic acid "encoding" a protein or polypeptide encompasses not
only cDNAs and other intronless nucleic acids, but also DNAs, such
as genomic DNA, with introns, on the assumption that the introns
included have appropriate splice donor and acceptor sites that will
ensure that the introns are spliced out of the corresponding
transcript when the transcript is processed in a eukaryotic cell.
Due to the degeneracy of the genetic code wherein more than one
codon can encode the same amino acid, multiple DNA sequences can
code for the same polypeptide. Such variant DNA sequences can
result from genetic drift or artificial manipulation (e.g.,
occurring during PCR amplification or as the product of deliberate
mutagenesis of a native sequence). Deliberate mutagenesis of a
native sequence can be carried out using numerous techniques well
known in the art. For example, oligonucleotide-directed
site-specific mutagenesis procedures can be employed, particularly
where it is desired to mutate a gene such that predetermined
restriction nucleotides or codons are altered by substitution,
deletion or insertion. Exemplary methods of making such alterations
are disclosed by Walder et al., Gene, 42:133 (1986); Bauer et al.,
Gene 37:73 (1985); Craik, BioTechniques, January 12-19 (1985);
Smith et al., Genetic Engineering: Principles and Methods, Plenum
Press, (1981); Kunkel (PNAS USA 82:488 (1985); Kunkel et al.,
Methods in Enzymol. 154.367 (1987). The present invention thus
encompasses any nucleic acid capable of encoding a protein of the
current invention.
[0057] In a peptide or protein, suitable conservative substitutions
of amino acids are known to those of skill in this art and may be
made generally without altering the biological activity of the
resulting molecule. Those of skill in this art recognize that, in
general, single amino acid substitutions in non-essential regions
of a polypeptide do not substantially alter biological activity
(see, e.g. Watson et al., Molecular Biology of the Gene, 4th
Edition, 1987, Benjamin/Cummings, p. 224). In particular, such a
conservative variant has a modified amino acid sequence, such that
the change(s) do not substantially alter the proteins structure
and/or activity, e.g., antibody activity, enzymatic activity, or
receptor activity. These include conservatively modified variations
of an amino acid sequence, i.e., amino acid substitutions,
additions or deletions of those residues that are not critical for
protein activity, or substitution of amino acids with residues
having similar properties (e.g., acidic, basic, positively or
negatively charged, polar or non-polar, etc.) such that the
substitutions of even critical amino acids does not substantially
alter structure and/or activity. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. For example, one exemplary guideline to select conservative
substitutions includes (original residue followed by exemplary
substitution): Ala/Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu;
Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro; His/Asn or Gln; Ile/Leu
or Val; Leu/Ile or Val; Lys/Arg or Gln or Glu; Met/Leu or Tyr or
Ile; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or
Phe; Val/Ile or Leu. An alternative exemplary guideline uses the
following six groups, each containing amino acids that are
conservative substitutions for one another: (1) alanine (A or Ala),
serine (S or Ser), threonine (T or Thr); (2) aspartic acid (D or
Asp), glutamic acid (E or Glu); (3) asparagine (N or Asn),
glutamine (Q or Gln); (4) arginine (R or Arg), lysine (K or Lys);
(5) isoleucine (I or Ile), leucine (L or Leu), methionine (M or
Met), valine (V or Val); and (6) phenylalanine (F or Phe), tyrosine
(Y or Tyr), tryptophan (W or Trp); (Creighton (1984) Proteins, W.
H. Freeman and Company; Schulz and Schimer (1979) Principles of
Protein Structure, Springer-Verlag). One of skill in the art will
appreciate that the above-identified substitutions are not the only
possible conservative substitutions. For example, for some
purposes, one may regard all charged amino acids as conservative
substitutions for each other whether they are positive or negative.
In addition, individual substitutions, deletions or additions that
alter, add or delete a single amino acid or a small percentage of
amino acids in an encoded sequence can also be considered
"conservatively modified variations" when the three-dimensional
structure and the function of the protein to be delivered are
conserved by such a variation.
[0058] As used herein, the term "isolated" with reference to a
nucleic acid molecule or polypeptide or other biomolecule means
that the nucleic acid or polypeptide has been separated from the
natural environment from which the polypeptide or nucleic acid were
obtained. It may also mean that the biomolecule has been altered
from the natural state. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated," as the tem
is employed herein. Thus, a polypeptide or polynucleotide produced
and/or contained within a recombinant host cell is considered
isolated. Also intended as an "isolated polypeptide" or an
"isolated polynucleotide" are polypeptides or polynucleotides that
have been purified, partially or substantially, from a recombinant
host cell or from a native source. For example, a recombinantly
produced version of a compound can be substantially purified by the
one-step method described in Smith et al., Gene 67:3140 (1998). The
terms "isolated" and "purified" are sometimes used
interchangeably.
[0059] Thus, by "isolated" it is meant that the nucleic acid is
free of the coding sequences of those genes that, in a
naturally-occurring genome, immediately flank the gene encoding the
nucleic acid of interest. Isolated DNA may be single-stranded or
double-stranded, and may be genomic DNA, cDNA, recombinant hybrid
DNA, or synthetic DNA. It may be identical to a native DNA
sequence, or may differ from such sequence by the deletion,
addition, or substitution of one or more nucleotides.
[0060] "Isolated" or "purified" also refer to preparations made
from biological cells or hosts and means any cell extract
containing the indicated DNA or protein including a crude extract
of the DNA or protein of interest. For example, in the case of a
protein, a purified preparation can be obtained following an
individual technique or a series of preparative or biochemical
techniques and the protein of interest can be present at various
degrees of purity in these preparations. Particularly for proteins,
the procedures may include for example, but are not limited to,
ammonium sulfate fractionation, gel filtration, ion exchange
chromatography, affinity chromatography, density gradient
centrifugation, electrofocusing, chromatofocusing, and
electrophoresis. As used herein, the term "substantially purified,"
when applied to a composition or extract derived from yeast, and
wherein the composition or extract contains an isoprenoid, is
hereby defined as containing at least about twice the concentration
of isoprenoid in proportion to yeast material, wherein yeast
material is defined as being selected from the group consisting of
yeast cell membrane, yeast organelle, yeast cytoplasm, yeast
microsomal fraction, yeast cell, and yeast extract.
[0061] A preparation of DNA or protein that is "substantially pure"
or "isolated" refers to a preparation free from naturally occurring
materials with which such DNA or protein is normally associated in
nature. "Essentially pure" means a "highly" purified preparation
that contains at least 95% of the DNA or protein of interest.
[0062] A cell extract that contains the DNA or protein of interest
should be understood to mean a homogenate preparation or cell-free
preparation obtained from cells that express the protein or contain
the DNA of interest. The term "cell extract" includes culture
media, for example, spent culture media from which the cells have
been removed.
[0063] A "vector" is a nucleic acid that is capable of transporting
another nucleic acid. Vectors may be, for example, plasmids,
viruses, cosmids or phage. An "expression vector" is a vector that
is capable of directing expression of a protein encoded by one or
more genes carried by the vector when it is present in the
appropriate environment. Examples of vectors are those that can
autonomously replicate and express structural gene products present
in the DNA segments to which they are operatively linked. Vectors,
therefore, can contain the replicons and selectable markers
described earlier. Vectors include, but are not necessarily limited
to, expression vectors.
[0064] As used herein with regard to nucleic acid molecules,
including DNA fragments, the phrase "operatively linked" means the
sequences or segments have been covalently joined, preferably by
conventional phosphodiester bonds, into one strand of DNA, whether
in single- or double-stranded form such that operatively linked
portions function as intended.
[0065] As used herein, the phrase "substantially identical" means
that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98%, or 99% identical to a given sequence. By way of
example, such sequences may be allelic variants, sequences derived
from various species, or they may be derived from the given
sequence by truncation, deletion, amino acid substitution or
addition. Percent identity between two sequences is determined by
standard alignment algorithms such as ClustalX when the two
sequences are in best alignment according to the alignment
algorithm.
[0066] In order to maximize production of farnesyl pyrophosphate
(FPP), novel mutants in the ERG9 gene of Saccharomyces cerevisiae
have been developed. The ERG9 gene is the gene encoding squalene
synthase. These mutants have reduced, but not eliminated, squalene
synthase activity. As such, they allow sufficient production of
squalene and subsequent sterols to allow growth, but are
sufficiently reduced in activity to allow accumulation of FPP and
overproduction of terpenes. This is done, unlike in previous
approaches, by generating and utilizing defective squalene synthase
genes, which when expressed, result in reduced cellular squalene
synthase activity rather than downregulating the transcription of a
normally active squalene synthase enzyme. This makes the reduced
squalene synthase activity independent of the activity of a
repressor.
[0067] Accordingly, one aspect of the present invention is an
isolated nucleic acid molecule that encodes a squalene synthase
enzyme that, when present and expressed in vivo in a eukaryotic
microbial host cell, catalyzes the synthesis of squalene at a
sufficiently high rate that supplementation of the eukaryotic
microbial host cell with a sterol is not required and that has a
reduced squalene synthase activity (referred to herein for
convenience as a variant squalene synthase enzyme). Typically, the
variant squalene synthase enzyme encoded by the isolated nucleic
acid molecule of the present invention has a reduced V.sub.max for
squalene synthesis. V.sub.max is the maximum rate of a reaction
being catalysed by an enzyme. Alternatively, the variant squalene
synthase enzyme encoded by the isolated nucleic acid molecule of
the present invention has an increased Michaelis constant (K.sub.m)
for its FPP substrate, in which case the enzyme is less active at a
given intracellular concentration of FPP than the wild-type enzyme.
The K.sub.m is a means of characterising an enzyme's affinity for a
substrate. The K.sub.m in an enzymatic reaction is the substrate
concentration at which the reaction rate is half its maximum speed.
Typically, the squalene synthase enzyme encoded by the isolated
nucleic acid molecule of the present invention, when expressed in
vivo in the eukaryotic microbial host cell, produces squalene at a
rate of less than 75% of the wild-type enzyme. Preferably, the
squalene synthase enzyme encoded by the isolated nucleic acid
molecule of the present invention, when expressed in vivo in the
eukaryotic microbial host cell, produces squalene at a rate of less
than 50% of the wild-type enzyme. More preferably, the squalene
synthase enzyme encoded by the isolated nucleic acid molecule of
the present invention, when expressed in vivo in the eukaryotic
microbial host cell, produces squalene at a rate of less than 25%
of the wild-type enzyme. The eukaryotic microbial host cell is
typically, but is not limited to, a fungal host cell. The fungal
host cell is typically, but is not limited to, a yeast host cell,
such as a Saccharomyces cerevisiae host cell or other host cells of
the genus Saccharomyces. Similarly, the variant squalene synthase
is not limited to a squalene synthase of Saccharomyces cerevisiae,
but can be a squalene synthase of another species of Saccharomyces
or a squalene synthase of any organism that has a gene that
catalyzes the conversion of farnesyl pyrophosphate into
squalene.
[0068] Isolated nucleic acid molecules according to the present
invention include, but are not limited to, the following isolated
nucleic acid molecules:
[0069] (1) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase enzyme as shown as Mutant 4 in Table 2
of the present invention;
[0070] (2) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 10 in Table
2 of the present invention;
[0071] (3) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 14 in Table
2 of the present invention;
[0072] (4) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 19 in Table
2 of the present invention;
[0073] (5) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 22 in Table
2 of the present invention;
[0074] (6) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 23 in Table
2 of the present invention;
[0075] (7) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 24 in Table
2 of the present invention;
[0076] (8) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 25 in Table
2 of the present invention;
[0077] (9) an isolated nucleic acid molecule encoding a mutated S.
cerevisiae squalene synthase enzyme as shown as Mutant 69 in Table
2 of the present invention;
[0078] (10) an isolated nucleic acid molecule identical to any of
(3), (4), (6), (7), or (8), above, except that one or more of the
silent mutations in nucleic acid molecules (3), (4), (6), (7), or
(8) are omitted;
[0079] (11) an isolated nucleic acid molecule encoding a variant S.
cerevisiae squalene synthase protein in which the wild-type S.
cerevisiae squalene synthase enzyme is mutated with the same amino
acid changes as in any of (1) through (10) above;
[0080] (12) an isolated nucleic acid encoding a squalene synthase
protein containing any of the amino acid changes in any of (1)
through (10). Although some of the mutations described above are
designated as "silent," meaning that they do not affect the amino
acid inserted into the polypeptide chain at that position, there is
evidence that such mutations may affect protein function in a
number of ways, including altering folding patterns due to effects
on translation due to codon utilization.
[0081] The invention includes an isolated nucleic acid molecule
encoding a squalene synthase enzyme that differs from the squalene
synthase enzyme encoded by the nucleic acid molecule in any of (1)
through (11), above, by one to three conservative amino acid
substitutions, in which a conservative amino acid substitution is
defined as one of the following substitutions: A.fwdarw.G or S;
R.fwdarw.K; N.fwdarw.Q or H; D.fwdarw.E; C.fwdarw.S; G.fwdarw.D;
G.fwdarw.A or P; H.fwdarw.N or Q; I.fwdarw.L or V; or V; K.fwdarw.R
or Q or E; M.fwdarw.L or Y or I; F.fwdarw.M or L or Y; S.fwdarw.T;
T.fwdarw.S; W.fwdarw.Y; Y.fwdarw.W or F; and V.fwdarw.I or L.
Preferably, the isolated nucleic acid molecule encodes a squalene
synthase protein that differs from the squalene synthase protein
encoded by the nucleic acid molecule in any of (1) through (10) by
one or two conservative amino acid substitutions. More preferably,
the isolated nucleic acid molecule encodes a squalene synthase
protein that differs from the squalene synthase protein encoded by
the nucleic acid molecule in any of (1) through (11) by one
conservative amino acid substitution.
[0082] The invention includes an isolated nucleic acid molecule
that is at least 95% identical to any of the isolated nucleic acid
molecules described above that encodes a mutated S. cerevisiae
squalene synthase enzyme, such that the isolated nucleic acid
molecule also encodes a mutated S. cerevisiae squalene synthase
enzyme that, when present and expressed in vivo in Saccharomyces
cerevisiae, catalyzes the synthesis of squalene at a sufficiently
high rate that supplementation of the S. cerevisiae with sterols is
not required and that has a reduced squalene synthase activity.
Typically, the isolated nucleic acid molecule is at least 97.5%
identical to any of the isolated nucleic acid molecules described
above. Preferably, the isolated nucleic acid molecule is at least
99% identical to any of the isolated nucleic acid molecules
described above. More preferably, the isolated nucleic acid
molecule is at least 99.5% identical to any of the isolated nucleic
acid molecules described above. Most preferably, the isolated
nucleic acid molecule is at least 99.8% identical to any of the
isolated nucleic acid molecules described above. For these
purposes. "identity" is defined according to the Needleman-Wunsch
algorithm (S. B. Needleman & C. D. Wunsch, "A General Method
Applicable to the Search for Similarities in the Amino Acid
Sequence of Two Proteins," J. Mol. Biol. 48: 443-453 (1970)).
[0083] Nucleic acid molecules according to the present invention
and having the desired degree of identity are not limited to
nucleic acid molecules derived from S. cerevisiae; they include
nucleic acid molecules derived from other species of Saccharomyces,
or, as described above, derived from any organism that has a gene
capable of catalyzing the conversion of farnesyl pyrophosphate into
squalene.
[0084] Additionally, isolated nucleic acid molecules according to
the present invention further include isolated nucleic acid
molecules, which encode a squalene synthase enzyme, and when
expressed in a eukaryotic microbial host in which no other squalene
synthase enzyme is expressed, result in a significant reduction of
conversion of farnesyl pyrophosphate to squalene as described
above. In one alternative, a nucleic acid molecule according to the
present invention, when expressed in viva, causes a host cell to
produce a greater concentration of an isoprenoid in grams of
isoprenoid per liter of culture than a corresponding host cell
expressing a wild-type nucleic acid molecule. These isolated
nucleic acid molecules have at least one change from a nucleic acid
molecule that includes the coding region for the wild-type ERG9
gene and its flanking sequences, including the sequences both
upstream and downstream from the coding region. This at least one
change reduces the squalene synthase activity, even though the
specific activity may potentially be unaltered. The reduction of
the activity of the squalene synthase enzyme can occur through one
or more of the following mechanisms: (1) reduction in transcription
so that less mRNA that can be translated into squalene synthase
enzyme is generated; (2) reduction of mRNA stability, again
reducing translation; and (3) reduction of enzyme stability brought
about by an increased rate of protein degeneration in viva. In
other words, either: (1) the specific activity of the resulting
squalene synthase enzyme is reduced through at least one change in
the amino acid sequence of the enzyme expressed from the nucleic
acid molecule; or (2) the in viva activity of the enzyme is reduced
through a reduction in transcription, a reduction in translation,
or a reduction of enzyme stability.
[0085] The nucleic acid described above can be DNA, RNA, or a
RNA-DNA hybrid, but is typically DNA. The nucleic acid described
above can be single-stranded or double-stranded. If the nucleic
acid is single-stranded, either the strand described or its
complement can be the coding strand and is within the scope of the
invention.
[0086] Also within the scope of the invention is an isolated
nucleic acid molecule that includes therein, as a discrete,
continuous nucleic acid segment, the isolated nucleic acid molecule
encoding the variant squalene synthase. This embodiment of the
invention can include, at either the 5'-terminus, the 3'-terminus,
or both, additional nucleic acid sequences such as linkers,
adaptors, restriction endonuclease cleavage sites, regulatory
sequences such as promoters, enhancers, or operators, or coding
sequences, to which the discrete, continuous nucleic acid segment
is operatively linked. In the event that the isolated nucleic acid
molecule includes additional coding sequences, the isolated nucleic
acid molecule can encode a fusion protein having S. cerevisiae
squalene synthase activity.
[0087] Also within the scope of the invention are vectors including
therein nucleic acid segments according to the present invention as
described above. The vectors can be capable of replication in
prokaryotes (bacteria) or in eukaryotes (yeast or cells of higher
organisms). In one alternative, the vectors are capable of
replication in yeast, for example, S. cerevisiae. In yeast, a
number of vectors containing constitutive or inducible promoters
may be used. For a review, see Current Protocols in Molecular
Biology, Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc.
& Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression
and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu
& Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544;
Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3;
and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in
Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152,
pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces,
1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and
II. A constitutive yeast promoter such as ADH 1 or LEU2 or an
inducible promoter such as GAL4 may be used (Cloning in Yeast, Ch.
3, Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. D M
Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may
be used which promote integration of foreign DNA sequences into the
yeast chromosome.
[0088] Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts are
described, for example, in Pouwels et al., Cloning Vectors. A
Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation
systems could also be employed to produce the disclosed
polypeptides using RNAs derived from DNA constructs disclosed
herein.
[0089] Examples of expression vectors that can be used in
prokaryotic host cells include those derived from commercially
available plasmids such as the cloning vector pET plasmids
(Novagen, Madison, Wis., USA) or pBR322 (ATCC 37017). The pBR322
vector contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed cells.
To construct an expression vector using pBR322, an appropriate
promoter and a DNA sequence encoding one or more of the
polypeptides of the invention are inserted into the pBR322 vector.
Other commercially available vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega
Biotec, Madison, Wis., USA). Other commercially available vectors
include those that are specifically designed for the expression of
proteins; these would include pMAL-p2 and pMAL-c2 vectors that are
used for the expression of proteins fused to maltose binding
protein (New England Biolabs, Beverly, Mass., USA).
[0090] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include the bacteriophage T7 promoter
(Studier and Moffatt, J. Mol, Biol. 189:113 (1986)),
.beta.-lactamase (penicillinase), lactose promoter system (Chang et
al., Nature 275:615, 1978; Goeddel et al., Nature 281:544 (1979)),
tryptophan (tip) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057 (1980); EP-A-36776), and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412(1982)). A particularly useful prokaryotic host cell expression
system employs a phage .lamda. PL promoter and a c1857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection (ATCC), which incorporate
derivatives of the P.sub.L promoter, include plasmid pHUB2
(resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident
in E. coli RR1 (ATCC 53082)).
[0091] As detailed below, nucleic acid segments according to the
present invention can also be incorporated in vectors suitable for
introduction into yeast cells, such as, for example, Saccharomyces
(particularly S. cerevisiae), Pichia (particularly P. pastoris),
and Kluyveromyces (particularly K. lactic). Yeast vectors will
often contain an origin of replication sequence from a 2.mu. yeast
plasmid, an autonomously replicating sequence (ARS), a promoter
region, sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Suitable promoter
sequences for yeast vectors include, among others, promoters for
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Biol. Chem. 255:2073 (1980)), or other glycolytic enzymes (Hess et
al., Adv. Enzyme Reg. 7:149 (1969); Holland et al., Biochem.
17:4900 (1978)), such as enolase, glyceraldehyde phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
described in Hitzeman; EPA-73,657; Fleer et al., Gene 107:285-295
(1991); and van den Berg et al., Bio/Technology, 8:135-139 (1990).
Another alternative is the glucose-repressible ADH2 promoter
described by Russell et al. (J. Biol. Chem. 258:2674 (1982)) and
Beier et al. (Nature 300:724 (1982)). Shuttle vectors replicable in
both yeast and E. Coli can be constructed by inserting DNA
sequences from pBR322 for selection and replication in E. soli
(Amp.sup.r gene and origin of replication) into the above-described
yeast vectors.
[0092] When the vectors are capable of replication in cells of
higher organisms, the higher organisms can be plants or animals,
including mammals. In cases where plant expression vectors are
used, the expression of a mutated S. cerevisiae squalene synthase
coding sequence according to the present invention may be driven by
any of a number of promoters. For example, viral promoters such as
the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., Nature
310:511-514 (1984)), or the coat protein promoter to TMV (Takamatsu
et al., EMBO J., 6:307-311 (1987)) may be used; alternatively,
plant promoters such as the small subunit of RUBISCO (Coruzzi et
al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science
224:838-843 (1984)); or heat shock promoters, e.g., soybean
hsp17.5-E or hsp17.3-B (Gurley et al., Mol. Cell. Biol. 6:559-565
(1986)) may be used. These constructs can be introduced into plant
cells using Ti plasmids, Ri plasmids, plant virus vectors, direct
DNA transformation, microinjection, electroporation, or other
techniques that are well known in the art. For reviews of such
techniques see, for example, Weissbach and Weissbach, Methods for
Plant Molecular Biology, Academic Press, NY, Section VIII, pp.
421-463 (1988); and Grierson and Corey, Plant Molecular Biology, 2d
Ed., Blackie, London, Ch. 7-9 (1988). Eukaryotic cells are
alternative host cells for the expression of mutated S. cerevisiae
squalene synthase coding sequences. Such host cell lines may
include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,
293, and W138.
[0093] Mammalian cell systems that utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the coding sequence of a
variant squalene synthase enzyme may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted into the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the variant squalene
synthase enzyme in infected hosts (Logan and Shenk, PNAS USA
81:3655-3659 (1984)). Alternatively, the vaccinia virus 7.5K
promoter may be used. (Mackett et al., PNAS USA, 79:7415-7419
(1982); Mackett et al., J. 49:857-864 (1984); Panicali, et al.,
PNAS USA, 79:4927-4931 (1982)). Of particular interest are vectors
based on bovine papilloma virus which have the ability to replicate
as extrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1:486
(1981)). Shortly after entry of this DNA into mouse cells, the
plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration of
the plasmid into the host's chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such as
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of the variant squalene synthase enzyme in host cells
(Cone and Mulligan, PNAS USA 81:6349-6353 (1984)). High level
expression may also be achieved using inducible promoters,
including, but not limited to, the metallothionein IIA promoter and
heat shock promoters.
[0094] Also within the scope of the invention are host cells
including a nucleic acid segment encoding the variant squalene
synthase according to the present invention, as described above.
These host cells are typically transformed or transfected with the
nucleic acid segment; methods for such transformation or
transfection are described above. The term "nucleic acid segment"
is used herein to include the following alternatives: (1) a vector
including therein the variant squalene synthase; or (2) a
chromosome of the host cell including therein the variant squalene
synthase. The vector or chromosome can include, as described above,
nucleic acid sequences either 5'-, 3'-, or both 5'- and 3'- to the
coding sequence of the mutated S. cerevisiae squalene synthase,
such as, but not limited to, linkers, adaptors, restriction
endonuclease cleavage sites, regulatory sequences such as
promoters, enhancers, or operators, or coding sequences. The host
cells can be prokaryotic cells, such as bacteria, or can be
eukaryotic cells, such as yeast cells, plant cells, or animal
cells. If the host cells are yeast cells, they are typically S.
cerevisiae, although other genera of yeast, such as Pichia (Pichia
pastoris) or Kluyveromyces (Kluyveromyces lactis) can also be
employed. If the cells are plant cells, many types of plant cells
are suitable host cells; one frequently employed host cell is
Arabidopsis thaliana. If the cells are animal cells, they can be
insect cells or mammalian cells.
[0095] In host cells according to the present invention including
therein a nucleic acid segment encoding the variant squalene
synthase as described above, the nucleic acid segment can be
incorporated into a vector as described above. Alternatively, the
nucleic acid segment can be integrated into a chromosome of the
host cell. Methods for integrating the nucleic acid segment
encoding the variant squalene synthase as described above into the
chromosome of a prokaryotic cell (i.e., a bacterium) or into one
chromosome of a eukaryotic cell are known in the art. As described
above, in this application, the nucleic acid is typically DNA.
[0096] DNA sequences encoding variant squalene synthase can be
obtained by several methods. For example, the DNA can be isolated
using hybridization procedures that are well known in the art.
These include, but are not limited to: (1) hybridization of probes
to genomic or cDNA libraries to detect shared nucleotide sequences;
(2) antibody screening of expression libraries to detect shared
structural features; and (3) synthesis by the polymerase chain
reaction (PCR). RNA sequences of the invention can be obtained by
methods known in the art (See, for example, Current Protocols in
Molecular Biology, Ausubel, et al., Eds. (1989)).
[0097] The development of specific DNA sequences encoding variant
squalene synthases of the invention can be obtained by: (1)
isolation of a double-stranded DNA sequence from the genomic DNA;
(2) chemical manufacture of a DNA sequence to provide the necessary
codons for the polypeptide of interest; and (3) in vitro synthesis
of a double-stranded DNA sequence by reverse transcription of mRNA
isolated from a eukaryotic donor cell. In the latter case, a
double-stranded DNA complement of mRNA is eventually formed which
is generally referred to as cDNA. Of these three methods for
developing specific DNA sequences for use in recombinant
procedures, the isolation of genomic DNA is the least common. This
is especially true when it is desirable to obtain the microbial
expression of eukaryotic polypeptides, such as yeast polypeptides,
due to the presence of introns. For obtaining nucleic acid
sequences encoding variant squalene synthases according to the
present invention, the synthesis of DNA sequences is frequently the
method of choice when the entire sequence of amino acid residues of
the desired polypeptide product is known. When the entire sequence
of amino acid residues of the desired polypeptide is not known, the
direct synthesis of DNA sequences is not possible and the method of
choice is the formation of cDNA sequences. Among the standard
procedures for isolating cDNA sequences of interest is the
formation of plasmid-carrying cDNA libraries which are derived from
reverse transcription of mRNA which is abundant in donor cells that
have a high level of genetic expression. When used in combination
with polymerase chain reaction technology, even rare expression
products can be clones. In those cases where significant portions
of the amino acid sequence of the polypeptide are known, the
production of labeled single or double-stranded DNA or RNA probe
sequences duplicating a sequence putatively present in the target
cDNA may be employed in DNA/DNA hybridization procedures which are
carried out on cloned copies of the cDNA which have been denatured
into a single-stranded form (Jay et al., Nucleic Acid Research
11:2325 (1983)).
[0098] Nucleotide sequences encompassed by the present invention
can also be incorporated into a vector as described above,
including, but not limited to, an expression vector, and used to
transfect or transform suitable host cells, as is well known in the
art. The vectors incorporating the nucleotide sequences that are
encompassed by the present invention are also within the scope of
the invention. Host cells that are transformed or transfected with
the vector or with polynucleotides or nucleotide sequences of the
present invention are also within the scope of the invention. The
host cells can be prokaryotic or eukaryotic; if eukaryotic, the
host cells can be mammalian cells, insect cells, or yeast cells. If
prokaryotic, the host cells are typically bacterial cells.
[0099] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as
Escherichia coli, competent cells which are capable of DNA uptake
can be prepared from cells harvested after exponential growth phase
and subsequently treated by the CaCl.sub.2 method by procedures
well known in the art. Alternatively, MgCl.sub.2 or RbCl can be
used. Transformation can also be performed after forming a
protoplast of the host cell or by electroporation.
[0100] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used.
[0101] A variety of host-expression vector systems may be utilized
to express the nucleic acid sequence encoding the variant squalene
synthase enzymes of the present invention. These include but are
not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing a variant squalene synthase enzyme coding
sequence; yeast transformed with recombinant yeast expression
vectors containing the variant squalene synthase enzyme coding
sequence; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing a variant squalene
synthase enzyme coding sequence; insect cell systems infected with
recombinant virus expression vectors (e.g., baculovirus) containing
a variant squalene synthase enzyme coding sequence; or animal cell
systems infected with recombinant virus expression vectors (e.g.,
retroviruses, adenovirus, vaccinia virus) containing a variant
squalene synthase enzyme coding sequence, or transformed animal
cell systems engineered for stable expression. In such cases where
glycosylation may be important, expression systems that provide for
translational and post-translational modifications may be used;
e.g., mammalian, insect, yeast or plant expression systems.
[0102] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements, for
example, constitutive and inducible promoters, transcription
enhancer elements, and transcription terminators, may be used in
the expression vector (Bitter et al., Meth. Enzymol. 153:516-544
(1987)). For example, when cloning in bacterial systems, inducible
promoters such as pL of bacteriophage .lamda., plac, ptrp, ptac
(ptrp-lac hybrid promoter) and the like may be used. When cloning
in mammalian cell systems, promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the retrovirus long terminal repeat; the adenovirus
late promoter; the vaccinia virus 7.5K promoter) may be used.
Promoters produced by recombinant DNA or synthetic techniques may
also be used to provide for transcription of the inserted variant
squalene synthase enzyme coding sequence.
[0103] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
variant squalene synthase enzyme expressed, and whether it is
desired to isolate the enzyme and in what state of purity. For
example, when large quantities are to be produced, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Those which are
engineered to contain a cleavage site to aid in recovering the
protein are preferred. Such vectors include, but are not limited
to, the Escherichia coli expression vector pUR278 (Ruttier et al.,
EMBO J. 2:1791 (1983)), in which the variant squalene synthase
enzyme coding sequence may be ligated into the vector in frame with
the lac Z coding region so that a hybrid variant squalene synthase
enzyme-lac Z protein is produced as well as pIN vectors (Inouye and
Inouye, Nucleic Acids Res. 13:3101-3109 (1985) and Van Heeke and
Schuster, J. Biol. Chem. 264:5503-5509 (1989)).
[0104] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review, see Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Bitter et
al., Expression and Secretion Vectors for Yeast, in Methods in
Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol.
153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press,
Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression in
Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.
Press, N.Y., Vol. 152, pp. 673-684 1987); and The Molecular Biology
of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring
Harbor Press, Vols. I and II (1982). A constitutive yeast promoter
such as ADH1 or LEU2 or an inducible promoter such as GAL4 may be
used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.
11, A Practical Approach, Ed. D M Glover, IRL Press, Wash., D.C.
(1986)). Alternatively, vectors may be used which promote
integration of foreign DNA sequences into the yeast chromosome.
[0105] In cases where plant expression vectors are used, the
expression of a variant squalene synthase enzyme coding sequence
may be driven by any of a number of promoters. For example, viral
promoters such as the 35S RNA and 19S RNA promoters of CaMV
(Brisson et al., Nature, 310:511-514 (1984)), or the coat protein
promoter to TMV (Takamatsu et al., EMBO J., 6:307-311 (1987)) may
be used; alternatively, plant promoters such as the small subunit
of RUBISCO (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et
al. Science 224:838-843 (1984)); or heat shock promoters, e.g.,
soybean hsp17.5-E Or hsp17.3-B (Gurley et al., Mol. Cell. Biol.,
6:559-565 (1986)) may be used. These constructs can be introduced
into plant cells using Ti plasmids, Ri plasmids, plant virus
vectors, direct DNA transformation, microinjection,
electroporation, or other techniques that are well known in the
art. (Weissbach & Weissbach, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp. 421-463 (1988);
Grierson and Corey, Plant Molecular Biology, 2d Ed., Blackie,
London, Ch. 7-9 (1988).
[0106] An alternative expression system that can be used to express
a variant squalene synthase enzyme of the present invention is an
insect system. In one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The variant
squalene synthase enzyme coding sequence may be cloned into
non-essential regions (in Spodoptera frugiperda, for example, the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter). Successful
insertion of the variant squalene synthase enzyme coding sequence
will result in inactivation of the polyhedrin gene and production
of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect cells in which the
inserted gene is expressed. (Smith et al., J. Virol. 46:584 (1983)
and U.S. Pat. No. 4,215,051).
[0107] Eukaryotic systems, and preferably mammalian expression
systems, allow for proper post-translational modifications of
expressed eukaryotic proteins to occur. Therefore, eukaryotic
cells, such as mammalian cells that possess the cellular machinery
for proper processing of the primary transcript, glycosylation,
phosphorylation, and advantageously, secretion of the gene product,
are the preferred host cells for the expression of a variant
squalene synthase enzyme. Such host cell lines may include, for
example CHO, VERO, BHK, HeLa, COS, MDCK, 293, and W138. Other
eukaryotic host cells are also suitable host cells for the
expression of a variant squalene synthase enzyme or of nucleic
acids according to the present invention, including, for example,
microalgal cells.
[0108] Mammalian cell systems that utilize recombinant viruses or
viral elements to direct expression may be engineered for use in
the present invention. For example, when using adenovirus
expression vectors, the coding sequence of a variant squalene
synthase enzyme may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted into the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the variant squalene
synthase enzyme in infected hosts (Logan and Shenk, PNAS USA
81:3655-3659 (1984)). Alternatively, the vaccinia virus 7.5K
promoter may be used (Mackett et al., PNAS USA 79:7415-7419 (1982);
Mackett et al., J. Virol. 49:857-864 (1984); Panicali et al., PNAS
USA 79:4927-4931 (1982)). Of particular interest are vectors based
on bovine papilloma virus which have the ability to replicate as
extrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1:486
(1981)). Shortly after entry of this DNA into mouse cells, the
plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration of
the plasmid into the host's chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such as
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of the variant squalene synthase enzyme in host cells
(Cone and Mulligan, PNAS USA 81:6349-6353 (1984)). High level
expression may also be achieved using inducible promoters,
including, but not limited to, the metallothionein IIA promoter and
heat shock promoters.
[0109] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with a cDNA controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines. For
example, following the introduction of foreign DNA, engineered
cells may be allowed to grow for 1-2 days in enriched media, and
then are switched to a selective medium. A number of selection
systems may be used, including but not limited, to the herpes
simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska and
Szybalski, PNAS USA, 48:2026 (1962)), and adenine
phosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) genes,
which can be employed in tk.sup.-, hgprt.sup.- or apr.sup.- cells,
respectively. Additionally, antimetabolite resistance-conferring
genes can be used as the basis of selection; for example, the genes
for dhfr, which confer resistance to methotrexate (Wigler et al.,
Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., PNAS USA,
78:1527 (1981)); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, PNA S USA, 78:2072 (1981)); neo, which confers
resistance to the aminoglycoside G418 (Colberre-Garapin et al., J.
Mol. Biol., 150:1 (1981)); and hygro, which confers resistance to
hygromycin (Santerre et al., Gene, 30:147 (1984)). Recently,
additional selectable genes have been described, namely trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman and
Mulligan, PNAS USA, 85:804 (1988)); and ODC (ornithine
decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue, In: Current Communications in Molecular Biology, Cold
Spring Harbor Laboratory ed., (1987)).
[0110] Another aspect of the present invention is a variant
squalene synthase enzyme encoded by a nucleic acid segment of the
present invention as described above. These squalene synthase
enzymes can be, for example, enzymes derived from S. cerevisiae or
other species of the genus Saccharomyces; they can also include
other squalene synthases derived from any organism that has a gene
that catalyzes the conversion of farnesyl pyrophosphate into
squalene.
[0111] When the variant squalene synthase enzymes are S. cerevisiae
squalene synthase enzymes, they include, for example, the
following:
[0112] (1) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 4 in Table 2;
[0113] (2) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 10 in Table 2;
[0114] (3) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 14 in Table 2;
[0115] (4) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 19 in Table 2;
[0116] (5) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 22 in Table 2;
[0117] (6) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 23 in Table 2;
[0118] (7) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 24 in Table 2;
[0119] (8) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 25 in Table 2;
[0120] (9) a variant S. cerevisiae squalene synthase enzyme as
shown as Mutant 69 in Table 2;
[0121] (10) a variant S. cerevisiae squalene synthase enzyme in
which the wild-type S. cerevisiae squalene synthase enzyme is
mutated with the same amino acid changes as in any of (1) through
(9) above;
[0122] (11) a variant S. cerevisiae squalene synthase enzyme
containing any of the amino acid changes as in any of (1) through
(10), above; and
[0123] (12) a variant S. cerevisiae squalene synthase enzyme in
which the squalene synthase enzyme differs from the variant
squalene synthase enzyme of any of (1) through (11) above by one to
three conservative amino acid substitutions, wherein a conservative
amino acid substitution is defined as one of the following
substitutions: A.fwdarw.G or S; R.fwdarw.K; N.fwdarw.Q or H;
D.fwdarw.E; C.fwdarw.S; Q.fwdarw.N; G.fwdarw.D; G.fwdarw.A or P;
H.fwdarw.N or Q; I.fwdarw.L or V; L.fwdarw.I or V; K.fwdarw.R or Q
or E; M.fwdarw.L or Y or I; F.fwdarw.M or L or Y; S.fwdarw.T;
T.fwdarw.S; W.fwdarw.Y; Y.fwdarw.W or F; and V.fwdarw.or L.
[0124] Furthermore, with respect to these alternatives, typically,
the variant squalene synthase enzyme has a reduced V.sub.max for
squalene synthesis. Alternatively, the variant squalene synthase
enzyme has an increased K.sub.m for its FPP substrate, in which
case the enzyme is less active at a given intracellular
concentration of FPP than the wild-type enzyme. Typically, the
variant squalene synthase enzyme, when expressed in vivo in a
suitable eukaryotic microbial host, as described above, produces
squalene at a rate of less than 75% of the wild-type enzyme.
Preferably, the variant squalene synthase enzyme, when expressed in
vivo in a suitable eukaryotic microbial host, as described above,
produces squalene at a rate of less than 50% of the wild-type
enzyme. More preferably, the variant squalene synthase enzyme, when
expressed in vivo in a suitable eukaryotic microbial host, produces
squalene at a rate of less than 25% of the wild-type enzyme.
[0125] With respect to (12) of these alternatives, preferably, the
variant S. cerevisiae squalene synthase enzyme differs from the
variant S. cerevisiae squalene synthase enzyme of any of (1)
through (10) above by one to two conservative amino acid
substitutions. More preferably, the variant S. cerevisiae squalene
synthase enzyme differs from the variant S. cerevisiae squalene
synthase enzyme of any of (1) through (10) above by one
conservative amino acid substitution.
[0126] Another aspect of the present invention is a host cell
containing and/or expressing a variant squalene synthase enzyme of
the present invention as described above. The host cell, in this
alternative, includes at least one copy of a nucleic acid sequence
encoding a variant squalene synthase enzyme. The at least one copy
of the nucleic acid sequence encoding a variant squalene synthase
enzyme can be present in the chromosome of a prokaryotic
(bacterial) cell or in one chromosome of a eukaryotic cell.
Alternatively, the at least one copy of the nucleic acid sequence
encoding a variant squalene synthase enzyme can be present in a
vector or plasmid that is present in the cell. The host cell, as
described above, can be a prokaryotic or eukaryotic cell. If it is
a prokaryotic cell, it can be a bacterial cell. If it is a
eukaryotic cell, it can be a yeast cell, a plant cell, or an animal
cell. Suitable host cells are described above.
[0127] Another aspect of the present invention is a method of
isolating a mutated ERG9 gene. The mutated ERG9 gene is typically a
S. cerevisiae gene, but can be a homologous gene from another
species as described above.
[0128] In general, a method of isolating a mutated ERG9 gene
according to the present invention comprises the steps of:
[0129] (1) isolating a wild-type ERG9 gene to produce an isolated
wild-type ERG9 gene;
[0130] (2) subjecting the isolated wild-type ERG9 gene to
mutagenesis to generate a pool of erg9 mutants;
[0131] (3) transforming mutants from the pool of erg9 mutants
generated in step (b) into a strain of a eukaryotic microbial host
that contains a plasmid expressing a terpene synthase gene that
produces a detectable and measurable terpene product, the strain of
the eukaryotic microbial host being transformed in such a manner
that replacement of the preexisting ERG9 allele with an erg9
mutation allows the strain to grow in a sterol-free medium; and
[0132] (4) isolating a transformant from step (c) that produces a
level of terpene product at least equivalent to the level of
terpene product produced by a strain of the eukaryotic microbial
host expressing the terpene synthase gene that requires a sterol in
the medium for growth.
[0133] In the present invention, the step of isolating a wild-type
ERG9 gene to produce an isolated wild-type ERG9 gene is typically
performed by amplifying a wild-type ERG9 gene by using a nucleic
acid amplification process to produce an amplified wild-type ERG9
gene; however, other isolation methods are known in the art and are
contemplated by this invention, it is not necessary to use PCR or
another nucleic acid amplification method. When a nucleic acid
amplification process is used, the nucleic acid amplification
method is typically PCR. However, other nucleic acid amplification
processes can be used that are well known in the art. In this
method, the ERG9 gene can be a fungal ERG9 gene, such as a
Saccharomyces cerevisiae ERG9 gene or an ERG9 gene of another
Saccharomyces species; alternatively, the ERG9 gene can be any
homologous ERG9 gene as described above. In this method, the
mutagenesis is typically performed using error-prone PCR, although
other mutagenesis methods are well known in the art and can
alternatively be used. Such mutagenesis methods include, for
example, ultraviolet (UV) radiation, ethyl methanesulfonate (EMS),
nitrosoguanidine, and other mutagens.
[0134] When error-prone PCR is used, the error-prone PCR is
typically performed using one or more DNA polymerase enzymes that
have higher misinsertion and misextension rates than wild-type
polymerase enzymes.
[0135] In the present invention, the terpene synthase gene that
produces a detectable and measurable terpene product can be, for
example, the Hyoscyamus muticus premnaspirodiene synthase (HPS)
gene. Preferably, the terpene synthase gene that produces a
detectable and measurable terpene product is one that produces a
product detectable and measurable by gas chromatography, although
other detection methods can be used.
[0136] In the present invention, a suitable strain of S. cerevisiae
that contains a plasmid expressing a terpene synthase gene that
produces a detectable and measurable terpene product is ALX7-95
(his3, trp1, erg9::HIS3, HMGcat/TRP1::rDNA, dpp1), a leucine
prototroph of strain CALI-5 (U.S. Pat. Nos. 6,531,303 and
6,689,593), containing a plasmid expressing the Hyoscyamus muticus
premnaspirodiene synthase (HPS) gene. Transformants of this strain
require histidine for growth; before transformation, this strain
requires supplementation with a sterol.
[0137] Although the invention can be used with isolates of
mutations in S. cerevisiae, analogous methods can be used with
other organisms, as described above.
[0138] Another aspect of the present invention is a method of
isolating a variant squalene synthase enzyme. The variant squalene
synthase enzyme to be isolated by these methods is as described
above.
[0139] In general, this method comprises the steps of:
[0140] (1) culturing a host cell that expresses a variant squalene
synthase enzyme or that contains a variant squalene synthase
enzyme; and
[0141] (2) isolating the variant squalene synthase enzyme from the
host cell.
[0142] Typically, this method further comprises the step of
purifying the isolated variant squalene synthase enzyme. Variant
squalene synthase enzymes according to the present invention can be
purified by conventional protein purification techniques,
including, for example, techniques such as precipitation with salts
such as ammonium sulfate, ion exchange chromatography, gel
filtration, affinity chromatography, electrophoresis, isoelectric
focusing, isotachophoresis, chromatofocusing, and other techniques
well known in the art and those described in R. K. Scopes, "Protein
Purification: Principles and Practice" (3rd ed., Springer-Verlag,
New York (1994)).
[0143] Yet another aspect of the present invention is a method of
producing an isoprenoid using a mutated ERG9 gene, in which the
defective ERG9 gene encodes a variant squalene synthase enzyme.
[0144] In one aspect of the invention, a host cell that includes a
mutated ERG9 gene encoding a variant squalene synthase enzyme
further includes at least one isoprenoid synthase gene, so that the
farnesyl pyrophosphate produced in the host cell, which is
available in greater concentrations for isoprenoid biosynthesis,
can be converted to an isoprenoid by the isoprenoid synthase
encoded by the isoprenoid synthase gene. For example, and not by
way of limitation, the isoprenoid synthase gene included in the
host cell can be a chimeric isoprenoid synthase gene such as those
described in U.S. Patent Application Publication No. 2008/0178354.
These chimeric isoprenoid synthase genes include derivatives of the
Hyoscyamus muticus vetispiradiene synthase gene and/or the
Nicotiana tabacum 5-epi-aristolochene synthase gene. Alternatively,
the isoprenoid synthase gene included in the host cell can be a
citrus valencene synthase gene as described in U.S. Patent
Application Publication No. 2006/0218661. As yet another
alternative, the isoprenoid synthase gene included in the host cell
can be a H. muticus premnaspirodiene synthase gene, such as those
described in Back and Chappell, J. Biol. Chem. 270:7375-7381
(1995); Back and Chappell, PNAS USA 93:6841-6845 (1996); and
Greenhagen et al., PNAS USA 103:9826-9831 (2006). As another
alternative, the isoprenoid synthase gene included in the host cell
can be an isoprenoid synthase gene such as those described in U.S.
Patent Application Publication No. 2005/0210549. These isoprenoid
synthase genes include 5-epi-aristolochene synthase from Capsicum
annuum, (E)-.beta.-farnesene synthase from Mentha piperita,
.delta.-selenene synthase and .gamma.-humulene synthase from Abies
grandis, 6-cadinene synthase from Gossypium arboreum,
E-.alpha.-bisabolene synthase from Abies grandis, germacrene C
synthase from Lycopersicon esculentum, epi-cedrol synthase and
amorpha-4,11-diene synthase from Artemisia annua, and germacrene A
synthases from Lactuca sativa, Cichorium intybus and Solidago
canadensis. Other suitable isoprenoid synthase genes are known in
the art. In addition, mutants and protein engineered variants of
these enzymes can be used. Methods for engineering variants of
terpene synthases are known in the art; such methods can, for
example, involve recombining domains from two or more terpene
synthases to generate a chimeric terpene synthase. Such methods are
described, for example, in U.S. Patent Application Publication No.
2006/0218661 and in U.S. Patent Application Publication No.
2008/0178354.
[0145] Accordingly, this aspect of the invention comprises the
steps of:
[0146] (1) providing a host cell including a mutated ERG9 gene
according to the present invention and at least one isoprenoid
synthase gene;
[0147] (2) allowing the host cell to produce farnesyl pyrophosphate
and to synthesize the isoprenoid from the farnesyl pyrophosphate;
and
[0148] (3) isolating the isoprenoid synthesized by the host
cell.
[0149] Methods for isolating the isoprenoid synthesized by the host
cell are well known in the art. For example, when the isoprenoid is
premnaspirodiene, the premnaspirodiene can be isolated by (i)
sequestering the premnaspirodiene by binding it to a hydrophobic
resin; (ii) and isolating the premnaspirodiene from the hydrophobic
resin. Preferably, the hydrophobic resin is Amberlite.RTM. XAD-16
hydrophobic resin. Other hydrophobic resins within the scope of the
present invention will be known to one of reasonable skill in the
art. Typically, premnaspirodiene is isolated from the hydrophobic
resin by methanol extraction. Other methods of isolating
premnaspirodiene that are within the scope of the present invention
will be known to one of reasonable skill in the art. Other
isoprenoids can be isolated by similar methods well known in the
art, making use of the fact that isoprenoids are hydrophobic and
bind to hydrophobic resins.
[0150] In an alternative for isolation of premnaspirodiene, a
two-phase system can be used with a non-polar solvent,
substantially immiscible with an aqueous phase, added to the
fermentation broth and the premnaspirodiene removed from the
non-polar phase by distillation. A preferred non-polar solvent is
an oil. A particularly preferred oil is a vegetable oil such as
soybean oil. Alternative non-polar solvents include, but are not
limited to, high molecular weight aliphatic hydrocarbons such as,
but not limited to, dodecane, tridecane, tetradecane, pentadecane,
and hexadecane; either straight-chain or branched-chain isomers can
be used; these high-molecular weight aliphatic hydrocarbons are
optionally substituted with one or more hydroxy or halogen
substituents as long as the substituted hydrocarbon remains
substantially immiscible with the aqueous phase.
[0151] In this method, where commercial production of the
isoprenoid is desired, a variety of fermentation methodologies may
be applied. For example, large scale production may be effected by
either batch or continuous fermentation. A classical batch
fermentation is a closed system where the composition of the medium
is set at the beginning of the fermentation and not subject to
artificial alterations during the fermentation. Thus, at the
beginning of the fermentation the medium is inoculated with the
desired microorganism or microorganisms and fermentation is
permitted to occur without further addition of nutrients.
Typically, the concentration of the carbon source in a batch
fermentation is limited, and factors such as pH and oxygen
concentration are controlled. In batch systems the metabolite and
biomass compositions of the system change constantly up to the time
the fermentation is stopped. Within batch cultures cells typically
modulate through a static lag phase to a high growth log phase and
finally to a stationary phase where growth rate is diminished or
halted. If untreated, cells in the stationary phase will eventually
die.
[0152] A variation on the standard batch system is the Fed-Batch
system. Fed-Batch fermentation processes are also suitable for use
in the present invention and comprise a typical batch system with
the exception that nutrients are added as the fermentation
progresses. Fed-Batch systems are useful when catabolite repression
is apt to inhibit the metabolism of the cells and where it is
desirable to have limited amounts of substrate in the medium. Also,
the ability to feed nutrients will often result in higher cell
densities in Fed-Batch fermentation processes compared to Batch
fermentation processes. Factors such as pH, dissolved oxygen,
nutrient concentrations, and the partial pressure of waste gases
such as CO.sub.2 are generally measured and controlled in Fed-Batch
fermentations. Batch and Fed-Batch fermentations are common and
well known in the art and examples may be found in Brock,
Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.;
Sinauer Associates: Sunderland, Mass. (1989); or Deshpande, Appl.
Biochem. Biotechnol. 36:227 (1992).
[0153] Commercial production of the isoprenoid may also be
accomplished with continuous fermentation. Continuous fermentation
is an open system where a defined fermentation medium is added
continuously to a bioreactor and an equal amount of conditioned
medium is removed simultaneously for processing. This system
generally maintains the cultures at a constant high density where
cells are primarily in their log phase of growth. Continuous
fermentation allows for modulation of any number of factors that
affect cell growth or end product concentration. For example, one
method will maintain a limiting nutrient such as the carbon source
or nitrogen level at a fixed rate and allow all other parameters to
moderate. In other systems a number of factors affecting growth can
be altered continuously while the cell concentration, measured by
the medium turbidity, is kept constant. Continuous systems strive
to maintain steady state growth conditions and thus the cell loss
due to the medium removal must be balanced against the cell growth
rate in the fermentation. Methods of modulating nutrients and
growth factors for continuous fermentation processes as well as
techniques for maximizing the rate of product formation are well
known in the art of industrial microbiology and a variety of
methods are detailed by Brock, supra.
[0154] Microorganism host cells useful in the present invention for
the production of the isoprenoid may include, but are not limited
to, bacteria, such as the enteric bacteria (Escherichia and
Salmonella for example) as well as Bacillus, Acinetobacter,
Streptomyces, Methylobacter, Rhodococcus and Pseudomonas;
Cyanobacteria, such as Rhodohacter and Synechocystis; yeasts, such
as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida,
Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis;
and filamentous fungi such as Aspergillus and Arthrobotrys; and
algae for example. Preferably, the host cell is a eukaryotic cell.
More preferably, the host cell is a yeast cell, which is a
eukaryotic microorganism host cell. Most preferably, the host cell
is a Saccharomyces cerevisiae cell.
[0155] Microbial expression systems and expression vectors
containing regulatory sequences that direct high level expression
of foreign proteins are well known to those skilled in the art.
These expression systems and expression vectors are known both for
prokaryotic organisms such as bacteria and for eukaryotic organisms
such as yeast. Similarly, vectors or cassettes useful for the
transformation of suitable microbial host cells are well known in
the art. These vectors and cassettes are known both for prokaryotic
organisms such as bacteria and for eukaryotic organisms such as
yeast. Typically, the vector or cassette contains sequences
directing expression of the relevant gene, a selectable marker, and
sequences allowing autonomous replication or chromosomal
integration. Suitable vectors comprise a region 5' of the gene
which harbors transcriptional initiation controls and a region 3'
of the DNA fragment which controls transcriptional termination.
[0156] Initiation control regions or promoters, which are useful to
drive expression of the relevant genes in the desired host cell are
numerous and familiar to those skilled in the art. Termination
control regions may also be derived from various genes native to
the preferred hosts.
[0157] Expression of cloned heterologous genes in yeast cells,
particularly cells of S. cerevisiae, is described in the following
references: Emir, Meth. Enzymol. 185: 231-233 (1991), is a general
overview of expression in yeast, including the possibility of
exploiting protein secretion and modification in yeast and
achieving stability of expressed proteins. Rose and Broach, Meth.
Enzymol. 185: 234-279 (1991), describes the use of 2-.mu.m
circle-based vectors for transfection of genes into yeast and for
expression of heterologous genes in yeast, including standard
2-.mu.m circle-based vectors, vectors for high copy propagation,
vectors for expression of cloned genes in yeast, and vectors for
specialized applications. Stearns et al., Meth. Enzymol. 185:
280-297 (1991), describes the use of yeast vector systems and
components, the use of homologous recombination to integrate
plasmids into the yeast host genome, and the use of centromere
plasmids. Mylin et al., Meth. Enzymol. 185: 297-308 (1991),
describes the use of galactose-inducible promoters to provide high
levels of production of cloned proteins in yeast. Price et al.,
Meth. Enzymol. 185: 308-318 (1991), describes the use of the
glucose-repressible ADH2 promoter to provide controllable, high
level expression of cloned proteins in yeast. Etcheveny, Meth.
Enzymol. 185: 319-329 (1991), describes the use of the yeast CUP1
promoter to drive controllable expression of cloned genes in yeast.
Kingman et al., Meth. Enzymol. 185: 329-341 (1991), describes the
use of the yeast PGK promoter to drive controllable expression of
cloned genes in yeast. Rosenberg et al., Meth. Enzymol. 185:
341-350 (1991), describes the use of expression cassette plasmids
utilizing the strong GAPDH-491 promoter for high levels of
heterologous protein production in yeast. Sledziewski et al., Meth.
Enzymol. 185: 351-366 (1991), describes the construction of
temperature-regulated variants of two strong yeast promoters, TPI1
and ADH2, and the use of these promoters for regulation of
expression and thus regulation of the extent of glycosylation of
proteins secreted by yeast. Donahue and Cigan, Meth. Enzymol. 185:
366-372 (1991), describes the significance of codon usage
variations between yeast and higher eukaryotes and the selection of
efficient leader sequences. Jones, Meth. Enzymol. 185: 372-386
(1991), describes the elimination of vacuolar protease activity in
yeast to maximize the yield of protein production from cloned
genes. Wilkinson, Meth. Enzymol. 185: 387-397 (1991), describes
methods for preventing ubiquitin-dependent protein degradation in
yeast, again to maximize the yield of protein production from
cloned genes. Kendall et al., Meth. Enzymol. 185: 398-407 (1991),
describes the cotranslational processing events that occur in yeast
at the amino-termini of nascent polypeptide genes and their effects
on heterologous gene expression and protein stability. Brake, Meth.
Enzymol. 185: 408-421 (1991), describes expression systems based on
the yeast .alpha.-factor leader. Hitzeman et al., Meth. Enzymol.
185: 421-440 (1991), describes the use of both heterologous and
homologous signal sequences for the production and secretion of
heterologous gene products in yeast. Chisholm et al., "Meth.
Enzymol. 185: 471-482 (1991), describes the use of an enhanced
secretion phenotype occurring among drug-resistant yeast mutants to
maximize secretion of cloned proteins in yeast.
[0158] General molecular biological techniques of gene cloning,
site-directed mutagenesis, and fusion protein construction can be
used to provide nucleic acid segments that include therein the
isoprenoid synthase gene. Typically, the nucleic acid segments are
DNA nucleic acid segments. Typically, as described above, the
isoprenoid synthase gene is operatively linked to at least one
nucleic acid expression control element, such as, but not limited
to, a promoter, an enhancer, or a site capable of binding a
repressor or activator. Such nucleic acid expression control
elements are well known in the art. Typically, as described above,
the isoprenoid synthase gene is included in a vector and, as such,
is again operatively linked to at least one nucleic acid expression
control element. Site-directed mutagenesis can be used, for
example, to provide optimum codon selection for expression in S.
cerevisiae, as described above. The isoprenoid synthase gene can,
in one alternative, be expressed in the form of a nucleic acid
segment encoding a fusion protein, such as a purification tag or
other detectable protein domain.
[0159] In another alternative method for producing an isoprenoid,
the method, in general, comprises the steps of:
[0160] (1) providing a host cell including a mutated ERGS gene
according to the present invention;
[0161] (2) allowing the host cell to produce farnesyl
pyrophosphate
[0162] (3) isolating farnesyl pyrophosphate from the host cell;
[0163] (4) reacting the farnesyl pyrophosphate in vitro with one or
more isoprenoid synthases to synthesize the isoprenoid; and
[0164] (5) isolating the isoprenoid.
[0165] As described above, a number of isoprenoid synthases are
available for in vitro use. These isoprenoid synthases have been
either cloned or isolated from plants. The step of isolating the
isoprenoid is performed as described above.
[0166] In both of these synthesis methods, additional reactions can
be performed on the isolated isoprenoid to transform the isolated
isoprenoid into another isoprenoid or related compound. These
additional reactions can be reactions such as oxidation,
hydroxylation, alkylation, halogenation, or other reactions well
known in the art. In particular, reactions such as hydroxylation or
oxidation can be carried out by cytochrome P450 enzymes. These
reactions, and methods of carrying them out by chemical or
enzymatic means, are well known in the art and need not be
described further here.
[0167] The present invention describes improved strains of
Saccharomyces cerevisiae that have a defective squalene synthase
enzyme. These strains have the ability to produce enough squalene
so that they do not need to be supplemented with sterols such as
ergosterol for growth. However, because these strains produce less
squalene than do wild-type strains, they have more farnesyl
pyrophosphate available for eventual isoprenoid synthesis, because
farnesyl pyrophosphate is a branch point for the steroid synthesis
and isoprenoid synthesis pathways. Therefore, these strains, as
well as the nucleic acid segments encoding the defective squalene
synthase enzyme and the defective squalene synthase enzymes
themselves, are useful for the improved production of isoprenoid
products because the strains of S. cerevisiae do not need to be
supplemented with sterols for growth and can produce high levels of
farnesyl pyrophosphate without such supplementation.
[0168] Because the eventual isoprenoid products have commercial
value as antibiotics, pest control agents, fragrances, flavors, and
anti-cancer agents, the nucleic acid segments, eukaryotic microbial
host cell strains, including S. cerevisiae strains, vectors and
host cells incorporating the nucleic acid segments, and the variant
squalene synthase enzymes have industrial utility.
[0169] The invention is illustrated by the following examples.
These examples are for illustrative purposes only, and are not
intended to limit the invention.
EXAMPLES
Example 1
Generation of Mutant ERG9 Genes
[0170] Chromosomal DNA isolated from Saccharomyces cerevisiae
strain ATCC28383 was used as the DNA template for PCR amplification
of the wild type ERG9 gene. The primers used for the amplification
were the upstream primer 7-162.1 5'-CCATCTTCAACAACAATACCG-3' (SEQ
ID NO: 1) (underlined nucleotides at the 5' end of the ERG9
sequence in FIG. 2) and the downstream primer 7-162.2
5'-GTACTTAGTTATTGTTCGG-3' (SEQ ID NO: 2) (underlined nucleotides at
the 3' end of ERG9). Using Taq polymerase (New England Biolabs),
amplification conditions were 94.degree. C. for 30 seconds,
45.degree. C. for 30 seconds, 72.degree. C. for 2 minutes for a
total of 30 cycles. The resulting ERG9 PCR product was sequenced
and verified to be identical to the published sequence for ERG9.
This DNA was used as the template for performing error prone PCR
using the GeneMorph kit from Stratagene and the same primers
described above. The error-prone PCR reaction was run using two
different DNA concentrations (.about.500 ng and .about.50 ng) to
generate a range of mutations per gene. This generated a pool of
ERG9 mutant genes.
Example 2
Isolation of erg9.sup.def Mutants
[0171] To isolate ERG9 mutants that make sufficient ergosterol to
restore growth, the PCR product from the mutagenic PCR reaction was
transformed into ALX7-95 (his3, trp1, erg9::HIS3,
HMGcat/TRP1::rDNA, dpp1), a leucine prototroph of strain CALI-5
(U.S. Pat. Nos. 6,531,303 and 6,689,593), containing a plasmid
expressing the Hyoscyamus muticus premnaspirodiene synthase (HPS)
gene. The HPS gene was cloned into the yeast shuttle expression
vector YEp-GW-URA-NheI/BamHI to give YEp-HPS-ura. This vector
contained the ADH1 promoter for initiating transcription of the HPS
gene. In addition, it contained the ADH1 terminator downstream of
the HPS gene. This vector was maintained in S. cerevisiae by
selecting media lacking uracil and it was maintained in E. coli by
selecting for resistance to ampicillin.
[0172] Transformation procedure of this strain with the ERG9 mutant
pool used the lithium acetate transformation kit from Sigma.
Transformants were selected for growth on YPD medium (10 g/L yeast
extract, 20 g/L peptone, 20 g/L glucose) without ergosterol. Since
the parent strain requires ergosterol for growth, transformants
that grew on YPD replaced the erg9::HIS3 replacement mutation with
a copy of ERG9 that made sufficient amount of ergosterol for
growth. This was verified by the fact that transformants had
obtained a requirement for histidine for growth.
[0173] To screen for premnaspirodiene production in strains
transformed with ERG9 mutant genes, a high-throughput screening
procedure using microvial cultures was employed. Transformant yeast
colonies were inoculated into individual wells of 96-well
microtiter plates filled with 200 .mu.L of SD (0.67 Bacto yeast
nitrogen base without amino acids, 2% glucose, 0.14% yeast
synthetic drop-out medium without uracil). The plate was grown for
two days at 28.degree. C. After growth to saturation, ten .mu.L
from each well was used to inoculate in duplicate two ml glass
vials containing 250 .mu.L of medium suitable for growth and
premnaspirodiene production. The vials were sealed with
serum-stoppered caps and then incubated with shaking for 3 days.
The products were extracted first by introducing 250 .mu.L of
acetone through the serum stopper and vortexing, followed by
addition of 500 .mu.l of n-hexane and vortexing. After phase
separation, the vials were placed on the sample tray of a gas
chromatography autosampler, which removed one microliter of the
organic phase for analysis of premnaspirodiene. The acetone and
hexane used for extraction were each spiked with internal standards
to aid in quantitation of the samples. The extracted samples were
analyzed by GC and the amount of premnaspirodiene was calculated
from the peak area.
[0174] Several mutant strains were identified that produced more or
similar amounts of premnaspirodiene as the control strain ALX7-95
containing expressed HPS. They were given the designations
ALX7-168.4, ALX7-168.10, ALX7-168.14, ALX7-168.19, ALX7-168.22,
ALX7-168.23, ALX7-168.24, ALX7-168.25, and ALX7-183.69. The ERG9
gene from these mutant strains was PCR amplified and the PCR
product was sequenced to determine the mutations within each
strain. A HIS3.sup.+ strain of ALX7-168.25 was constructed and is
designated ALX7-175.1.
Example 3
Production of Premnaspirodiene in Fermentors
[0175] As described in Example 2, strain ALX7-175.1 was constructed
for the production of premnaspirodiene. Production of
premnaspirodiene in this strain was compared to that of strain
ALX7-95 HPS, which is completely lacking in squalene synthase
activity.
[0176] Production of premnaspirodiene was carried out in a 3-L
fermentation tank (New Brunswick Bioflow 110). One liter of
fermentation medium was prepared and autoclaved in the fermentation
tank (20 g (NH.sub.4).sub.2SO.sub.4, 20 g KH.sub.2PO.sub.4, 1 g
NaCl, MgSO.sub.4.7H.sub.2O, 4 g yeast extract (Difco). Afterward
the following components were added: 20 ml mineral solution (0.028%
FeSO.sub.4.7H.sub.2O, 0.029% ZnSO.sub.4.7H.sub.2O, 0.008%
CuSO.sub.4.5H.sub.2O, 0.024% Na.sub.2MoO.sub.4.2H.sub.2O, 0.024%
CoCl.sub.2.6H.sub.2O, 0.017% MnSO.sub.4.H.sub.2O, 1 mL HCl); 10 mL
50% glucose; 30 mL vitamin solution (0.001% biotin; 0.012% calcium
pantothenate, 0.06% inositol, 0.012% pyridoxine-HCl, 0.012%
thiamine-HCl); 10 mL 10% CaCl.sub.2, and 20 mL autoclaved soybean
oil (purchased from local groceries). For ALX7-95 HPS, 1 mL of 50
mg/mL cholesterol in 100% ethanol was added.
[0177] The seed culture for inoculating the fermentation medium was
prepared by inoculating 50 mL of SD medium for ERG9 transformant
strains. Non-transformant control cultures were grown in SDE medium
(SD medium supplemented with 40 mg/L ergosterol). This culture was
grown until early stationary phase (24-48 hr). One mL of this
culture was inoculated into 500 mL of SD or SDE medium, as
appropriate, and grown for 24 hr. A 50-mL aliquot (5% inoculum) was
used to inoculate the one liter of medium.
[0178] The fermentor was maintained at 26.degree. C. The air flow
was 4.5 L/min and the dO.sub.2 was maintained above 30% by
adjusting the rpm. Furthermore, the pH was maintained at 4.5 using
acetic acid and NaOH.
[0179] Once the glucose concentration was below 1 g/L, a feeding
regimen was initiated such that the glucose in the fermentor was
kept between 0 and 1 g/L. The glucose feed was made by mixing 1400
mL of 60% glucose and 328 mL of 12.5% yeast extract.
[0180] After 5 days, the air and agitation were turned off, and the
oil was allowed to rise to the top of the tank and decanted.
Example 4
Comparison of Premnaspirodiene Production in erg9 Mutants and
erg9::HIS3 Strains in Microvial Cultures
[0181] As described in Example 2, initial screening of mutants was
conducted using microvial cultures. Mutants were further screened
in microvial cultures by growing in medium with or without 40 mg/L
ergosterol supplementation and compared to ALX7-95 BPS grown with
ergosterol (ALX7-95 BPS will not grow without ergosterol).
Twenty-five isolates are compared in FIG. 1. In FIG. 1, the
concentration of premnaspirodiene in mg/L is shown for each of the
25 isolates with and without ergosterol supplementation. Several of
these isolates produced more or comparable amounts of
premnaspirodiene as the control culture ALX7-95 HPS. In general,
these strains produced more premnaspirodiene in the absence of
ergosterol than in its presence. Isolates 4, 10, 14, 19, 22, 23,
24, 25, and 69 were given strain designations ALX7-168.4,
ALX7-168.10, ALX7-168.14, ALX7-168.19, ALX7-168.22, ALX7-168.23,
ALX7-168.24, ALX7-168.25, and ALX7-83.69 respectively.
Example 5
Comparison of Premnaspirodiene Production in erg9 Mutants and
erg9::HIS3 Strains in Fermentation Cultures
[0182] In this example, strains ALX7-95 HPS (erg9::HIS3) and
ALX7-175.1, a histidine prototroph of ALX7-168.25 (erg9.sup.def)
were grown in fermentors using the same protocol except for the
presence or absence of cholesterol in the medium. At the end of the
fermentation, premnaspirodiene was assayed by gas chromatography.
As indicated in Table 1, the yields of premnaspirodiene in the
fermentors was similar. However, because of faster growth and
growth to higher density of the erg9.sup.def strain, more glucose
was fed and consumed during the course of the fermentation. Because
of the resultant higher volume, more total premnaspirodiene was
produced by the erg9.sup.def culture grown under the same starting
conditions.
TABLE-US-00001 TABLE 1 Cell Premna- Final ERG9 Density, spirodiene
Volume, Total Yield, Strain Allele OD.sub.600 Titer, g/L Liters
grams ALX7-95 erg9::HIS3 48 3.9 1.1 4.3 HPS ALX7- erg9.sup.def 25
193 3.7 1.7 6.2 175.1
Example 6
Sequences of erg9.sup.def Mutants
[0183] The sequence of the wild type ERG9 gene and sequences 245
base pairs upstream are shown in FIG. 2. The ERG9 alleles from
strains described in Examples 2 and 4 were obtained by PCR
amplification of genomic DNA from strains designations ALX7-168.4,
ALX7-168.10, ALX7-168.14, ALX7-168.19, ALX7-168.22, ALX7-168.23,
ALX7-168.24, ALX7-168.25, and ALX7-183.69. The resulting DNA was
sequenced, and the sequences corresponding to those mutants are
shown in Table 2. All mutant genes contain mutations in the ERG9
coding region. Alleles 10, 23, 24, and 25 contain, in addition,
mutations in the 245 base pair noncoding region of upstream of the
gene.
TABLE-US-00002 TABLE 2 Nucleotide Amino Acid Affect of position and
position and change on Mutant change change amino acid 4 691
A.fwdarw.G 149 E.fwdarw.G 748 G.fwdarw.T 168 G.fwdarw.V 786
T.fwdarw.A 181 Y.fwdarw.N 1114 A.fwdarw.T 290 Q.fwdarw.L 1213
T.fwdarw.C 323 I.fwdarw.T 1290 T.fwdarw.C 349 L.fwdarw.L silent 10
72 C.fwdarw.A non-coding 110 .DELTA. A non-coding 801 G.fwdarw.A
186 V.fwdarw.I 14 989 T.fwdarw.A 248 P.fwdarw.P silent 1112
G.fwdarw.A 289 E.fwdarw.E silent 1220 G.fwdarw.A 325 K.fwdarw.K
silent 1233 T.fwdarw.C 330 Y.fwdarw.H 19 786 T.fwdarw.A 181
Y.fwdarw.N 1025 A.fwdarw.G 260 Q.fwdarw.Q silent 1056 T.fwdarw.A
271 L.fwdarw.I 1068 A.fwdarw.G 275 S.fwdarw.G 1203 A.fwdarw.G 320
N.fwdarw.D 22 886 T.fwdarw.C 214 M.fwdarw.T 969 A.fwdarw.G 242
I.fwdarw.V 1075 T.fwdarw.C 277 V.fwdarw.A 1114 A.fwdarw.T 290
Q.fwdarw.L 23 84 T.fwdarw.A non-coding 283 A.fwdarw.T 13 E.fwdarw.V
424 T.fwdarw.C 60 L.fwdarw.P 440 A.fwdarw.G 65 R.fwdarw.R silent
1076 T.fwdarw.C 277 V.fwdarw.V silent 24 619 A.fwdarw.T 125
D.fwdarw.V 634 T.fwdarw.C 130 L.fwdarw.P 962 C.fwdarw.T 239
P.fwdarw.P silent 25 150 A.fwdarw.T non-coding 410 T.fwdarw.G 55
A.fwdarw.A silent 411 G.fwdarw.T 56 V.fwdarw.L 1248 T.fwdarw.C 335
S.fwdarw.P 69 510 C.fwdarw.T 89 H.fwdarw.Y 573 T.fwdarw.C 110
F.fwdarw.L 918 A.fwdarw.G 224 R.fwdarw.G 997 A.fwdarw.G 251
K.fwdarw.G
[0184] As used in this specification and in the appended claims,
the singular forms include the plural forms. For example the terms
"a," "an," and "the" include plural references unless the content
clearly dictates otherwise. Additionally, the term "at least"
preceding a series of elements is to be understood as referring to
every element in the series. The inventions illustratively
described herein can suitably be practiced in the absence of any
element or elements, limitation or limitations, not specifically
disclosed herein. Thus, for example, the terms "comprising,"
"including," "containing," etc. shall be read expansively and
without limitation. Additionally, the terms and expressions
employed herein have been used as terms of description and not of
limitation, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the future shown and
described or any portion thereof, and it is recognized that various
modifications are possible within the scope of the invention
claimed.
[0185] Although the present invention has been described in
considerable detail with reference to certain preferred
embodiments, other embodiments are possible. The steps disclosed
for the present methods, for example, are not intended to be
limiting nor are they intended to indicate that each step is
necessarily essential to the method, but instead are exemplary
steps only. Therefore, the scope of the appended claims should not
be limited to the description of preferred embodiments contained in
this disclosure. All references cited herein are incorporated by
reference in their entirety.
Sequence CWU 1
1
4121DNAArtificial SequencePrimer 1ccatcttcaa caacaatacc g
21219DNAArtificial SequencePrimer 2gtacttagtt attgttcgg
1931631DNASaccharomyces cerevisiaeCDS(246)..(1580) 3gcccatcttc
aacaacaata ccgacttacc atcctatttg ctttgccctt tttcttttcc 60actgcacttt
gcatcggaag gcgttatcgg ttttgggttt agtgcctaaa cgagcagcga
120gaacacgacc acgggctata taaatggaaa gttaggacag gggcaaagaa
taagagcaca 180gaagaagaga aaagacgaag agcagaagcg gaaaacgtat
acacgtcaca tatcacacac 240acaca atg gga aag cta tta caa ttg gca ttg
cat ccg gtc gag atg aag 290 Met Gly Lys Leu Leu Gln Leu Ala Leu His
Pro Val Glu Met Lys 1 5 10 15 gca gct ttg aag ctg aag ttt tgc aga
aca ccg cta ttc tcc atc tat 338Ala Ala Leu Lys Leu Lys Phe Cys Arg
Thr Pro Leu Phe Ser Ile Tyr 20 25 30 gat cag tcc acg tct cca tat
ctc ttg cac tgt ttc gaa ctg ttg aac 386Asp Gln Ser Thr Ser Pro Tyr
Leu Leu His Cys Phe Glu Leu Leu Asn 35 40 45 ttg acc tcc aga tcg
ttt gct gct gtg atc aga gag ctg cat cca gaa 434Leu Thr Ser Arg Ser
Phe Ala Ala Val Ile Arg Glu Leu His Pro Glu 50 55 60 ttg aga aac
tgt gtt act ctc ttt tat ttg att tta agg gct ttg gat 482Leu Arg Asn
Cys Val Thr Leu Phe Tyr Leu Ile Leu Arg Ala Leu Asp 65 70 75 acc
atc gaa gac gat atg tcc atc gaa cac gat ttg aaa att gac ttg 530Thr
Ile Glu Asp Asp Met Ser Ile Glu His Asp Leu Lys Ile Asp Leu 80 85
90 95 ttg cgt cac ttc cac gag aaa ttg ttg tta act aaa tgg agt ttc
gac 578Leu Arg His Phe His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe
Asp 100 105 110 gga aat gcc ccc gat gtg aag gac aga gcc gtt ttg aca
gat ttc gaa 626Gly Asn Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr
Asp Phe Glu 115 120 125 tcg att ctt att gaa ttc cac aaa ttg aaa cca
gaa tat caa gaa gtc 674Ser Ile Leu Ile Glu Phe His Lys Leu Lys Pro
Glu Tyr Gln Glu Val 130 135 140 atc aag gag atc acc gag aaa atg ggt
aat ggt atg gcc gac tac atc 722Ile Lys Glu Ile Thr Glu Lys Met Gly
Asn Gly Met Ala Asp Tyr Ile 145 150 155 tta gat gaa aat tac aac ttg
aat ggg ttg caa acc gtc cac gac tac 770Leu Asp Glu Asn Tyr Asn Leu
Asn Gly Leu Gln Thr Val His Asp Tyr 160 165 170 175 gac gtg tac tgt
cac tac gta gct ggt ttg gtc ggt gat ggt ttg acc 818Asp Val Tyr Cys
His Tyr Val Ala Gly Leu Val Gly Asp Gly Leu Thr 180 185 190 cgt ttg
att gtc att gcc aag ttt gcc aac gaa tct ttg tat tct aat 866Arg Leu
Ile Val Ile Ala Lys Phe Ala Asn Glu Ser Leu Tyr Ser Asn 195 200 205
gag caa ttg tat gaa agc atg ggt ctt ttc cta caa aaa acc aac atc
914Glu Gln Leu Tyr Glu Ser Met Gly Leu Phe Leu Gln Lys Thr Asn Ile
210 215 220 atc aga gat tac aat gaa gat ttg gtc gat ggt aga tcc ttc
tgg ccc 962Ile Arg Asp Tyr Asn Glu Asp Leu Val Asp Gly Arg Ser Phe
Trp Pro 225 230 235 aag gaa atc tgg tca caa tac gct cct cag ttg aag
gac ttc atg aaa 1010Lys Glu Ile Trp Ser Gln Tyr Ala Pro Gln Leu Lys
Asp Phe Met Lys 240 245 250 255 cct gaa aac gaa caa ctg ggg ttg gac
tgt ata aac cac ctc gtc tta 1058Pro Glu Asn Glu Gln Leu Gly Leu Asp
Cys Ile Asn His Leu Val Leu 260 265 270 aac gca ttg agt cat gtt atc
gat gtg ttg act tat ttg gcc ggt atc 1106Asn Ala Leu Ser His Val Ile
Asp Val Leu Thr Tyr Leu Ala Gly Ile 275 280 285 cac gag caa tcc act
ttc caa ttt tgt gcc att ccc caa gtt atg gcc 1154His Glu Gln Ser Thr
Phe Gln Phe Cys Ala Ile Pro Gln Val Met Ala 290 295 300 att gca acc
ttg gct ttg gta ttc aac aac cgt gaa gtg cta cat ggc 1202Ile Ala Thr
Leu Ala Leu Val Phe Asn Asn Arg Glu Val Leu His Gly 305 310 315 aat
gta aag att cgt aag ggt act acc tgc tat tta att ttg aaa tca 1250Asn
Val Lys Ile Arg Lys Gly Thr Thr Cys Tyr Leu Ile Leu Lys Ser 320 325
330 335 agg act ttg cgt ggc tgt gtc gag att ttt gac tat tac tta cgt
gat 1298Arg Thr Leu Arg Gly Cys Val Glu Ile Phe Asp Tyr Tyr Leu Arg
Asp 340 345 350 atc aaa tct aaa ttg gct gtg caa gat cca aat ttc tta
aaa ttg aac 1346Ile Lys Ser Lys Leu Ala Val Gln Asp Pro Asn Phe Leu
Lys Leu Asn 355 360 365 att caa atc tcc aag atc gaa cag ttt atg gaa
gaa atg tac cag gat 1394Ile Gln Ile Ser Lys Ile Glu Gln Phe Met Glu
Glu Met Tyr Gln Asp 370 375 380 aaa tta cct cct aac gtg aag cca aat
gaa act cca att ttc ttg aaa 1442Lys Leu Pro Pro Asn Val Lys Pro Asn
Glu Thr Pro Ile Phe Leu Lys 385 390 395 gtt aaa gaa aga tcc aga tac
gat gat gaa ttg gtt cca acc caa caa 1490Val Lys Glu Arg Ser Arg Tyr
Asp Asp Glu Leu Val Pro Thr Gln Gln 400 405 410 415 gaa gaa gag tac
aag ttc aat atg gtt tta tct atc atc ttg tcc gtt 1538Glu Glu Glu Tyr
Lys Phe Asn Met Val Leu Ser Ile Ile Leu Ser Val 420 425 430 ctt ctt
ggg ttt tat tat ata tac act tta cac aga gcg tga 1580Leu Leu Gly Phe
Tyr Tyr Ile Tyr Thr Leu His Arg Ala 435 440 agtctgcgcc aaataacata
aacaaacaac tccgaacaat aactaagtac t 16314444PRTSaccharomyces
cerevisiae 4Met Gly Lys Leu Leu Gln Leu Ala Leu His Pro Val Glu Met
Lys Ala 1 5 10 15 Ala Leu Lys Leu Lys Phe Cys Arg Thr Pro Leu Phe
Ser Ile Tyr Asp 20 25 30 Gln Ser Thr Ser Pro Tyr Leu Leu His Cys
Phe Glu Leu Leu Asn Leu 35 40 45 Thr Ser Arg Ser Phe Ala Ala Val
Ile Arg Glu Leu His Pro Glu Leu 50 55 60 Arg Asn Cys Val Thr Leu
Phe Tyr Leu Ile Leu Arg Ala Leu Asp Thr 65 70 75 80 Ile Glu Asp Asp
Met Ser Ile Glu His Asp Leu Lys Ile Asp Leu Leu 85 90 95 Arg His
Phe His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe Asp Gly 100 105 110
Asn Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr Asp Phe Glu Ser 115
120 125 Ile Leu Ile Glu Phe His Lys Leu Lys Pro Glu Tyr Gln Glu Val
Ile 130 135 140 Lys Glu Ile Thr Glu Lys Met Gly Asn Gly Met Ala Asp
Tyr Ile Leu 145 150 155 160 Asp Glu Asn Tyr Asn Leu Asn Gly Leu Gln
Thr Val His Asp Tyr Asp 165 170 175 Val Tyr Cys His Tyr Val Ala Gly
Leu Val Gly Asp Gly Leu Thr Arg 180 185 190 Leu Ile Val Ile Ala Lys
Phe Ala Asn Glu Ser Leu Tyr Ser Asn Glu 195 200 205 Gln Leu Tyr Glu
Ser Met Gly Leu Phe Leu Gln Lys Thr Asn Ile Ile 210 215 220 Arg Asp
Tyr Asn Glu Asp Leu Val Asp Gly Arg Ser Phe Trp Pro Lys 225 230 235
240 Glu Ile Trp Ser Gln Tyr Ala Pro Gln Leu Lys Asp Phe Met Lys Pro
245 250 255 Glu Asn Glu Gln Leu Gly Leu Asp Cys Ile Asn His Leu Val
Leu Asn 260 265 270 Ala Leu Ser His Val Ile Asp Val Leu Thr Tyr Leu
Ala Gly Ile His 275 280 285 Glu Gln Ser Thr Phe Gln Phe Cys Ala Ile
Pro Gln Val Met Ala Ile 290 295 300 Ala Thr Leu Ala Leu Val Phe Asn
Asn Arg Glu Val Leu His Gly Asn 305 310 315 320 Val Lys Ile Arg Lys
Gly Thr Thr Cys Tyr Leu Ile Leu Lys Ser Arg 325 330 335 Thr Leu Arg
Gly Cys Val Glu Ile Phe Asp Tyr Tyr Leu Arg Asp Ile 340 345 350 Lys
Ser Lys Leu Ala Val Gln Asp Pro Asn Phe Leu Lys Leu Asn Ile 355 360
365 Gln Ile Ser Lys Ile Glu Gln Phe Met Glu Glu Met Tyr Gln Asp Lys
370 375 380 Leu Pro Pro Asn Val Lys Pro Asn Glu Thr Pro Ile Phe Leu
Lys Val 385 390 395 400 Lys Glu Arg Ser Arg Tyr Asp Asp Glu Leu Val
Pro Thr Gln Gln Glu 405 410 415 Glu Glu Tyr Lys Phe Asn Met Val Leu
Ser Ile Ile Leu Ser Val Leu 420 425 430 Leu Gly Phe Tyr Tyr Ile Tyr
Thr Leu His Arg Ala 435 440
* * * * *