U.S. patent application number 16/464104 was filed with the patent office on 2019-10-31 for control of protein to protein interactions of acid decarboxylase.
The applicant listed for this patent is CATHAY R&D CENTER CO., LTD., CIBT America Inc.. Invention is credited to Ling Chen, Howard Chou, Xiucai Liu, Wenqiang Lu.
Application Number | 20190330613 16/464104 |
Document ID | / |
Family ID | 62194569 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330613 |
Kind Code |
A1 |
Chou; Howard ; et
al. |
October 31, 2019 |
CONTROL OF PROTEIN TO PROTEIN INTERACTIONS OF ACID
DECARBOXYLASE
Abstract
This invention provides acid decarboxylase-prion subunit fusion
polypeptides, nucleic acid sequences, expression vectors, and host
cells expression such fusion polypeptides to produce various amino
acids and derivatives of the amino acids such as polyamines.
Inventors: |
Chou; Howard; (Shanghai,
CN) ; Chen; Ling; (Shanghai, CN) ; Lu;
Wenqiang; (Shanghai, CN) ; Liu; Xiucai;
(Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CATHAY R&D CENTER CO., LTD.
CIBT America Inc. |
Shanghai
Newark |
DE |
CN
US |
|
|
Family ID: |
62194569 |
Appl. No.: |
16/464104 |
Filed: |
November 24, 2016 |
PCT Filed: |
November 24, 2016 |
PCT NO: |
PCT/CN2016/107083 |
371 Date: |
May 24, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 2319/00 20130101; C12Y 401/01018 20130101; C12N 9/88 20130101;
C12N 15/70 20130101 |
International
Class: |
C12N 9/88 20060101
C12N009/88; C07K 14/47 20060101 C07K014/47; C12N 15/70 20060101
C12N015/70 |
Claims
1. A product, which is one of the following products I) through
IV): I) a genetically modified host cell comprising a nucleic acid
encoding an acid decarboxylase fusion protein comprising an acid
decarboxylase polypeptide joined to a prion subunit fused to the
carboxyl end of the acid decarboxylase polypeptide, wherein acid
decarboxylase fusion protein has increased activity relative to the
acid decarboxylase polypeptide not joined to the prion subunit; II)
an acid decarboxylase fusion protein comprising an acid
decarboxylase polypeptide fused to a prion subunit, wherein the
fusion protein has improved acid decarboxylase activity in vitro as
measured by the production of polyamines at alkaline pH, relative
to a counterpart fusion protein lacking the prion subunit; III) a
polynucleotide encoding a fusion protein of II); IV) an expression
vector comprising the polynucleotide of III), operably linked to a
promoter.
2. A product of claim 1, which is I) the genetically modified host
cell, wherein the prion subunit is at least 50 amino acids in
length, at least 75 amino acids in length or at least 100 amino
acids in length, but 500 amino acids or fewer in length.
3. A product of claim 1, which is I) the genetically modified host
cell of claim 1, wherein the prion subunit comprises an amino acid
composition having at least 20% glutamine and/or asparagine
residues.
4. A product of claim 1, which is I) the genetically modified host
cell, where the prion subunit: (i) has at least 70%, 75%, 80%, 85%,
90%, or 95% identity to a Sup35, New1, Ure2, or Rnq1 amino acid
sequence; (ii) comprises a Sup35, New1, Ure2, or Rnq1 amino acid
sequence; (iii) has at least 70%, 75%, 80%, 85%, 90%, or 95%
identity to an amino acid sequence set forth in SEQ ID NO:3 or SEQ
ID NO:4; (iv) comprises SEQ ID NO:3 or SEQ ID NO:4; or (v) has at
least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid
sequence of the prion subunit region of any one of SEQ ID NOS: 7,
8, 11, 13, 14, 15, 17, or 19.
5. (canceled)
6. (canceled)
5. A product of claim 1, which is I) the genetically modified host
cell, wherein the prion subunit is joined at the carboxyl terminus
to a BST fragment, .lamda.CI fragment, or RecA fragment.
6. A product of claim 1, which is I) the genetically modified host,
wherein prion subunit: (a) is joined at the C-terminal end to a BST
fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity
to the amino acid sequence of SEQ ID NO:15, excluding the linker
region; or comprises the amino acid sequence to SEQ ID NO:15,
excluding the linker region; (b) is joined at the C-terminal end to
a .DELTA.CI fragment and has at least 70%, 75%, 80%, 85%, 90%, or
95% identity to the amino acid sequence of SEQ ID NO:16, excluding
the linker region; or comprises the amino acid sequence to SEQ ID
NO:16, excluding the linker region; (c) is joined at the C-terminal
end to a RecA fragment and has at least 70%, 75%, 80%, 85%, 90%, or
95% identity to the amino acid sequence of SEQ ID NO:19, excluding
the linker region; or comprises the amino acid sequence to SEQ ID
NO:19, excluding the linker region; or (d) is joined at the
carboxyl terminus to an amino acid sequence RRFGEASSAF, ASQWPEETFG,
or EGVAETNEDF.
9. (canceled)
7. A product of claim 1, which is I) the genetically modified host
cell, wherein the acid decarboxylase is a lysine decarboxylase,
ornithine decarboxylase, arginine decarboxylase, or glutamate
decarboxylase.
8. A product of claim 1, which is I) the genetically modified host
cell of, wherein the acid decarboxylase is a CadA, LdcC, AdiA,
SpeA, SpeC, SpeF, GadA, or GadB polypeptide.
12. (canceled)
13. (canceled)
9. A product of claim 8, wherein the lysine decarboxylase is a CadA
lysine decarboxylase or a LdcC lysine decarboxylase.
10. A product of claim 8, wherein the host cell is genetically
modified to over express one or more lysine biosynthesis
polypeptides.
11. A product of claim 1, which is I) the genetically modified host
cell, wherein the nucleic acid encoding the acid decarboxylase
fusion protein is encoded by an expression vector introduced into
the cell, wherein the expression vector comprises the nucleic acid
encoding the acid decarboxylase fusion protein operably linked to a
promoter, and/or the nucleic acid encoding the acid decarboxylase
fusion protein is integrated into the host chromosome.
17. (canceled)
12. A product of claim 1, which is I) the genetically modified host
cell, wherein the nucleic acid encoding the acid decarboxylase
fusion polypeptide comprises the nucleic acid sequence of any one
of SEQ ID NOS:1, 5, 6, 9, 10, 16, 18, or 20.
13. A product of claim 1, which is I) the genetically modified host
cell, wherein the host cell is a bacterium.
14. A product of claim 13, wherein the host cell is from the genus
Escherichia or Hafnia.
15. A product of claim 14, wherein the host cell is Escherichia
coli or Hafnia alvei.
16. A method, which is one of the following methods I) through
III): (I) a method for producing an acid decarboxylase fusion
protein comprising cultivating a host cell of claim 1 I) under
conditions in which the acid decarboxylase fusion protein is
expressed; (II) a method of producing an amino acid or an amino
acid derivative, the method comprising culturing a host cell of
claim 1 I) under conditions in which the acid decarboxylase fusion
protein is expressed; (III) a method of improving acid
decarboxylase activity in vitro under alkaline pH comprising fusing
a prion subunit to the carboxyl terminus of an acid decarboxylase,
and subjecting the recombinant protein to alkaline pH.
23. (canceled)
24. (canceled)
25. (canceled)
17. A product of claim 1, which is II) the fusion protein, wherein
the prion subunit: (i) is 30 amino acids in length, at least 50
amino acids in length, at least 75 amino acids in length or at
least 100 amino acids in length, but 1200 amino acids or fewer in
length; (ii) comprises an amino acid composition having at least
20% glutamine and/or asparagine residues; (iii) has at least 70%,
75%, 80%, 85%, 90%, or 95% identity to a Sup35, New1, Ure2, or Rnq1
amino acid sequence; (iv) comprises a Sup35, New1, Ure2, or Rnq1
amino acid sequence; (v) has at least 70%, 75%, 80%, 85%, 90%, or
95% identity to an amino acid sequence set forth in SEQ ID NO: 3 or
SEQ ID NO: 4; (vi) comprises SEQ ID NO: 3 or SEQ ID NO: 4; or (vii)
has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino
acid sequence of the prion subunit region of any one of SEQ ID NOS:
7, 8, 11, 13, 14, 15, 17, or 19.
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
18. A product of claim 1, which is II) the fusion protein wherein
the prion subunit: (i) is joined to the C-terminus of the acid
decarboxylase; (ii) is joined at the carboxyl terminus to a BST
fragment, .DELTA.CI fragment, or RecA fragment; (iii) is joined the
carboxyl terminus to an amino acid sequence RRFGEASSAF, ASQWPEETFG,
or EGVAETNEDF; (iv) is joined at the C-terminal end to a BST
fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity
to the amino acid sequence of SEQ ID NO: 15, excluding the linker
region; or comprises the amino acid sequence to SEQ ID NO: 15,
excluding the linker region; (v) is joined at the C-terminal end to
a .DELTA.CI fragment and has at least 70%, 75%, 80%, 85%, 90%, or
95% identity to the amino acid sequence of SEQ ID NO: 16, excluding
the linker region; or comprises the amino acid sequence to SEQ ID
NO: 16, excluding the linker region; or (vi) is joined at the
C-terminal end to a RecA fragment and has at least 70%, 75%, 80%,
85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:
19, excluding the linker region; or comprises the amino acid
sequence to SEQ ID NO: 19, excluding the linker region.
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
19. A product of claim 1, which is II) the fusion protein, wherein
the acid decarboxylase is a CadA, LdcC, AdiA, SpeA, SpeC, SpeF,
GadA, or GadB polypeptide.
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
20. A product of claim 1, which is II) the fusion protein, wherein
the fusion protein is immobilized to a solid support.
42. (canceled)
43. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Most enzymes function optimally within a narrow pH range,
because they are amphoteric molecules. The pH of the surrounding
environment directly affects the charges on the acidic and basic
groups of the amino acids that make up the enzyme. These changes in
charge affect the net charge of the enzyme, the pKa of the active
site, and the charge distribution across the surface of the enzyme.
As a result, changes in pH can affect the activity, solubility, and
stability of an enzyme.
[0002] The class of proteins known as acid decarboxylases is a
group of enzymes that catalyze the decarboxylation reaction of
amino acids, e.g., basic amino acids such as lysine, arginine,
ornithine, in order to generate products, e.g., polyamines, as part
of the acid stress response in many microorganisms. Escherichia
coli has six pyridoxal 5'-phosphate (PLP)-inducible acid
decarboxylases: CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, and GadB.
All of these enzymes function within a narrow pH range, and the
enzyme's activity decreases significantly outside of that pH range
(Kanjee et al., Biochemistry 50, 9388-9398, 2011). It has been
previously observed that these PLP-dependent decarboxylases
dimerize in order to form a complete active site. In some cases,
such as CadA, the dimers form decamers that aggregate into higher
molecular weight protein complexes required for optimal function.
The inhibition of higher molecular weight protein complex formation
(e.g., in conditions outside of the optimal pH) leads to a
significant decrease in function (Kanjee et al., The EMBO Journal
30, 931-944, 2011).
[0003] Previous studies on the production of polyamines focused on
the overexpression of various acid decarboxylases. However, there
has not been any study on increasing the stability of the enzyme's
activity under various stresses such as alkaline pH. Tolerance to
alkaline pH by acid decarboxylases is important, because the
polyamines they generate as a product increase the pH of the
reaction environment. Therefore, the activity of the acid
decarboxylase usually decreases as more polyamines are generated,
which can cause the decarboxylation reaction to stop prematurely
when the pH of the reaction environment surpasses the pH range
tolerated by the acid decarboxylase.
[0004] The typical process to produce polyamines (e.g., cadaverine)
uses a process and fermentation medium similar to those used to
produce amino acids (e.g., lysine) (Qian et al., Biotechnol.
Bioeng. 108, 93-103, 2010). For example, ammonium sulfate is the
major nitrogen source due to its ability to provide nitrogen and
being slightly acidic (0.1M solution has a pH 5.5). The acidic pH
preserves the pH range for an acid decarboxylase, such as lysine
decarboxylases, e.g., CadA, to function. However, the use of
ammonium sulfate leaves sulfate ions in the medium, which becomes a
byproduct that is a salt waste during the fermentation process. The
ability to tolerate alkaline pH allows for the use alternative
nitrogen sources, and the production of less salt waste during the
fermentation process.
[0005] Prions were identified as the infectious agent that causes
transmissible spongiform encephalopathy (similar to "mad cow
disease", sheep scrapie, human kuru, and Creutzfeldt, Jacob
disease). For a review on prions, see Derkatch & Liebman, Prion
1:3, 161-169, 2007. Prions are protein conformations that are
infectious. The protein may have other roles in the cell when they
are not in the prion conformation. Proteins that form the prion
conformation are not homologous, but some are rich in glutamine or
asparagine residues. Prion aggregates are highly ordered, and
typically form through intermolecular interactions between
beta-strands. Therefore, prion conformations are beta-sheet rich,
and assemble into structures that resemble amyloid fibers. See,
e.g., Derkatch & Liebman, Prion 1:3, 161-169, 2007,
[0006] Prions have also been identified in yeast, and were first
observed in two yeast determinants: [PSI.sup.] and [URE3]
(Kushnirov & Ter-Avanesyan, Cell 94, 13-16, 1998). It was
observed that the formation of prions could be induced by
overexpression of the proteins Sup35 or Ure2. It was found that the
Sup35 and Ure2 proteins in the yeast cells that have [PSI.sup.+] or
[URE3] phenotypes show increase protease resistance and are found
in a high-molecular weight aggregated state. In addition to the
Sup35 and Ure2 proteins, two other proteins were identified in
yeast with the ability to form prion conformations--New1 and Rnq1
(Osherovich et al., PLOS Biology 2, 442-451, 2004) LikeSup35 and
Ure2, New1 and Rnq1 also have long series of sequences rich in
glutamine and asparagine. Previously, Sup35 has been fused to GST
in pGEX-4T-3 (a 25 kD protein) (Ono et al., Biosci. Biotechnol.
Biochem. 70, 2813-2823, 2006), and Sup35, New1, and Rnq1 have been
fused to GFP (a 27 kD protein) (Garrity et al., PNAS 107,
10596-10601, 2010). These proteins are relatively small, however.
An acid decarboxylase monomer is about 81 kD (more than three times
larger than GST) and forms higher molecular weight structures,
e.g., that are larger than 1620 kD (more than sixty times larger
than GST), in order to function. There have been no studies
evaluating whether prions can be fused to much larger proteins
without affecting function.
BRIEF SUMMARY OF ASPECTS OF THE DISCLOSURE
[0007] This invention is based, in part, on the surprising
discovery that fusing a prion protein to an acid decarboxylase
increases the stability of the enzyme's activity under various
stresses that typically cause the protein complex to transition
from a high oligomerization state to a low oligomerization state
(e.g., alkaline pH and high temperature).
[0008] In one aspect, the disclosure thus provides a genetically
modified host cell comprising a nucleic acid encoding an acid
decarboxylase fusion protein comprising an acid decarboxylase
polypeptide joined to a prion subunit fused to the carboxyl end of
the acid decarboxylase polypeptide, wherein acid decarboxylase
fusion polypeptide has increased activity relative to the acid
decarboxylase polypeptide not joined to the prion subunit. In some
embodiments, the prion subunit is at least 50 amino acids in
length, at least 75 amino acids in length or at least 100 amino
acids in length, but 500 amino acids or fewer in length. The prion
subunit typically has an amino acid composition of 10% or greater
glutamine and/or asparagine residues. In some embodiments, the
prion subunit comprises an amino acid composition having at least
20% glutamine and/or asparagine residues. In some embodiments, the
prion subunit has at least 70%, 75%, 80%, 85%, 90%, or 95% identity
to a Sup35, New1, Ure2, or Rnq1 amino acid sequence; or comprises a
Sup35, New1, Ure2, or Rnq1 amino acid sequence. In some
embodiments, the prion subunit has at least 70%, 75%, 80%, 85%,
90%, or 95% identity to an amino acid sequence set forth in SEQ ID
NO:3 or SEQ ID NO:4; or comprises the amino acid sequence of SEQ ID
NO:3 or SEQ ID NO:4. In further embodiments, the prion subunit is
joined at the carboxyl terminus to a BST fragment, .lamda.CI
fragment, or RecA fragment, for example a fragment having the amino
acid sequence RRFGEASSAF, ASQWPEETFG, or EGVAETNEDF. In some
embodiments, the prion subunit is joined at the C-terminal end to a
BST fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95%
identity to the amino acid sequence of SEQ ID NO:15, excluding the
linker region; or comprises the amino acid sequence to SEQ ID
NO:15, excluding the linker region. In some embodiments, the prion
subunit is joined at the C-terminal end to a .lamda.CI fragment and
has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino
acid sequence of SEQ ID NO:16, excluding the linker region; or
comprises the amino acid sequence to SEQ ID NO:16, excluding the
linker region. In some embodiments, the prion subunit is joined at
the C-terminal end to a RecA fragment and has at least 70%, 75%,
80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID
NO:19, excluding the linker region; or comprises the amino acid
sequence to SEQ ID NO:19, excluding the linker region. In some
embodiments, the acid decarboxylase is a lysine decarboxylase,
ornithine decarboxylase, glutamate decarboxylase, or arginine
decarboxylase. In some embodiments, the acid decarboxylase is a
CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, or GadB polypeptide. For
example, in some embodiments, the acid decarboxylase is a lysine
decarboxylase, such as a CadA lysine decarboxylase polypeptide or a
LdcC polypeptide. In some embodiments where the acid decarboxylase
is a lysine decarboxylase, the host cell is genetically modified to
over express one or more lysine biosynthesis polypeptides. In some
embodiments, the nucleic acid encoding the acid decarboxylase
fusion protein is encoded by an expression vector introduced into
the cell, wherein the expression vector comprises the nucleic acid
encoding the acid decarboxylase fusion protein operably linked to a
promoter. In alternative embodiments, the nucleic acid encoding the
acid decarboxylase fusion protein is integrated into the host
chromosome. The host cell may be a bacterium, such as a bacterium
from the genus Escherichia or Hafnia. In some embodiments, the host
cell is Escherichia coli or Hafnia alvei.
[0009] In a further aspect, the invention provides a method for
producing an acid decarboxylase fusion protein comprising
cultivating a host cell as described in the preceding paragraph
under conditions in which the acid decarboxylase fusion protein is
expressed. In another aspect, the invention provides a method of
producing an amino acid or an amino acid derivative, the method
comprising culturing a host cell as described in the preceding
paragraph under conditions in which the acid decarboxylase fusion
polypeptide is expressed.
[0010] The invention additionally provides a method of improving
acid decarboxylase activity in vitro under alkaline pH and/or high
temperature. In some embodiments, the method comprises fusing a
prion subunit to the carboxyl terminus of an acid decarboxylase and
subjecting the fusion protein to alkaline pH. In some embodiments,
the method comprises fusing a prion subunit to the carboxyl
terminus of an acid decarboxylase and subjecting the fusion protein
to high temperature.
[0011] In an additional aspect, the invention provides an acid
decarboxylase fusion protein comprising an acid decarboxylase
polypeptide fused to a prion subunit, wherein the fusion protein
has improved acid decarboxylase activity in vitro as measured by
the production of polyamines at elevated temperature and/or
alkaline pH, relative to a counterpart fusion protein lacking the
prion subunit. In some embodiments, the prion subunit is 30 amino
acids in length, at least 50 amino acids in length, at least 75
amino acids in length or at least 100 amino acids in length, but
1200 amino acids or fewer in length. In some embodiments, the prion
subunit comprises an amino acid composition having at least 20%
glutamine and/or asparagine residues. In further embodiments, the
prion subunit has at least 70%, 75%, 80%, 85%, 90%, or 95% identity
to a Sup35, New1, Ure2, or Rnq1 amino acid sequence; or comprises a
Sup35, New1, Ure2, or Rnq1 amino acid sequence. In still other
embodiments, the prion subunit has at least 70%, 75%, 80%, 85%,
90%, or 95% identity to the amino acid sequence set forth in SEQ ID
NO:3 or SEQ ID NO:4; or comprises the amino acid sequence of SEQ ID
NO:3 or SEQ ID NO:4. In other embodiments, the prion subunit has at
least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid
sequence of the prion subunit region of SEQ ID NO:7, 8, 11, 12, 13,
14, 15, or 19; or comprises the prion subunit region of SEQ ID
NO:7, 8, 11, 12, 13, 14, 15, or 19. In some embodiments, the prion
subunit is joined to the C-terminus of the acid decarboxylase. In
some embodiments, the prion subunit is joined at the carboxyl
terminus to a stability fragment, e.g., a BST fragment, .lamda.CI
fragment, or RecA fragment, such as a fragments having the amino
acid sequence RRFGEASSAF, ASQWPEETFG, or EGVAETNEDF. In some
embodiments, the prion subunit is joined at the C-terminal end to a
BST fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95%
identity to the amino acid sequence of SEQ ID NO:15, excluding the
linker region; or comprises the amino acid sequence to SEQ ID
NO:15, excluding the linker region. In some embodiments, the prion
subunit is joined at the C-terminal end to a .lamda.CI fragment and
has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino
acid sequence of SEQ ID NO:16, excluding the linker region; or
comprises the amino acid sequence to SEQ ID NO:16, excluding the
linker region. In some embodiments, the prion subunit is joined at
the C-terminal end to a RecA fragment and has at least 70%, 75%,
80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID
NO:19, excluding the linker region; or comprises the amino acid
sequence to SEQ ID NO:19, excluding the linker region. In some
embodiments, the acid decarboxylase is a lysine decarboxylase,
ornithine decarboxylase, arginine decarboxylase, or glutamate
decarboxylase. In still other embodiments, the acid decarboxylase
is a CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, or GadB polypeptide.
In some embodiments, the acid decarboxylase is a lysine
decarboxylase, such as a CadA lysine decarboxylase and the fusion
protein has improved lysine decarboxylase activity in vitro as
measured by the production of cadaverine at elevated temperature
and/or alkaline pH, relative to a counterpart fusion protein
lacking the prion subunit. In some embodiments, the fusion protein
is immobilized to a solid support.
[0012] In further aspects the invention provides a polynucleotide
encoding a fusion protein as described herein and expression
vectors that comprise such polynucleotides.
[0013] Other aspects of the invention are further described herein
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. SDS-PAGE results showing the soluble (s) and
precipitate (p) fractions of lysed cell cultures from H. avlei
transformed with either pCIB128 or pCIB222. The results from two
different colonies are shown for each transformant.
[0015] FIG. 2. SDS-PAGE results showing the total protein of lysed
cell cultures from E. coli BL21 transformed with either pCIB222 or
one of its truncated variants. The results from two different
colonies are shown for each transformant.
DETAILED DESCRIPTION OF ASPECTS OF THE DISCLOSURE
[0016] Before the present invention is described, it is to be
understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting.
[0017] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications and accession numbers mentioned herein are
incorporated herein by reference to disclose and describe the
methods and/or materials in connection with which the publications
are cited.
Terminology
[0018] A "prion" refers to an infectious agent comprised of protein
material that can trigger normal proteins to fold into multiple,
structurally distinct conformations (For a review on prions, see
Derkatch & Liebman, Prion 1:3, 161-169, 2007). Illustrative
examples of yeast prion proteins isolated from S. cerevisiae
include Sup35, Ure2, New1, Rnq1, Swi1, Cyc8, Mot3, Spf1 and Mod5.
An illustrative example of a filamentous fungal prion protein
isolated from Podospora anserine is Het-s. The term "prion variant"
as commonly used in the art refers to prion isolates with different
properties despite being based on a prion protein with the same
sequence. A "prion sequence variant" as used in the present
invention refers to a prion amino acid sequence that differs from
the amino acid sequence of a native prion amino acid sequence put
retains activity of the native prion amino acid sequence when fused
to an acid decarboxylase.
[0019] As used herein, the term "prion subunit" refers to a minimal
amino acid sequence of a prion protein that is fused to an acid
decarboxylase in accordance with the invention and increases the
activity of the acid decarboxylase compared to the acid
decarboxylase when it is not fused to the prion subunit. The
minimal amino acid sequence (and corresponding polynucleotide
sequence) comprising the prion subunit can generally be found in
the N-terminal region of prion proteins, such as Ure2, Sup35, New1
or Rnq1. In one embodiment, the prion subunit is at least 30 amino
acids in length, at least 40 amino acids in length, at least 50
amino acids in length, at least 60 amino acids in length, at least
70 amino acids in length, at least 75 amino acids in length, at
least 100 amino acids in length, at least 150 amino acids in
length, at least 200 amino acids in length, or at least 300 amino
acids in length, or more, but 1200 amino acids or fewer in length.
In some embodiments, a prion subunit is 1000 amino acids or fewer
in length. In some embodiments, a prion submit is 500 amino acids
or fewer in length. In another embodiment, the prion subunit is
between 30 and 200 amino acids in length, between 40 and 150 amino
acids in length, or between 50 and 120 amino acids in length. A
prion subunit when fused to an acid decarboxylase improves activity
of the acid decarboxylase in response to stress conditions, such as
alkaline pH or elevated temperature compared to the acid
decarboxylase that is not fused to the prion subunit.
[0020] As used in the context of the present disclosure, an "acid
decarboxylase" refers to a polypeptide that catalyzes the
decarboxylation reaction of basic amino acids (e.g., lysine,
arginine, ornithine, glutamate) to generate polyamines. Acid
decarboxylases include lysine decarboxylases, e.g., CadA, LdcD;
arginine decarboxylases, e.g., AdiA; ornithine decarboxylases,
e.g., SpeC, SpeF; and glutamate decarboxylases, e.g., GadA, GadB;
that are part of the prokaryotic ornithine decarboxylase subclass
of Fold Type I pyridoxal 5'-phosphate (PLP)-dependent
decarboxylases. This class of proteins typically contains a
N-terminal wing domain, a core domain, and a C-terminal domain. The
core domain contains a PLP-binding subdomain. The acid
decarboxylase SpeA is also a PLP-dependent decarboxylase, but
belongs to a different fold family of the PLP-dependent
decarboxylases that contain a TIM barrel domain, .beta.-sandwich,
insert, and C-terminal domain (Forouhar, et al., Acta. Cryst. F66,
1562-1566, 2010). Acid decarboxylase monomers may form multimers of
various sizes, depending on the acid decarboxylase. For example,
the acid decarboyxlases CadA, LdcC, and AdiA form a two-fold
symmetric dimer that completes the active site of each monomer.
Five dimers associate to form a decamer having a double-ringed
structure with five-fold symmetry. The decamerscan associate with
other decamers to form higher-order oligomers under favorable pH
conditions. Not all acid decarboxylases form decamers to function.
For example, the acid decarboxylases GadA and GadB form hexamers,
and SpeA forms tetramers. According to Kanjee et al., 2011 the acid
decarboxylases CadA, LdcC, AdiA, SpeC, and SpeF share the same
structural fold and exist at minimum as homodimers. Crystal
structure analysis indicates that GadA and GadB also share the same
Type I fold of PLP-dependent enzymes, such as CadA, LdcC, AdiA,
SpeC, and SpeF (Capitani, et al., The EMBO Journal 22, 4027-4037,
2003). Similarity between LdcC decamer and CadA decamer is
described in Kandia, et al., Sci. Rep. 6, 24601, 2016. AdiA decamer
formation is described in Boeker E A & Snell E E, J. Biol.
Chem. 243, 1678-1684, 1968 and Andrell, et al., Biochemistry 48,
3915-3927, 2009. A structural description of GadA and GadB is
described in Capitani, et al., The EMBO Journal 22, 4027-4037,
2003. The protein data bank IDs for structures of illustrative acid
decarboxylases are: 3N75 (CadA), 5FKZ (LdcCd), and 2VYC (AdiA).
Other E. coli acid decarboxylases such as SpeC and SpeF form
homodimers. SpeA forms homotetramer (PDB ID: 3NZQ), while GadA (PDB
ID: 1XEY) and GadB (PDB ID: 1PMM) form homohexamers.
[0021] The term "acid decarboxylase" encompasses biologically
active variants, alleles, mutants, and interspecies homologs to the
specific polypeptides described herein. A nucleic acid that encodes
an acid decarboxylase refers to a gene, pre-mRNA, mRNA, and the
like, including nucleic acids encoding variants, alleles, mutants,
and interspecies homologs of the particular amino acid sequences
described herein.
[0022] An "acid decarboxylase fusion polypeptide" as used herein
refers to a polypeptide comprising an acid decarboxylase fused to a
prion subunit. An "acid decarboxylase fusion polynucleotide" or
"acid decarboxylase fusion gene" refers to a nucleic acid that
encodes an acid decarboxylase fusion polypeptide.
[0023] A lysine decarboxylase refers to an enzyme that converts
L-lysine into cadaverine. The enzyme is classified as E.C.
4.1.1.18. Lysine decarboxylase polypeptides are well characterized
enzymes, the structures of which are well known in the art (see,
e.g., Kanjee, et al., EMBO J. 30: 931-944, 2011; and a review by
Lemmonier & Lane, Microbiology 144; 751-760, 1998; and
references described therein). Illustrative lysine decarboxylase
sequences are CadA homologs from Klebsiella sp., WP 012968785.1;
Enterobacter aerogenes, YP 004592843.1; Salmonella enterica, WP
020936842.1; Serratia sp., WP 033635725.1; and Raoultella
ornithinolytica, YP 007874766.1; and LdcC homologs from Shigella
sp., WP 001020968.1; Citrobacter sp., WP 016151770.1; and
Salmonella enterica, WP 001021062.1. As used herein, a lysine
decarboxylase includes variants of native lysine decarboxylase
enzymes that have lysine decarboxylase enzymatic activity.
Additional lysine decarboxylase enzyme are described in
PCT/CN2014/080873 and PCT/CN2015/072978.
[0024] A "cadA" polypeptide refers to an Escherichia coli cadA
polypeptide having the amino acid sequence of SEQ ID NO:2, or a
biologically active variant thereof that has acid decarboxylase
activity. Biologically active variants include alleles, mutants,
and interspecies homologs of the E. coli cadA polypeptide. CadA
contains an N-terminal wind domain, a core domain, and a C-terminal
domain. Illustrative cadA polypeptides from other species include
Salmonella enterica, protein sequence accession number WP
001021062.1. In some embodiments, a "CadA" polypeptide has at least
60% amino acid sequence identity, preferably at least 65%, 70%,
75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% or greater amino acid sequence identity, preferably over
a region of at least about 50, 100, or more, amino acids, or over
the length of the cadA polypeptide of SEQ ID NO:2. A "CadA
polynucleotide" as used herein refers to a polynucleotide that
encodes a CadA polypeptide.
[0025] As used herein, the term "alkaline pH" refers to a solution
or surrounding environment having a pH of greater than 7.5. In one
embodiment, alkaline pH refers to a solution or surrounding
environment having a pH of at least 8.0 or at least 8.5, or
higher.
[0026] As used herein, the term "elevated temperature" or "high
temperature" refers to a temperature about 35.degree. C. or
greater. In some embodiments, a higher temperature is at least
37.degree. C., at least 40.degree. C., at least 42.degree. C., at
least 45.degree. C., at least 48.degree. C., at least 50.degree.
C., at least 52.degree. C., at least 55.degree. C., or greater. In
one embodiment, elevated temperature refers to a temperature of at
least 42.degree. C. but less than 60.degree. C.
[0027] The term "enhanced" or "improved" in the context of the
production of an amino acid, e.g., lysine, or a lysine derivative,
e.g., cadaverine, as used herein refers to an increase in the
production of an amino acid or the amino acid derivative produced
by a host cell that expresses an acid decarboxylase fusion
polypeptide comprising an acid decarboxylase polypeptide fused to a
prion subunit, e.g., at the carboxyl end of the acid decarboxylase
polypeptide, in comparison to a control counterpart cell, such as a
cell of the wildtype strain or a cell of the same strain that
expresses the acid decarboxylase protein, but is not fused to the
prion subunit. In one embodiment, acid decarboxylase activity of
the acid decarboxylase fusion protein, e.g., where the prion
subunit is fused to the carboxyl end of the acid decarboxylase, is
improved by at least 5%, typically at least 10%, 15% 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, or greater compared to the acid
decarboxylase activity of a counterpart cell expressing an acid
decarboxylase lacking the prion subunit, where activity is assessed
by measuring the production of polyamines, such as cadaverine, and
lysine produced by the host cell and control cell under identical
conditions. In some embodiments, activity of a lysed extract from a
host cell culture is measured at an elevated temperature. In some
embodiments, activity of a lysed extract from a host cell culture
is measure under alkaline conditions. For example, activity of an
acid decarboxylase fusion polypeptide of the invention can be
assessed by evaluating an aliquot of a culture of host cells
transformed with the acid decarboxylase fusion polypeptide compared
to a corresponding aliquot from a culture of counterpart host cells
of the same strain that expresses the acid decarboxylase without
fusion to the prion subunit. By way of illustration, the activity
of a lysine decarboxylase fusion polypeptide of the invention
compared to the counterpart lysine decarboxylase not fused to the
prion subunit can be determined by evaluating the reaction rates of
a lysed sample, e.g., from a 100 ml sample, at a pH of 8.0.
Reaction rates can be measured using NMR by sampling the amount of
lysine converted in the presence of PLP into cadaverine about every
1.5 minutes for a total of 20 minutes, and taking the slope of the
linear portion of the yield curve. The samples are diluted so that
the reaction rate per volume (U) of lysed sample measured at pH 6.0
and 35.degree. C. is the same. The kinetic constants Vmax and Km
for lysine of each lysed samples is measured using the same U at an
initial pH of 8. By normalizing for U, the concentration of active
enzyme in each sample is the same.
[0028] The terms "numbered with reference to", or "corresponding
to," or "determined with reference to" when used in the context of
the numbering of a given amino acid or polynucleotide sequence,
refers to the numbering of the residues of a specified reference
sequence when the given amino acid or polynucleotide sequence is
compared to the reference sequence. For example, a segment of a
prion subunit polypeptide sequence "corresponds to" a segment in
SEQ ID NO:4 when the segment aligns with SEQ ID NO:4 in a maximal
alignment.
[0029] The terms "polynucleotide" and "nucleic acid" are used
interchangeably and refer to a single or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the
3' end. A nucleic acid as used in the present invention will
generally contain phosphodiester bonds, although in some cases,
nucleic acid analogs may be used that may have alternate backbones,
comprising, e.g., phosphoramidate, phosphorothioate,
phosphorodithioate, or O-methylphosphoroamidite linkages (see
Eckstein, Oligonucleotides and Analogues: A Practical Approach,
Oxford University Press); positive backbones; non-ionic backbones,
and non-ribose backbones. Nucleic acids or polynucleotides may also
include modified nucleotides that permit correct read-through by a
polymerase. "Polynucleotide sequence" or "nucleic acid sequence"
includes both the sense and antisense strands of a nucleic acid as
either individual single strands or in a duplex. As will be
appreciated by those in the art, the depiction of a single strand
also defines the sequence of the complementary strand; thus the
sequences described herein also provide the complement of the
sequence. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences, as
well as the sequence explicitly indicated. The nucleic acid may be
DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid
may contain combinations of deoxyribo- and ribo-nucleotides, and
combinations of bases, including uracil, adenine, thymine,
cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,
isoguanine, etc.
[0030] The term "substantially identical," used in the context of
two nucleic acids or polypeptides, refers to a sequence that has at
least 40%, 45%, or 50% sequence identity with a reference sequence.
Percent identity can be any integer from 50% to 100%. Some
embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared
to a reference sequence using the programs described herein;
preferably BLAST using standard parameters, as described below.
[0031] Two nucleic acid sequences or polypeptide sequences are said
to be "identical" if the sequence of nucleotides or amino acid
residues, respectively, in the two sequences is the same when
aligned for maximum correspondence as described below. The terms
"identical" or percent "identity," in the context of two or more
nucleic acids or polypeptide sequences, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of amino acid residues or nucleotides that are the same,
when compared and aligned for maximum correspondence over a
comparison window, as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
When percentage of sequence identity is used in reference to
proteins or peptides, it is recognized that residue positions that
are not identical often differ by conservative amino acid
substitutions, where amino acids residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art. Typically this involves scoring a conservative
substitution as a partial rather than a full mismatch, thereby
increasing the percentage sequence identity. Thus, for example,
where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1.
[0032] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0033] An algorithm that may be used to determine whether an acid
decarboxylase fusion polypeptide has sequence identity to a
sequence, e.g., SEQ ID NO:2; or any one of SEQ ID NOS:21-28, or
another polypeptide reference sequence, is the BLAST algorithm,
which is described in Altschul et al., 1990, J. Mol. Biol.
215:403-410, which is incorporated herein by reference. Software
for performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (on the worldwide web
at ncbi.nlm.nih.gov/). For amino acid sequences, the BLASTP program
uses as defaults a word size (W) of 3, an expectation (E) of 10,
and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989,
Proc. Natl. Acad. Sci. USA 89:10915). Other programs that may be
used include the Needleman-Wunsch procedure, J. MoI. Biol. 48:
443-453 (1970), using BLOSUM62, a Gap start penalty of 7 and gap
extend penalty of 1; and gapped BLAST 2.0 (see Altschul, et al.
1997, Nucleic Acids Res., 25:3389-3402) both
[0034] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0035] Nucleic acid or protein sequences that are substantially
identical to a reference sequence include "conservatively modified
variants." With respect to particular nucleic acid sequences,
conservatively modified variants refers to those nucleic acids
which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine) can be modified to yield a
functionally identical molecule. Accordingly, each silent variation
of a nucleic acid which encodes a polypeptide is implicit in each
described sequence.
[0036] The term "polypeptide" as used herein includes reference to
polypeptides containing naturally occurring amino acids and amino
acid backbones as well as non-naturally occurring amino acids and
amino acid analogs.
[0037] As to amino acid sequences, one of skill will recognize that
individual substitutions, in a nucleic acid, peptide, polypeptide,
or protein sequence which alters a single amino acid or a small
percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Examples of amino
acid groups defined in this manner can include: a "charged/polar
group" including Glu (Glutamic acid or E), Asp (Aspartic acid or
D), Asn (Asparagine or N), Gln (Glutamine or Q), Lys (Lysine or K),
Arg (Arginine or R) and His (Histidine or H); an "aromatic or
cyclic group" including Pro (Proline or P), Phe (Phenylalanine or
F), Tyr (Tyrosine or Y) and Trp (Tryptophan or W); and an
"aliphatic group" including Gly (Glycine or G), Ala (Alanine or A),
Val (Valine or V), Leu (Leucine or L), Ile (Isoleucine or I), Met
(Methionine or M), Ser (Serine or S), Thr (Threonine or T) and Cys
(Cysteine or C). Within each group, subgroups can also be
identified. For example, the group of charged/polar amino acids can
be sub-divided into sub-groups including: the "positively-charged
sub-group" comprising Lys, Arg and His; the "negatively-charged
sub-group" comprising Glu and Asp; and the "polar sub-group"
comprising Asn and Gln. In another example, the aromatic or cyclic
group can be sub-divided into sub-groups including: the "nitrogen
ring sub-group" comprising Pro, His and Trp; and the "phenyl
sub-group" comprising Phe and Tyr. In another further example, the
aliphatic group can be sub-divided into sub-groups including: the
"large aliphatic non-polar sub-group" comprising Val, Leu and Ile;
the "aliphatic slightly-polar sub-group" comprising Met, Ser, Thr
and Cys; and the "small-residue sub-group" comprising Gly and Ala.
Examples of conservative mutations include amino acid substitutions
of amino acids within the sub-groups above, such as, but not
limited to: Lys for Arg or vice versa, such that a positive charge
can be maintained; Glu for Asp or vice versa, such that a negative
charge can be maintained; Ser for Thr or vice versa, such that a
free --OH can be maintained; and Gln for Asn or vice versa, such
that a free --NH2 can be maintained. The following six groups each
contain amino acids that further provide illustrative conservative
substitutions for one another. 1) Ala, Ser, Thr; 2) Asp, Glu; 3)
Asn, Gln; 4) Arg, Lys; 5) Ile, Leu, Met, Val; and 6) Phe, Try, and
Trp (see, e.g., Creighton, Proteins (1984)).
[0038] The term "promoter," as used herein, refers to a
polynucleotide sequence capable of driving transcription of a DNA
sequence in a cell. Thus, promoters used in the polynucleotide
constructs of the invention include cis- and trans-acting
transcriptional control elements and regulatory sequences that are
involved in regulating or modulating the timing and/or rate of
transcription of a gene. For example, a promoter can be a
cis-acting transcriptional control element, including an enhancer,
a repressor binding sequence and the like. These cis-acting
sequences typically interact with proteins or other biomolecules to
carry out (turn on/off, regulate, modulate, etc.) gene
transcription. Most often the core promoter sequences lie within
1-2 kb of the translation start site, more often within 1 kbp and
often within 500 bp or 200 bp or fewer, of the translation start
site. By convention, promoter sequences are usually provided as the
sequence on the coding strand of the gene it controls. In the
context of this application, a promoter is typically referred to by
the name of the gene for which it naturally regulates expression. A
promoter used in an expression construct of the invention is
referred to by the name of the gene. Reference to a promoter by
name includes a wild type, native promoter as well as variants of
the promoter that retain the ability to induce expression.
Reference to a promoter by name is not restricted to a particular
species, but also encompasses a promoter from a corresponding gene
in other species.
[0039] A "constitutive promoter" in the context of this invention
refers to a promoter that is capable of initiating transcription
under most conditions in a cell, e.g., in the absence of an
inducing molecule. An "inducible promoter" initiates transcription
in the presence of an inducer molecule.
[0040] A polynucleotide is "heterologous" to an organism or a
second polynucleotide sequence if it originates from a foreign
species, or, if from the same species, is modified from its
original form. For example, when a polynucleotide encoding a
polypeptide sequence is said to be operably linked to a
heterologous promoter, it means that the polynucleotide coding
sequence encoding the polypeptide is derived from one species
whereas the promoter sequence is derived from another, different
species; or, if both are derived from the same species, the coding
sequence is not naturally associated with the promoter (e.g., is a
genetically engineered coding sequence, e.g., from a different gene
in the same species, or an allele from a different ecotype or
variety). Similarly, a polypeptide is "heterologous" to a host cell
if the native wildtype host cell does not produce the
polypeptide.
[0041] The term "exogenous" refers generally to a polynucleotide
sequence or polypeptide that does not naturally occur in a
wild-type cell or organism, but is typically introduced into the
cell by molecular biological techniques, i.e., engineering to
produce a recombinant microorganism. Examples of "exogenous"
polynucleotides include vectors, plasmids, and/or man-made nucleic
acid constructs encoding a desired protein or enzyme.
[0042] The term "endogenous" refers to naturally-occurring
polynucleotide sequences or polypeptides that may be found in a
given wild-type cell or organism. In this regard, it is also noted
that even though an organism may comprise an endogenous copy of a
given polynucleotide sequence or gene, the introduction of a
plasmid or vector encoding that sequence, such as to over-express
or otherwise regulate the expression of the encoded protein,
represents an "exogenous" copy of that gene or polynucleotide
sequence. Any of the pathways, genes, or enzymes described herein
may utilize or rely on an "endogenous" sequence, which may be
provided as one or more "exogenous" polynucleotide sequences, or
both.
[0043] "Recombinant nucleic acid" or "recombinant polynucleotide"
as used herein refers to a polymer of nucleic acids wherein at
least one of the following is true: (a) the sequence of nucleic
acids is foreign to (i.e., not naturally found in) a given host
cell; (b) the sequence may be naturally found in a given host cell,
but in an unnatural (e.g., greater than expected) amount; or (c)
the sequence of nucleic acids comprises two or more subsequences
that are not found in the same relationship to each other in
nature. For example, regarding instance (c), a recombinant nucleic
acid sequence will have two or more sequences from unrelated genes
arranged to make a new functional nucleic acid.
[0044] The term "operably linked" refers to a functional
relationship between two or more polynucleotide (e.g., DNA)
segments. Typically, it refers to the functional relationship of a
transcriptional regulatory sequence to a transcribed sequence. For
example, a promoter or enhancer sequence is operably linked to a
DNA or RNA sequence if it stimulates or modulates the transcription
of the DNA or RNA sequence in an appropriate host cell or other
expression system. Generally, promoter transcriptional regulatory
sequences that are operably linked to a transcribed sequence are
physically contiguous to the transcribed sequence, i.e., they are
cis-acting. However, some transcriptional regulatory sequences,
such as enhancers, need not be physically contiguous or located in
close proximity to the coding sequences whose transcription they
enhance.
[0045] The term "expression cassette" or "DNA construct" or
"expression construct" refers to a nucleic acid construct that,
when introduced into a host cell, results in transcription and/or
translation of an RNA or polypeptide, respectively. In the case of
expression of transgenes, one of skill will recognize that the
inserted polynucleotide sequence need not be identical, but may be
only substantially identical to a sequence of the gene from which
it was derived. As explained herein, these substantially identical
variants are specifically covered by reference to a specific
nucleic acid sequence. One example of an expression cassette is a
polynucleotide construct that comprises a polynucleotide sequence
encoding a polypeptide of the invention protein operably linked to
a promoter, e.g., its native promoter, where the expression
cassette is introduced into a heterologous microorganism. In some
embodiments, an expression cassette comprises a polynucleotide
sequence encoding a polypeptide of the invention where the
polynucleotide that is targeted to a position in the genome of a
microorganism such that expression of the polynucleotide sequence
is driven by a promoter that is present in the microorganism.
[0046] The term "host cell" as used in the context of this
invention refers to a microorganism and includes an individual cell
or cell culture that can be or has been a recipient of any
recombinant vector(s) or isolated polynucleotide(s) of the
invention. Host cells include progeny of a single host cell, and
the progeny may not necessarily be completely identical (in
morphology or in total DNA complement) to the original parent cell
due to natural, accidental, or deliberate mutation and/or change. A
host cell includes cells into which a recombinant vector or a
polynucleotide of the invention has been introduced, including by
transformation, transfection, and the like.
[0047] The term "isolated" refers to a material that is
substantially or essentially free from components that normally
accompany it in its native state. For example, an "isolated
polynucleotide," as used herein, may refer to a polynucleotide that
has been isolated from the sequences that flank it in its
naturally-occurring or genomic state, e.g., a DNA fragment that has
been removed from the sequences that are normally adjacent to the
fragment, such as by cloning into a vector. A polynucleotide is
considered to be isolated if, for example, it is cloned into a
vector that is not a part of the natural environment, or if it is
artificially introduced in the genome of a cell in a manner that
differs from its naturally-occurring state. Alternatively, an
"isolated peptide" or an "isolated polypeptide" and the like, as
used herein, refers to a polypeptide molecule that is free of other
components of the cell, i.e., it is not associated with in vivo
substances.
[0048] The invention employs various routine recombinant nucleic
acid techniques. Generally, the nomenclature and the laboratory
procedures in recombinant DNA technology described below are
commonly employed in the art. Many manuals that provide direction
for performing recombinant DNA manipulations are available, e.g.,
Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd
Ed, 2001); and Current Protocols in Molecular Biology (Ausubel, et
al., John Wiley and Sons, New York, 2009-2016).
SUMMARY OF CERTAIN ASPECTS OF THE DISCLOSURE
[0049] The present disclosure is based, in part, on the discovery
that fusing a prion subunit to an acid decarboxylase, e.g., at the
carboxyl terminus, to produce an acid decarboxylase fusion protein
and expressing the fusion protein in a host cell improves the
activity of the acid decarboxylase fusion protein as measured by
the production of polyamines under various stress conditions, such
as alkaline pH or elevated temperature, as compared to the
expression of an acid decarboxylase lacking the prion subunit.
[0050] Additionally, the present disclosure is based, in part, on
the discovery that fusion of a prion subunit to an acid
decarboxylase, e.g., at the carboxyl terminus, to produce a fusion
protein increases the stability of the acid decarboxylase as
measured by oligomerization of the fusion protein at elevated
temperature or alkaline pH when compared to a counterpart acid
decarboxylase lacking the prion subunit.
[0051] Further, the present disclosure is based, in part, on the
discovery that fusion of a prion subunit to an acid decarboxylase,
e.g., at the carboxyl terminus, to produce a fusion protein
increases the solubility of the acid decarboxylase at elevated
temperature or alkaline pH. Accordingly, fusion proteins of the
present disclosure typically have improved solubility relative to
an acid decarboxylase protein lacking the corresponding prion
subunit as measured by acid decarboxylase activity at an alkaline
pH.
[0052] The ability of an acid decarboxylase fusion protein of the
present invention to tolerate alkaline pH also allows the use of
alternative nitrogen sources that have higher pH values, such as
urea and ammonia (1M solution has a pH 11.6) in fermentation
reactions to generate the desired product, e.g., polyamines. These
alternative nitrogen sources generate less salt waste
byproduct.
Prion Subunit
[0053] Prions are self-propagating and transmissible protein
isoforms. A normal cellular protein (PrPc) having an altered
confirmation can be infectious, resulting in disease. While there
is no protein with homology to PrPc in yeast, several yeast prion
proteins have been identified.
[0054] The first prions identified in yeast, [PSI+] and [URE3],
were determined to be prion forms of the Ure2 and Sup35 proteins,
respectively. Since that time, other yeast prions have been
identified in Saccharomyces cerevisiae including [PIN+]/[RNQ+],
[SWI+], [OCT+], [MOT+], [ISP+], [BETA], [MOD+] and a fungal prion,
[Het-s], identified in Podospora anserina (see, Wickner et al.,
Microbiology and Molecular Biology Reviews, 2015).
[0055] In some embodiments, a prion subunit can defined as a prion
polypeptide or fragment thereof where the percent composition of
asparagine (N) and glutamine (Q) is 10% or greater. For example,
the percent of Q/N in Sup35 prion subunit is 25% of 154 amino
acids, and that of New1 is 27% of 253 amino acids. The 10% is
determined with reference to the portion of the fused polypeptide
that is considered to have prion activity, i.e., is determined
considered in the context of the prion subunit sequence only.
[0056] Prion polypeptide sequences suitable for use in the
invention as a prion subunit include amino acid sequences of a
prion polypeptide as illustrated in SEQ ID NO:3 or 4, or
substantially identical sequence variants thereof. Such a sequence
variant typically has at least 50%, or at least 60%, 70%, 75%, 80%,
85%, or 90% identity to one of SEQ ID NOS: 3 or 4, or e.g., a
homolog of SEQ ID NO: 3 or 4. In some embodiments, a prion subunit
comprises the amino acid sequence of the prion region of SEQ ID
NO:7, 8, 11, 12, 13, or 14; or has at least 50%, or has at least
60%, 70%, 75%, 80%, 85%, or 90% identity to the prion region of SEQ
ID NO:7, 8, 11, 12, 13, or 14. As used herein, the term "sequence
variant" encompasses biologically active polypeptides having one or
more substitutions, deletions, or insertions relative to a prion
polypeptide reference sequence, such as SEQ ID NO: 3, or 4. Thus,
the term "sequence variant" includes biologically active fragments
as well as substitution variants.
[0057] In one embodiment, prion subunit polypeptide sequences
suitable for use in the invention include amino acid sequences
encoding Ure2, Sup35, New1, Rnq1, Swi1, Cyc8, Mot3 or Sfp1 or
substantially identical variants thereof. Illustrative examples of
Ure2 polypeptides include those from S. cerevisiae protein sequence
accession number AAM93184; Candida albicans protein sequence
accession number AAM91946; S. bayanus protein sequence accession
number AAM91939; and Eremothecium gossypii protein sequence
accession number AAM91943. Illustrative examples of Sup35
polypeptides include those from S. cerevisiae protein sequence
accession number AJV18122; S. boulardii protein sequence accession
number KOH51638; and S. bayanus protein sequence accession number
AAL15027. Illustrative examples of New1 polypeptides include those
from S. cerevisiae protein sequence accession number AHY77957; S.
boulardii protein sequence accession number KOH47591; and
Sugiyamella lignohabitans protein sequence accession number
ANB11767. Illustrative examples of Rnq1 polypeptides include those
from S. cerevisiae protein sequence accession number AFU61310; and
S. boulardii protein sequence accession number KOH52602.
Illustrative examples of Swi1 polypeptides include those from S.
cerevisiae protein sequence accession number AJP42124; C. albicans
protein sequence accession number AOW28823; and Sugiyamella
lignohabitans protein sequence accession number ANB13699.
Illustrative examples of Cyc8 polypeptides include those from S.
cerevisiae protein sequence accession number CAA85069; and
Aspergillus nomius protein sequence accession number KNG81485.
Illustrative examples of Mot3 polypeptides include those from S.
cerevisiae protein sequence accession number AAC49982; and S.
boulardii protein sequence accession number KQC41827. Illustrative
examples of Sfp1 polypeptides include those from S. cerevisiae
protein sequence accession number AAB82343; S. boulardii protein
sequence accession number KOH49283; and S. arboricola protein
sequence accession number EJS42621. In one embodiment, polypeptide
sequences suitable for use in the invention include amino acid
sequences encoding a prion polypeptide that are capable of inducing
protein oligomerization in vivo or in vitro.
[0058] In some embodiments, polynucleotide sequences suitable for
use in the invention include nucleic acid sequences encoding one or
more of the following prion proteins: Ure2, Sup35, New1, Rnq1,
Swi1, Cyc8, Mot3 or Sfp1 or homologs thereof. Moreover, suitable
polynucleotides for use in the invention include nucleic acid
sequences that encode any one of the illustrative prion
polypeptides provided herein. In another embodiment, suitable
polynucleotides include nucleic acid sequences that encode any one
or more of the illustrative prion polypeptides disclosed herein
that are capable of inducing protein oligomerization in vivo or in
vitro. In one embodiment, polynucleotide sequences suitable for use
in the invention include nucleic acid sequences that encode a prion
polypeptide as illustrated in SEQ NOs: 5 or 6, or substantially
identical variants thereof. Such a variant typically has at least
60%, or at least 70%, 75%, 80%, 85%, or 90% identity to one of SEQ
ID NOS: 5 or 6.
[0059] In one embodiment, the invention relates to a genetically
modified host cell having a nucleic acid sequence encoding an acid
decarboxylase fusion protein, where the acid decarboxylase fusion
protein comprises or consists of an acid decarboxylase polypeptide
joined to a prion subunit, e.g., at the carboxyl terminus of the
acid decarboxylase polypeptide, and where the fusion protein has
improved acid decarboxylase activity as measured by the production
of polyamines relative to a counterpart host cell that expresses
the acid decarboxylase polypeptide not joined to the prion subunit.
In one embodiment, the prion subunit is at least 30 amino acids in
length, at least 50 amino acids in length, at least 75 amino acids
in length or at least 100 amino acids in length, but 1200 amino
acids or fewer in length. In some embodiments, a prion subunit
comprises an amino acid composition having at least 20% Q or N
residues. In another embodiment, the prion subunit has at least
70%, 75%, 80%, 85%, 90%, or 95% identity to a Sup35, New1, Ure2, or
Rnq1 amino acid sequence; or comprises a Sup35, New1, Ure2, or Rnq1
amino acid sequence.
Structural Organization of Prions
[0060] Generally, yeast prions are intrinsically disordered in
solution and QN-rich. Scrambled PrD's of Sup35 and Ure2 maintaining
amino acid composition but not exact sequence, were found to be
capable of both generating amyloid in vitro and prions in vivo, and
of propagating the prion state, indicating that the amino acid
composition plays an important role in prion properties (Ross et
al., 2005). Thus, variants of yeast prion sequences of use in the
invention as prion subunits include fragments of Sup35 or Ure2 that
have less than 50% sequence identity to Sup35 and Ure2, but have a
QN composition of 10% or greater.
Sup35
[0061] Yeast protein Sup35 (685 aa) is a subunit of the translation
termination factor and terminates translation at stop codons, and
residues 254-685 (Sup35C) have been observed to be sufficient to
carry out the essential translation termination function. Residues
1-253 (Sup35NM) were observed to regulate general mRNA turnover
through interactions with the poly(A) --binding protein and the
poly(A)-degrading enzyme. Residues 1-114 (Sup35N) are sufficient to
propagate the original [PSI+] variant, while residues 1-61 are
sufficient to propagate several variants of this prion (Chang et
al., PNAS, 2008). The N-proximal PrD region of Sup35 includes an
N-terminal QN-rich region located within the first 40 amino acids,
and a region of 5.5 imperfect oligopeptide repeats (ORs) located at
positions 41 and 97. The PrD fragment required for aggregation is
shorter than the fragment needed for propagation of the prion state
and is primarily confined to the QN-rich region (Osherovich et al,
2004).
[0062] Parts of the Sup35M domain (residues 115-253), up to residue
137, were observed as necessary for propagation of some strong and
weak [PSI+] variants, and deletions and substitutions within the M
domain were observed to alter the character of [PSI+] variant
significantly (Liu et al., PNAS, 2002). Solid-state nuclear
magnetic resonance (ss-NMR) experiments with Sup35NM filaments
showed that Tyrosine (Tyr) residues, all of which are within the N
terminal, are in an in-register parallel .beta.-sheet structure
(Shewmaker, PNAS, 2006). Additionally, it was observed that there
are eight leucine (Leu) residues, i.e., residues 110, 126, 144,
146, 154, 212, 218 and 238. The ss-NMR data suggests that four of
these Leu residues are in an in-register parallel structure
(Shewmaker et al., Biochemistry, 2009).
Ure2
[0063] Yeast protein Ure2 (354aa) acts to regulate nitrogen
catabolism. The part of Ure2 whose overproduction induces the
formation of [URE3] was found to be the N-terminal 65 residues and
this region proved to be sufficient to propagate [URE3] in the
absence of the remainder of the molecule (Masison et al., Science,
1997). The N-terminal prion domain normally functions to stabilize
Ure2 against degradation (Shewmaker et al., Genetics, 2007).
Rnq1
[0064] In the case of the yeast protein Rnq1 (405 aa), four QN-rich
regions were found within the PrD (Kadnar et al., 2010). While none
were essential for prion propagation, two of the four stretches
were each found to support prion maintenance if retained alone.
New1
[0065] Yeast protein New1 (1196 aa) consists of a N-terminal prion
region (New1N) and a C-terminal region homologous to a translation
elongation factor with two ATP-binding motifs.
[0066] Generally, a prion capable of inducing protein aggregation
requires a glutamine (Q) and/or asparagine (N) or (NQ)-rich region.
For example, the prion protein, New1, contains the sequence
"QQQRNWKQGGNYQQYQSYN" and "SNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ". In the
prion protein Sup35, the N-terminus contains a NQ-rich region
followed by the N domain repeat (NR) region, which contains five
complete copies (R1-R5) and one partial copy (R6) of the imperfect
oligopeptide repeating sequence "PQGGYQQN".
[0067] In one embodiment, the prion subunit can comprise or consist
of a nucleic acid encoding a prion protein selected from the group
consisting of Ure2, Sup35, New1, Rnq1, Swi1, Cyc8, Mot3 and Sfp1.
In another embodiment, the prion subunit can comprise or consist of
a nucleic acid encoding a polypeptide derived from Ure2, Sup35,
New1, Rnq1, Swi1, Cyc8, Mot3 or Sfp1 capable of inducing protein
oligomerization in vivo or in vitro. In yet another embodiment, the
prion subunit can consist of or comprise an amino acid sequence of
at least 30 amino acids, at least 40 amino acids, at least 50 amino
acids, at least 60 amino acids, at least 70 amino acids, at least
80 amino acids, at least 90 amino acids, at least 100 amino acids,
at least 200 amino acids, at least 300 amino acids, but less than
1200 amino acids that when fused to the carboxyl end of an acid
decarboxylase improves decarboxylation by the acid decarboxylase
under alkaline pH and/or elevated temperature as compared to a
counterpart acid decarboxylase lacking the prion subunit under the
same conditions. In one embodiment, decarboxylation is measured by
the production of polyamines by the acid decarboxylase.
[0068] In one embodiment, the prion subunit can consist of or
comprise an amino acid sequence of at least 30 amino acids, at
least 40 amino acids, at least 50 amino acids, at least 60 amino
acids, at least 70 amino acids, at least 80 amino acids, at least
90 amino acids, at least 100 amino acids, at least 200 amino acids,
at least 300 amino acids, but less than 1200 amino acids that when
fused to the carboxyl end of a lysine decarboxylase improves
decarboxylation by the lysine decarboxylase under alkaline pH
and/or elevated temperature as compared to a counterpart lysine
decarboxylase lacking the prion subunit under the same conditions.
In one embodiment, decarboxylation is measured by the production of
cadaverine by the lysine decarboxylase.
[0069] In one embodiment, the prion subunit can consist of or
comprise an amino acid sequence of at least 30 amino acids, at
least 40 amino acids, at least 50 amino acids, at least 60 amino
acids, at least 70 amino acids, at least 80 amino acids, at least
90 amino acids, at least 100 amino acids, at least 200 amino acids,
at least 300 amino acids, but less than 1200 amino acids that when
fused to the carboxyl end of a arginine decarboxylase improves
decarboxylation by the arginine decarboxylase under alkaline pH
and/or elevated temperature as compared to a counterpart arginine
decarboxylase lacking the prion subunit under the same conditions.
In one embodiment, decarboxylation is measured by the production of
putrescine by the arginine decarboxylase.
[0070] In one embodiment, the prion subunit can consist of or
comprise an amino acid sequence of at least 30 amino acids, at
least 40 amino acids, at least 50 amino acids, at least 60 amino
acids, at least 70 amino acids, at least 80 amino acids, at least
90 amino acids, at least 100 amino acids, at least 200 amino acids,
at least 300 amino acids, but less than 1200 amino acids that when
fused to the carboxyl end of an ornithine decarboxylase improves
decarboxylation by the ornithine decarboxylase under alkaline pH
and/or elevated temperature as compared to a counterpart ornithine
decarboxylase lacking the prion subunit under the same conditions.
In one embodiment, decarboxylation is measured by the production of
spermine by the ornithine decarboxylase.
[0071] In one embodiment, the prion subunit can consist of or
comprise an amino acid sequence of at least 30 amino acids, at
least 40 amino acids, at least 50 amino acids, at least 60 amino
acids, at least 70 amino acids, at least 80 amino acids, at least
90 amino acids, at least 100 amino acids, at least 200 amino acids,
at least 300 amino acids, but less than 1200 amino acids that when
fused to the carboxyl end of a glutamate decarboxylase improves
decarboxylation by the glutamate decarboxylase under alkaline pH
and/or elevated temperature as compared to a counterpart glutamate
decarboxylase lacking the prion subunit under the same conditions.
In one embodiment, decarboxylation is measured by the production of
gamma-aminobutyric acid (GABA) by the glutamate decarboxylase.
[0072] A prion subunit has at least 10% Q and N residues, and
typically at least 20%, 30%, 40%, 50% or more Q and N residues. In
another embodiment, the prion subunit has at least 10% Q residues,
and typically at least 20%, 30%, 40%, 50% or more Q residues. In
yet another embodiment, the prion subunit has at least 10% N
residues, and typically at least 20%, 30%, 40%, 50% or more N
residues. In one embodiment, the prion subunit contains a higher
percentage of N residues as compared to Q residues. In another
embodiment, the prion subunit contains a higher percentage of Q
residues as compared to N residues. In another embodiment, the
percentage of Q and N residues present in the fusion protein is
such that the prion subunit capable of causing protein
oligomerization contains at least 10% Q or N residues.
[0073] In one embodiment, the prion subunit of the fusion protein
can comprise or consist of a nucleic acid sequence encoding a prion
protein selected from the group consisting of Ure2, Sup35, New1,
Rnq1, Swi1, Cyc8, Mot3 or Sfp1. In another embodiment, the prion
subunit can comprise or consist of a nucleic acid sequence encoding
a polypeptide derived from Ure2, Sup35, New1, Rnq1, Swi1, Cyc8,
Mot3 or Sfp1 capable of inducing protein oligomerization in vivo or
in vitro. In yet another embodiment, the prion subunit can consist
of or comprise an amino acid sequence of at least 30 amino acids,
at least 40 amino acids, at least 50 amino acids, at least 60 amino
acids, at least 70 amino acids, at least 80 amino acids, at least
90 amino acids, at least 100 amino acids, at least 200 amino acids,
at least 300 amino acids, but less than 1200 amino acids that when
fused to the carboxyl end of an acid decarboxylase increases
decarboxylation by the acid decarboxylase under alkaline pH and/or
elevated temperature as compared to a counterpart acid
decarboxylase lacking the prion subunit under the same conditions.
In one embodiment, decarboxylation is measured by the production of
polyamines by the acid decarboxylase.
[0074] In another embodiment, the prion subunit represents the
minimally required amino acid sequence (and corresponding
polynucleotide sequence) necessary to improve acid decarboxylase
activity as measured by the production of polyamines by the fusion
protein. In one embodiment, the prion subunit can be at least 30
amino acids in length, or at least 40, 50, 60, 70, 80, 90, 100,
200, 300 or more amino acids, but less than 1200 amino acids in
length.
[0075] In one embodiment, a prion subunit can be linked to another
short amino acid sequence that confers stability. In one
embodiment, the short peptide is selected from the group consisting
of a BST fragment, a RecA fragment and a .lamda.CI fragment. In
another embodiment, the linker polypeptide can comprise the amino
acid sequence RRFGEASSAF, ASQWPEETFG, or EGVAETNEDF.
[0076] In one embodiment, the prion subunit represents the
minimally required amino acid sequence (and corresponding
polynucleotide sequence) necessary to improve acid decarboxylase
activity as measured by the production of polyamines by the fusion
protein relative to an acid decarboxylase lacking the prion
subunit. In one embodiment, the prion subunit can be at least 30
amino acids in length, or at least 40, 50, 60, 70, 80, 90, 100,
200, 300 or more amino acids, but less than 1200 amino acids in
length.
[0077] In one embodiment, the prion subunit is capable of inducing
protein aggregation (oligomerization) and the prion subunits forma
.beta.-sheet structure, such as an in-register parallel (3-sheet
structure. In one embodiment, the prion subunit may be capable of
inducing protein aggregation of one or more protein monomers fused
to the prion subunit and the prion subunits form into a
.beta.-helix structure.
[0078] A prion subunit may be fused to an acid decarboxylase at the
N-terminus, the C-terminus of an acid decarboxylase, or may be
introduced at a surface region of the acid decarboxylase protein.
In certain embodiments, the prion subunit is fused at the
C-terminus of the acid decarboxylase. A prion subunit is typically
joined to the acid decarboxylase by a linker, such as flexible
linker comprising amino acids such as Gly, Ser, Ala, and the
like.
Acid Decarboxylases
[0079] Various acid decarboxylase activity have been well
characterized, both structurally and functionally. These include
CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, GadB, and their homologs.
The optimal pH for CadA is between 5 and 6, LdcC is between 7 and
8, AdiA is between 4.5 and 5.5, SpeC is between 7.5 and 8.5, and
SpeF is between 7 and 8 (Kanjee et al., Biochemistry 50, 9388-9398,
2011). GadA and GadB are activated when the pH of the environment
is between 2 and 2.5 (Castanie-Cornet et al., J. Bacteriol. 181,
3525-3535, 1999). However, the decarboxylation of basic amino acids
lysine, arginine, ornithine, and glutamateleads to the production
of cadaverine, putrescine, spermine, and GABA; and their formation
involves the consumption of protons. These are basic molecules that
tend to increase the pH of the medium. For example, the pKa's of
cadaverine are 9.1 and 10.2, and that of putrescine are 9.7 and
11.2. Therefore, the production of these basic molecules quickly
increases the pH of the reaction medium to a pH that is outside of
the optimal pH of the acid decarboxylase.
[0080] Acid decarboxylases that are fused to a prion subunit as
described herein include lysine decarboxylases, arginine
decarboxylases, ornithine decarboxylases, and glutamate
decarboxylases, which as detailed above, are part of the
prokaryotic ornithine decarboxylase subclass of Fold Type I
pyridoxal 5'-phosphate (PLP)-dependent decarboxylases. This class
of proteins typically contains a N-terminal wing domain, a core
domain, and a C-terminal domain. The core domain contains the
PLP-binding subdomain and subdomain 4. The acid decarboxylase SpeA
belongs to a different fold family of the PLP-dependent
decarboxylases. These share little sequence identity, but the
structures are well known. The Protein Data Bank Identification
numbers for illustrative acid decarboxylase structures are CadA:
3N75, LdcC: 5FKZ, AdiA: 2VYC, SpeA: 3NZQ, GadA: 1XEY, and GadB:
1PMM.
[0081] For CadA, the N-terminal wind domain (residues 1 to 129 as
determined with reference to SEQ ID NO:2) has a flavodoxin-like
fold consisting of five-stranded parallel beta-sheets sandwiched
between two sets of amphipathic alpha-helices. The core domain
(residues 130 to 563 as determined with reference to 563 of SEQ ID
NO:2) includes a linker region (amino acid residues 130 to 183 of
SEQ ID NO:2) that form a short helical bundle, the PLP-binding
subdomain (amino acids 184 to 417 of SEQ ID NO:2) that form a
seven-stranded beta-sheet core surrounded by three sets of
alpha-helices, and subdomain 4 consists of amino acid residues 418
to 563 that form a four stranded antiparallel beta-sheet core with
three alpha-helices facing outward. The C-terminal domain
(corresponding to amino acid residues 564 to 715 as determined with
referenced to SEQ ID NO:2) forms two sets of beta sheets with an
alpha-helical outer surface (Kanjee et al., The EMBO Journal 30,
931-944 2011).
[0082] The CadA protein forms a two-fold symmetric dimer that
completes the active site of each monomer. Five dimers associate to
form a decamer that consist of a double-ringed structure with
five-fold symmetry. The decamer associates with other decamers to
form higher-order oligomers. It has been shown that in acidic
conditions (pH 5), CadA predominantly exists in the oligomeric
state, and less oligomers and decamers are found as the environment
becomes more basic. It was estimated that 25% of the enzymes exist
as dimers and 75% exist as decamers at pH 6.5, while 95% of the
enzymes exist as dimers at pH 8.0 (Kanjee et al., The EMBO Journal
30, 931-944 2011). This decrease in oligomer formation coincides
with the decrease in decarboxylase activity observed as the pH of
the environment of the enzyme increases above 5.0, suggesting that
the decrease in oligomer formation is one of the causes of the
decrease in decarboxylase activity.
[0083] Any acid decarboxylase, e.g., lysine decarboxylase, arginine
decarboxylase, glutamate decarboxylase, or ornithine decarboxylase,
may be fused to a prion protein in accordance with the invention.
Suitable acid decarboxylases include CadA, LdcC, AdiA, SpeA, SpeC,
SpeF, GadA, GadB, and their homologs. As used herein: a lysine
decarboxylase refers to an enzyme that converts L-lysine into
cadaverine; the enzyme is classified as E.C. 4.1.1.18; an arginine
decarboxylase refers to an enzyme that converts L-arginine to
agmatine, which can be further converted to purtrescine through the
activity of agmatinase, the enzyme is classified as E.C. 4.1.1.19;
an ornithine decarboxylase refers to an enzyme that converts
ornithine into putrescine, which can be further converted into
spermidine and spermine, the enzyme is classified as E.C. 4.1.1.17;
and a glutamate decarboxylase refers to an enzyme that converts
glutamate to gamma-aminobutyrate (GABA), the enzyme is classified
as 4.1.1.15.
[0084] In some embodiments, the lysine decarboxylase is CadA from
E. coli or a CadA homolog from another species, e.g., Klebsiella
sp.; Enterobacter aerogenes; Salmonella enterica; Serratia sp.; and
Raoultella ornithinolytica. In some embodiments, the lysine
decarboxylase is LdcC from E. coli or an LdcC homologs from
Shigella sp., Citrobacter sp., and Salmonella enterica. As used
herein, a lysine decarboxylase includes variants of native lysine
decarboxylase enzymes that have lysine decarboxylase enzymatic
activity. Additional lysine decarboxylase enzymes are described in
PCT/CN2014/080873 and PCT/CN2015/072978.
[0085] In some embodiments, a lysine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the length of, the cadA polypeptide of SEQ ID NO:2.
[0086] In some embodiments, a lysine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity; preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of a cadA polypeptide.
[0087] In some embodiments, a lysine decarboxylase polypeptide
suitable for in the invention has at least 60% amino acid sequence
identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%,
preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater
amino acid sequence identity, preferably over a region of at least
about 50, at least 100, at least 200, at least 300, at least 400,
or at least 500 or more amino acids in length, or over the length
of, the LdcC polypeptide of SEQ ID NO:21.
[0088] In some embodiments, a lysine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity; preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of an LdcC polypeptide. Illustrative
homologs and accession numbers for the sequences of the
polypeptides are: Shigella multispecies (WP 001020996.1),
Escherichia fergusonii (WP 001021009.1), Achromobacter sp ATCC35328
(CUJ86682.1), Enterobacteriaceae multispecies (WP 058668594.1),
Citrobacter multispecies (WP 016151770.1), Gammoprobacteria
multispecies (WP 046401634.1), and Salmonella bongori (WP
038390535.1).
[0089] In some embodiments, an arginine decarboxylase polypeptide
suitable for in the inventionis an Adi or a homolog therefore from
another species. In some embodiments, an arginine decarboxylase
polypeptide has at least 60% amino acid sequence identity,
preferably at least 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid
sequence identity, preferably over a region of at least about 50,
at least 100, at least 200, at least 300, at least 400, or at least
500 or more amino acids in length, or over the full-length of, SEQ
ID NO:22.
[0090] In some embodiments, an arginine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of an AdiA polypeptide. Illustrative
homologs and accession numbers for the sequences of the
polypeptides are: Shigella multispecies (WP 000978677.1),
Escherichia multispecies (WP 000978709.1), Enterobacteriaceae
multispecies (WP 032934133.1), Citrobacter multispecies (WP
008786969.1), Klebsiella oxytoca (SAP84601.1), and Salmonella
enterica (WP 048668294.1).
[0091] In some embodiments, an arginine decarboxylase polypeptide
suitable for in the inventionis an SpeA or a homolog therefore from
another species. In some embodiments, an SpeA polypeptide has at
least 60% amino acid sequence identity, preferably at least 65%,
70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or greater amino acid sequence identity, preferably
over a region of at least about 50, at least 100, at least 200, at
least 300, at least 400, or at least 500 or more amino acids in
length, or over the full-length of, SEQ ID NO:23.
[0092] In some embodiments, an arginine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of an SpeA polypeptide. Illustrative
homologs and accession numbers for the sequences of the
polypeptides are: Shigella multispecies (WP 005096955.1),
Escherichia multispecies (WP 010350365.1), Gammaproteobacteria
multispecies (WP 042998051.1), Salmonella multispecies (WP
001278580.1), Achromobacter sp. ATCC35328 (CUJ95389.1), Citrobacter
farmeri (WP 042324083.1), Citrobacter koseri (WP 024130934.1), and
Citrobacter amalonaticus (WP 052746994.1).
[0093] In some embodiments, an acid decarboxylase polypeptide
suitable for in the inventionis an ornithine decarboxylase, SpeC or
a homolog thereof, from another species. In some embodiments, the
ornithine decarboxylase has at least 60% amino acid sequence
identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%,
preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater
amino acid sequence identity, preferably over a region of at least
about 50, at least 100, at least 200, at least 300, at least 400,
or at least 500 or more amino acids in length, or over the
full-length of, SEQ ID NO:24.
[0094] In some embodiments, an ornithinedecarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of an SpeC polypeptide. Illustrative
homologs and accession numbers for the sequences homologs are:
Shigella multispecies (WP 005085661.1), Escherichia multispecies
(WP 010352539.1), Citrobacter multispecies (WP 044255681.1),
Enterobacteriaceae (WP 047357853.1), Gammaproteobacteria
multispecies (WP 044327655.1), Achromobacter sp. ATCC35328
(CUJ95194.1), Klebsiella oxytoca (SBL12331.1), and Klebsiella
pneumonia (CDK72259.1).
[0095] In some embodiments, an ornithine decarboxylase polypeptide
suitable for in the inventionis SpeF or a homolog thereof, from
another species. In some embodiments, the ornithine decarboxylase
has at least 60% amino acid sequence identity, preferably at least
65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% or greater amino acid sequence identity,
preferably over a region of at least about 50, at least 100, at
least 200, at least 300, at least 400, or at least 500 or more
amino acids in length, or over the full-length of, SEQ ID
NO:25.
[0096] In some embodiments, an ornithine decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or at least 500 or more amino acids in length, or over
the full-length of, a homolog of an SpeF polypeptide. Illustrative
homologs and the accession number of polypeptide sequences are:
Shigella multispecies (WP 000040203.1), Enterobacteriaceae
multispecies (WP 049009856.1), Escherichia multispecies (WP
001292417.1), Gammaproteobacteria multispecies (WP 046401512.1),
Citrobacter koseri (WP 024130539.1), Citrobacter amalonaticus (WP
046274704.1), Citrobacter braakii (WP 047501716.1), and Salmonella
enterica (WP 023220629.1).
[0097] In some embodiments, an glutamate decarboxylase polypeptide
suitable for in the invention is GadA or a homolog thereof, from
another species. In some embodiments, the glutamate decarboxylase
has at least 60% amino acid sequence identity, preferably at least
65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% or greater amino acid sequence identity,
preferably over a region of at least about 50, at least 100, at
least 200, at least 300, at least 400, or more amino acids in
length, or over the full-length of, SEQ ID NO:26.
[0098] In some embodiments, an glutamate decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or more amino acids in length, or over the full-length
of, a homolog of an GadA polypeptide. Illustrative homologs and the
accession numbers of the polypeptide sequences are: Escherichia
multispecies (WP 001517297.1), Shigella multispecies (WP
000358931.1), Yersinia multispecies (WP 050085789.1), Achromobacter
sp. ATCC35328 (WP 054518524.1), and Rhodococcs gingshengii
(KDQ00107.1).
[0099] In some embodiments, an glutamate decarboxylase polypeptide
suitable for in the invention is GadB or a homolog thereof, from
another species. In some embodiments, the glutamate decarboxylase
has at least 60% amino acid sequence identity, preferably at least
65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% or greater amino acid sequence identity,
preferably over a region of at least about 50, at least 100, at
least 200, at least 300, at least 400, or more amino acids in
length, or over the full-length of, SEQ ID NO:27.
[0100] In some embodiments, an glutamate decarboxylase polypeptide
suitable for use in the invention has at least 60% amino acid
sequence identity, preferably at least 65%, 70%, 75%, 80%, 85%,
90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or
greater amino acid sequence identity, preferably over a region of
at least about 50, at least 100, at least 200, at least 300, at
least 400, or more amino acids in length, or over the full-length
of, a homolog of an GadB polypeptide. Illustrative homologs and the
accession numbers of the polypeptide sequences are: Shigella
multispecies (WP 000358931.1), Escherichia multispecies (WP
016248697.1), Yersinia multispecies (WP 050085789.1), Achromobacter
sp. ATCC35328 (WP 054518524.1), Rhodococcs gingshengii
(KDQ00107.1)
Nucleic Acids Encoding Prion Subunits and Acid Decarboxylases
[0101] Isolation or generation of acid decarboxylase polynucleotide
sequences can be accomplished by a number of techniques In some
embodiments, oligonucleotide probes based on the sequences
disclosed here can be used to identify the desired polynucleotide
in a cDNA or genomic DNA library from a desired bacteria species.
Probes may be used to hybridize with genomic DNA or cDNA sequences
to isolate homologous genes from different organisms, e.g., fungal
species or plant species.
[0102] Alternatively, the nucleic acids of interest can be
amplified from nucleic acid samples using routine amplification
techniques. For instance, PCR may be used to amplify the sequences
of the genes directly from mRNA, from cDNA, from genomic libraries
or cDNA libraries. PCR and other in vitro amplification methods may
also be useful, for example, to clone nucleic acid sequences that
code for proteins to be expressed, to make nucleic acids to use as
probes for detecting the presence of the desired mRNA in samples,
for nucleic acid sequencing, or for other purposes.
[0103] Appropriate primers and probes for identifying an acid
decarboxylase polynucleotide in bacteria can be generated from
comparisons of the sequences provided herein. For a general
overview of PCR see PCR Protocols: A Guide to Methods and
Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T.,
eds.), Academic Press, San Diego (1990). Illustrative primer
sequences are shown in the Table of Primers in the Examples
section.
[0104] Nucleic acid sequences encoding an acid decarboxylase
polypeptide for use in the disclosure includes genes and gene
products identified and characterized by techniques such as
hybridization and/or sequence analysis using illustrative nucleic
acid sequences, e.g., a cadA polynucleotide sequence of SEQ ID
NO:1. In some embodiments, a host cell is genetically modified by
introducing a nucleic acid sequence having at least 60% identity,
or at least 70%, 75%, 80%, 85%, or 90% identity, or 100% identity,
to an acid decarboxylase polynucleotide, e.g., a cadA
polynucleotide of SEQ ID NO:1.
[0105] Nucleic acid sequences encoding an acid decarboxylase fusion
protein in accordance with the invention that confers increased
production of an amino acid, e.g., lysine, or an amino acid-derived
product, e.g., cadaverine, to a host cell, may additionally be
codon-optimized for expression in a desired host cell. Methods and
databases that can be employed are known in the art. For example,
preferred codons may be determined in relation to codon usage in a
single gene, a set of genes of common function or origin, highly
expressed genes, the codon frequency in the aggregate protein
coding regions of the whole organism, codon frequency in the
aggregate protein coding regions of related organisms, or
combinations thereof. See e.g., Henaut and Danchin in "Escherichia
coli and Salmonella," Neidhardt, et al. Eds., ASM Pres, Washington
D.C. (1996), pp. 2047-2066; Nucleic Acids Res. 20:2111-2118;
Nakamura et al., 2000, Nucl. Acids Res. 28:292).
Preparation of Recombinant Vectors
[0106] Recombinant vectors for expression of an acid decarboxylase
fusion protein can be prepared using methods well known in the art.
For example, a DNA sequence encoding an acid decarboxylase fusion
polypeptide (described in further detail below), can be combined
with transcriptional and other regulatory sequences which will
direct the transcription of the sequence from the gene in the
intended cells, e.g., bacterial cells such as E. coli. In some
embodiments, an expression vector that comprises an expression
cassette that comprises the gene encoding the acid decarboxylase
fusion polypeptide further comprises a promoter operably linked to
the nucleic acid sequence encoding the acid decarboxylase fusion
polypeptide. In other embodiments, a promoter and/or other
regulatory elements that direct transcription of acid decarboxylase
fusion polypeptide sequence gene are endogenous to the host cell
and an expression cassette comprising the acid decarboxylase fusion
gene is introduced, e.g., by homologous recombination, such that
the exogenous gene is operably linked to an endogenous promoter and
is expression driven by the endogenous promoter.
[0107] As noted above, expression of the polynucleotide encoding an
acid decarboxylase fusion protein can be controlled by a number of
regulatory sequences including promoters, which may be either
constitutive or inducible; and, optionally, repressor sequences, if
desired. Examples of suitable promoters, especially in a bacterial
host cell, are the promoters obtained from the E. coli lac operon
and other promoters derived from genes involved in the metabolism
of other sugars, e.g., galactose and maltose. Additional examples
include promoters such as the trp promoter, bla promoter
bacteriophage lambda PL, and T5. In addition, synthetic promoters,
such as the tac promoter (U.S. Pat. No. 4,551,433), can be used.
Further examples of promoters include Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xylA and xylB genes.
Suitable promoters are also described in Ausubel and Sambrook &
Russell, both supra. Additional promoters include promoters
described by Jensen & Hammer, Appl. Environ. Microbiol. 64:82,
1998; Shimada, et al., J. Bacteriol. 186:7112, 2004; and Miksch et
al., Appl. Microbiol. Biotechnol. 69:312, 2005.
[0108] In some embodiments, a promoter that influences expression
of an acid decarboxylase polypeptide may be modified to increase
expression. For example, an endogenous acid decarboxylase promoter
may be replaced by a promoter that provides for increased
expression compared to the native promoter.
[0109] An expression vector may also comprise additional sequences
that influence expression of a polynucleotide encoding the acid
decarboxylase fusion polypeptide. Such sequences include enhancer
sequences, a ribosome binding site, or other sequences such as
transcription termination sequences, and the like.
[0110] A vector expressing a polynucleotide encoding an acid
decarboxylase fusion polypeptide of the invention may be an
autonomously replicating vector, i.e., a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid, an extrachromosomal
element, a minichromosome, or an artificial chromosome. The vector
may contain any means for assuring self-replication. Alternatively,
the vector may be one which, when introduced into the host, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Thus, an
expression vector may additionally contain an element(s) that
permits integration of the vector into the host's genome.
[0111] An expression vector of the invention preferably contains
one or more selectable markers which permit easy selection of
transformed hosts. For example, an expression vector may comprise a
gene that confers antibiotic resistance (e.g., ampicillin,
kanamycin, chloramphenicol or tetracycline resistance) to the
recombinant host organism, e.g., a bacterial cell such as E.
coli.
[0112] Although any suitable expression vector may be used to
incorporate the desired sequences, readily available bacterial
expression vectors include, without limitation: plasmids such as
pSC101, pBR322, pBBR1MCS-3, pUR, pET, pEX, pMR100, pCR4, pBAD24,
p15a, pACYC, pUC, e.g., pUC18 or pUC19, or plasmids derived from
these plasmids; and bacteriophages, such as Ml 3 phage and .lamda.
phage. One of ordinary skill in the art, however, can readily
determine through routine experimentation whether any particular
expression vector is suited for any given host cell. For example,
the expression vector can be introduced into the host cell, which
is then monitored for viability and expression of the sequences
contained in the vector.
[0113] Expression vectors of the invention may be introduced into
the host cell using any number of well-known methods, including
calcium chloride-based methods, electroporation, or any other
method known in the art.
Host Cells
[0114] The present invention provides for a genetically modified
host cell that is engineered to express an acid decarboxylase
fusion polypeptide. A genetically modified host strain of the
present invention typically comprises at least one additional
genetic modification to enhance production of an amino acid or
amino acid derivative relative to a control strain that does not
have the one additional genetic modification, e.g., a wildtype
strain or a cell of the same strain without the one additional
genetic modification. An "additional genetic modification to
enhance production of an amino acid or amino acid derivative" can
be any genetic modification. In some embodiments, the genetic
modification is the introduction of a polynucleotide that expresses
an enzyme involved in the synthesis of the amino acid or amino acid
derivative. In some embodiments, the host cell comprises multiple
modifications to increase production, relative to a wildtype host
cell, of an amino acid or amino acid derivative.
[0115] In some aspects, genetic modification of a host cell to
express an acid decarboxylase fusion polypeptide is performed in
conjunction with modifying the host cell to overexpress one or more
lysine biosynthesis polypeptides.
[0116] In some embodiments, a host cell may be genetically modified
to express one or more polypeptides that affect lysine
biosynthesis. Examples of lysine biosynthesis polypeptides include
the E. coli genes SucA, Ppc, AspC, LysC, Asd, DapA, DapB, DapD,
ArgD, DapE, DapF, LysA, Ddh, PntAB, CyoABE, GadAB, YbjE, GdhA,
GltA, SucC, GadC, AcnB, PflB, ThrA, AceA, AceB, GltB, AceE, SdhA,
MurE, SpeE, SpeG, PuuA, PuuP, and YgjG, or the corresponding genes
from other organisms. Such genes are known in the art (see, e.g.,
Shah et al., J. Med. Sci. 2:152-157, 2002; Anastassiadia, S. Recent
Patents on Biotechnol. 1: 11-24, 2007). See, also, Kind, et al.,
Appl. Microbiol. Biotechnol. 91: 1287-1296, 2011 for a review of
genes involved in cadaverine production. Illustrative genes
encoding lysine biosynthesis polypeptides are provided below.
TABLE-US-00001 EC GenBank Protein Gene Number Accession No.
.alpha.-ketogultarate sucA 1.2.4.2 YP_489005.1 dehydrogenase (SucA)
Phosphoenolpyruvate ppc 4.1.1.31 AAC76938.1 carboxylase (PPC)
aspartate transaminase (AspC) aspC 2.6.1.1 AAC74014.1 aspartate
kinase (LysC) lysC 2.7.2.4 NP_418448.1 aspartate semialdehyde asd
1.2.1.11 AAC76458.1 dehydrogenase (Asd) dihydrodipicolinate
synthase dapA 4.3.3.7 NP_416973.1 (DapA) dihydropicolinate
reductase dapB 1.17.1.8 AAC73142.1 (DapB) tetrahydrodipicoinate
dapD 2.3.1.117 AAC73277.1 succinylase (DapD)
N-succinyldiaminopimelate argD 2.6.1.11 AAC76384.1 aminotransferase
(ArgD) N-succinyl-L-diaminopimelate dapE 3.5.1.18 AAC75525.1
deacylase (DapE) diaminopimelate epimerase dapF 5.1.1.7 AAC76812.2
(DapF) diaminopimelate decarboxylase lysA 4.1.1.20 AAC75877.1
(LysA) meso-diaminopimelate ddh NA P04964.1 dehydrogenase (Ddh)
pyridine nucleotide pntAB NA AAC74675.1, transhydrogenase (PntAB)
AAC74674.1 cytochrome O oxidase cyoABE 1.10.3.10 AAC73535.1,
(CyoABE) AAC73534.1, AAC73531.1 glutamate decarboxylase gadAB
4.1.1.15 AAC76542.1, (GadAB) AAC74566.1 L-amino acid efflux
transporter ybjE NA AAC73961.2 (YbjE) glutamate dehydrogenase gdhA
1.4.1.4 AAC74831.1 (GdhA) citrate synthase (GltA) gltA 2.3.3.1/
AAC73814.1 2.3.3.16 succinyl-coA synthase (SucC) sucC 6.2.1.5
AAC73822.1 glutamate-GABA antiporter gadC NA AAC74565.1 (GadC)
aconitase B (AcnB) acnB 4.2.1.99 AAC73229.1 pyruvate-formate lyase
(PflB) pflB NA AAC73989.1 aspartate kinase/homoserine thrA 2.7.2.4
AAC73113.1 dehydrogenase (ThrA) isocitrate lyase (AceA) aceA
4.1.3.1 AAC76985.1 malate synthase (AceB) aceB 2.3.3.9 AAC76984.1
glutmate synthase (GltB) gltB 1.4.1.13 AAC76244.2 pyruvate
dehydrogenase (AceE) aceE 1.2.4.1 AAC73225.1 succinate
dehydrogenase sdhA 1.3.5.1 AAC73817.1 (SdhA)
UDP-N-acetylmuramoyl-L- murE 6.3.2.13 AAC73196.1
alanyl-D-glutamate:meso- diaminopimelate ligase (MurE)
putrescine/cadaverine speE 2.5.1.16 AAC73232.1
aminopropyltransferase (SpeE) spermidine acetyltransferase speG NA
AAC74656.1 (SpeG) glutamate-putrescine/glutamate- puuA NA
AAC74379.2 cadaverine ligase (PuuA) putrescine importer (PuuP) puuP
NA AAC74378.2 putrescine/cadaverine ygjG 2.6.1.82 AAC76108.3
aminotransferase (YgjG)
[0117] In some embodiments, a host cell may be genetically modified
to attenuate or reduce the expression of one or more polypeptides
that affect lysine biosynthesis. Examples of such polypeptides
include the E. coli genes Pck, Pgi, DeaD, CitE, MenE, PoxB, AceA,
AceB, AceE, RpoC, and ThrA, or the corresponding genes from other
organisms. Such genes are known in the art (see, e.g., Shah et al.,
J. Med. Sci. 2:152-157, 2002; Anastassiadia, S. Recent Patents on
Biotechnol. 1: 11-24, 2007). See, also, Kind, et al., Appl.
Microbiol. Biotechnol. 91: 1287-1296, 2011 for a review of genes
attenuated to increase cadaverine production. Illustrative genes
encoding polypeptides whose attenuation increases lysine
biosynthesis are provided below.
TABLE-US-00002 EC GenBank Protein Gene Number Accession No. PEP
carboxykinase (Pck) pck 4.1.1.49 NP_417862 Glucose-6-phosphate pgi
5.3.1.9 NP_418449 isomerase (Pgi) DEAD-box RNA deaD NP_417631
helicase (DeaD) citrate lyase (CitE) citE 4.1.3.6/ NP_415149
4.1.3.34 o-succinylbenzoate-CoA menE 6.2.1.26 NP_416763 ligase
(MenE) pyruvate oxidase (PoxB) poxB 1.2.2.2 NP_415392 isocitrate
lyase (AceA) aceA 4.1.3.1 NP_418439 malate synthase A (AceB) aceB
2.3.3.9 NP_418438 pyruvate dehydrogenase (aceE) aceE 1.2.4.1
NP_414656 RNA polymerase b' subunit rpoC 2.7.7.6 NP_418415 (RpoC)
aspartokinase I (ThrA) thrA 2.7.2.4/ NP_414543 1.1.1.3
[0118] Nucleic acids encoding a lysine biosynthesis polypeptide may
be introduced into the host cell along with acid decarboxylase
fusion polynucleotide, e.g., encoded on a single expression vector,
or introduced in multiple expression vectors at the same time.
Alternatively, the host cell may be genetically modified to
overexpress one or more lysine biosynthesis polypeptides before or
after the host cells genetically modified express an acid
decarboxylase fusion polypeptide.
[0119] A host cell engineered to express an acid decarboxylase
fusion polypeptideis typically a bacterial host cell. In typical
embodiments, the bacterial host cell is a Gram-negative bacterial
host cell. In some embodiments of the invention, the bacterium is
an enteric bacterium. In some embodiments of the invention, the
bacterium is a species of the genus Corynebacterium, Escherichia,
Pseudomonas, Zymomonas, Shewanella, Salmonella, Shigella,
Enterobacter, Citrobacter, Cronobacter, Erwinia, Serratia, Proteus,
Hafnia, Yersinia, Morganella, Edwardsiella, or Klebsiella
taxonomical classes. In some embodiments, the host cells are
members of the genus Escherichia, Hafnia, or Corynebacterium. In
some embodiments, the host cell is an Escherichia coli, Hafnia
alvei, or Corynebacterium glutamicum host cell.
[0120] In some embodiments, the host cell is a gram-positive
bacterial host cell, such as a Bacillus sp., e.g., Bacillus
subtilis or Bacillus licheniformis; or another Bacillus sp. such as
B. alcalophilus, B. aminovorans, B. amyloliquefaciens, B.
caldolyticus, B. circulans, B. stearothermophilus, B.
thermoglucosidasius, B. thuringiensis or B. vulgatis.
[0121] Host cells modified in accordance with the invention can be
screened for increased production of lysine or a lysine derivative,
such as cadaverine, as described herein.
[0122] In some embodiments, an acid decarboxylase fusion protein of
the present invention may be recovered from a host cell that
expresses the fusion protein. In some embodiments, the recovered
fusion protein may be immobilized onto a solid substrate or inert
material to form an immobilized enzyme. In one embodiment, the
immobilized enzyme may have improved thermal stability and/or
operational stability than the soluble form of the fusion protein.
In one embodiment, the fusion protein comprises a lysine, arginine,
ornithine, or glutamate decarboxylase fused at the C-terminal end
to a prion subunit.
Methods of Producing Lysine or a Lysine Derivative.
[0123] A host cell genetically modified to express an acid
decarboxylase fusion polypeptide can be employed to produce lysine
or a derivative of lysine. In some embodiments, the host cell
produces cadaverine. To produce lysine or the lysine derivative, a
host cell genetically modified to express an acid decarboxylase
fusion polypeptide as described herein can be cultured under
conditions suitable to allow expression of the polypeptide and
expression of genes that encode the enzymes that are used to
produce lysine or the lysine derivative. A host cell modified in
accordance with the invention to express an acid decarboxylase
fusion polypeptide provides a higher yield of lysine or lysine
derivatives relative to a non-modified counterpart host cell that
expresses the acid decarboxylase that is not fused to a prion
subunit.
[0124] Host cells may be cultured using well known techniques (see,
e.g., the illustrative conditions provided in the examples
section.
[0125] In some embodiments, host cells are cultured using nitrogen
sources that are not salts (e.g., ammonium sulfate or ammonium
chloride), such as ammonia or urea. Host cells may be cultured at
an alkaline pH during cell growth or enzyme production.
[0126] The lysine or lysine derivative then be separated and
purified using known techniques. Lysine or lysine derivatives,
e.g., cadverine, produced in accordance with the invention may then
be used in any known process, e.g., to produce a polyamide.
[0127] In some embodiments, lysine may be converted to caprolactam
using chemical catalysts or by using enzymes and chemical
catalysts.
[0128] The present invention will be described in greater detail by
way of specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters, which can be changed or modified
to yield essentially the same results.
EXAMPLES
Example 1: Construction of Plasmid Vectors that Encode CadA
[0129] A plasmid vector containing wild-type E. coli cadA (SEQ ID
NO: 1), which encodes the lysine decarboxylase CadA (SEQ ID NO: 2),
was amplified from the E. coli MG1655 K12 genomic DNA using the PCR
primers cadA-F and cadA-R (FIG. 1), digested using the restriction
enzymes SacI and XbaI, and ligated into pUC18 to generate the
plasmid pCIB60. The 5' sequence upstream of the cadA gene was
optimized using the PCR primers cadA-F2 and cadA-R2 to create
pCIB71. The chloramphenicol resistance gene cat was amplified using
the primers cat-HindIII-F and cat-NdeI-R, and cloned behind cadA in
pCIB71 to create pCIB128.
Example 2: Synthesis of Codon Optimized New1 and Sup35 Prion
Fragments
[0130] The minimal polypeptide fragment necessary for New1 and
Sup35 prion activity was determined based on Osherovich et al.,
PLOS Biology 2:4, 2004. Therefore, amino acids 2-100 of New1 (SEQ
ID NO: 3) and 2-103 of Sup35 (SEQ ID NO: 4) were codon optimized
for heterologous expression in E. coli (SEQ ID NO: 5 and 6). In
addition, a short polypeptide linker sequence that consists of the
amino acids GSGSG was added to the beginning of SEQ ID NO: 3 and 4
(SEQ ID NO: 7 and 8), and their corresponding DNA sequences are
presented in SEQ ID NO: 9 and 10. Codon optimization and DNA
assembly was performed according to Hoover D M & Lubkowski J,
Nucleic Acids Research 30:10, 2002.
Example 3: Construction of a Polynucleotide Encoding a Fragment of
New1 Fused 3' of CadA
[0131] The stop codon of cadA in pCIB128 was removed using the
primers cadAt-XbaI-R and cat-XbaI-F to create the plasmid pCIB138.
The DNA fragment that consists of the prion domain of New1 with the
polypeptide linker was amplified using the primers New1-XbaI-F and
New1-HindIII-R, digested using the restriction enzymes XbaI and
HindIII, and ligated into pCIB138 to make pCIB222.
Example 4: Construction of a Polynucleotide Encoding a Fragment of
Sup35 Fused 3' of CadA
[0132] The DNA fragment that consists of the prion domain of Sup35
with the polypeptide linker was amplified using the primers
35-XbaI-F and 35-HindIII-R, digested using the restriction enzymes
XbaI and HindIII, and ligated into pCIB138 to make pCIB223.
Example 5: Lysine Decarboxylase Activity of Novel Polypeptide
Consisting of Either the New1 or Sup35 Prion Domain Fragment Fused
3' of CadA
[0133] H. alvei was transformed with pCIB128, pCIB222, and pCIB223.
Three single colonies from each transformation were grown overnight
at 37.degree. C. in 4 mL of LB medium with ampicillin (100
.mu.g/mL). The following day, 0.7 mL of each overnight culture was
added to 0.3 mL of lysine-HCl and PLP to a final concentration of
120 g/L and 0.1 mM, respectively. Each mixture was incubated at
37.degree. C. for 2 hours. Cadaverine production from each sample
was quantified using NMR, and yield/OD was calculated by dividing
the molar amount of cadaverine produced by the molar amount of
lysine added. The yield from each sample after 2 hours is presented
in Table 1.
TABLE-US-00003 TABLE 1 Production of cadaverine H. alvei strains
overproducing a fusion polypeptide consisting of a prion domain
fragment and a lysine decarboxylase. Cadaverine Yield Strain
Plasmid (%) H. alvei pCIB128 67.4 .+-. 4.7 pCIB222 48.4 .+-. 5.5
pCIB223 50.3 .+-. 4.2
[0134] As shown in Table 1, both polypeptides that consisted of
either a New1 or Sup35 prion domain fragment fused 3' of CadA
showed significant lysine decarboxylase activity. The lysine
decarboxylase activity was lower than the control, where the lysine
decarboxylase was expressed by itself without being fused to a
prion domain fragment. The decrease in activity could be caused by
many factors, some of which are a decrease cell density and total
protein, a decrease in lysine decarboxylase activity, a decrease in
the amount of functional soluble enzyme, or an increase of the
insoluble protein fraction as a result of the fusion polypeptide
having difficulty folding.
Example 6: Construction of a Polynucleotide Encoding a Fragment of
New1 Fused 5' of CadA
[0135] The cadA gene was amplified using the primers cadA-XbaI-F
and cadA-HindIII-R, digested using the restriction enzymes XbaI and
HindIII, and ligated into pCIB128 to create the plasmid pCM146
having two copies of the cadA gene. The SacI restriction site was
added 5' of the first cadA gene after the promoter using the
primers rbs2-SacI-F and rbs2-SacI-R to construct pCIB149. The New1
prion fragment was amplified using the primers New1-SacI-F and
New1-XbaI-R, digested using the restriction enzymes SacI and XbaI,
and ligated into pCM149 to create pCIB241.
Example 7: Construction of a Polynucleotide Encoding a Fragment of
Sup35 Fused 5' of CadA
[0136] The Sup35 prion fragment was amplified using the primers
Sup35-SacI-F and Sup35-XbaI-R, digested using the restriction
enzymes SacI and XbaI, and ligated into pCM149 to create
pCIB242.
Example 8: Lysine Decarboxylase Activity of Novel Polypeptide
Consisting of Either the New1 or Sup35 Prion Domain Fragment Fused
5' of CadA
[0137] H. alvei was transformed with pCIB128, pCIB241, and pCIB242.
Three single colonies from each transformation were grown overnight
at 37.degree. C. in 4 mL of LB medium with ampicillin (100
.mu.g/mL). The following day, 0.7 mL of each overnight culture was
added to 0.3 mL of lysine-HCl and PLP to a final concentration of
120 g/L and 0.1 mM, respectively. Each mixture was incubated at
37.degree. C. for 2 hours. Cadaverine production from each sample
was quantified using NMR, and yield/OD was calculated by dividing
the molar amount of cadaverine produced by the molar amount of
lysine added. The yield from each sample after 2 hours is presented
in Table 2.
TABLE-US-00004 TABLE 2 Production of cadaverine H. alvei strains
overproducing a fusion polypeptide consisting of a prion domain
fragment and a lysine decarboxylase. Cadaverine Yield Strain
Plasmid (%) H. alvei pCIB128 68.8 .+-. 3.5 pCIB241 5.2 .+-. 1.6
pCIB242 8.3 .+-. 3.2
[0138] As shown in Table 2, both polypeptides that consisted of
either a New1 or Sup35 prion domain fragment fused 5' of CadA
showed very little lysine decarboxylase activity. The lysine
decarboxylase activity was lower than the control, where the lysine
decarboxylase was expressed by itself without being fused to a
prion domain fragment. The lysine decarboxylase activities of these
enzymes were also lower compared to those where the prion domain
fragments were fused 3' of CadA. This indicates that the synthetic
fusion polypeptide is more functional when the prion domain is
fused 3' of the acid decarboxylase after the C-terminal domain, and
less functional when the prion domain is fused 5' of the acid
decarboxylase in front of the N-terminal wing domain.
Example 9: Cell Density and Enzyme Production by H. Alvei
Overexpressing a New1 Prion Domain Fused to CadA
[0139] To determine the cause for the decrease in observed total
activity when the New1 prion domain fragment was fused 3' of the
lysine decarboxylase, the cell density of an overnight culture was
measured at Abs600, and the culture was lysed in order to determine
the total protein content using the Bradford assay. The lysed cell
culture was analyzed using SDS-PAGE in order to determine how much
of the total protein consisted of the synthetic fusion polypeptide.
Cell lysis was performed using a combination of freeze thaw and
lysozyme. Lysed samples were treated with DNAse in order to remove
most DNA. The OD and total protein data is presented in Table 3.
The result of the SDS-PAGE is shown in FIG. 1.
TABLE-US-00005 TABLE 3 OD and total protein data of a fusion
polypeptide consisting of a prion domain fragment and a lysine
decarboxylase. OD Total protein Strain Plasmid (Abs.sub.600)
(mg/mL) H. alvei pCIB128 4.1 .+-. 0.5 1.05 pCIB222 3.4 .+-. 0.1
0.65
[0140] As shown in Table 3, both the observed OD and total protein
are lower when H. alvei is producing the fusion polypeptide
(pCIB222) compared to producing the lysine decarboxylase alone
(pCIB128). The OD decreased by 17%, while the total protein
decreased by 38%. The SDS-PAGE result indicates that the background
protein did not change significantly; instead, the target protein
decreased significantly (the dark band at 80 kDa). Furthermore,
most of the target protein is found in the soluble fraction, which
indicates that protein solubility is not a problem. Therefore, the
decrease in total activity is not due to accumulation of inactive
protein that is insoluble.
Example 10: Construction of Different Sized Fragments of the New1
Prion Domain Fused to CadA
[0141] It was observed that the fusion of the prion domain fragment
to lysine decarboxylase affects the growth of the host cell, and
decreases the final cell density (OD measured at Abs600) achieved
when compared to the control without the prion domain fragment. As
demonstrated above, the decrease in cell density decreases the
amount of soluble enzyme and total activity produced. The decrease
in OD is not due to the accumulation of poorly folded enzyme, as
indicated in the SDS-PAGE result. However, the reduction in cell
density could be caused by challenges in folding of the new fusion
polypeptide (e.g., slower kinetics).
[0142] Different sized fragments of the New1 prion domain were
generated to determine whether truncating a portion of the prion
domain would decrease the burden on the cell and improve growth and
the final cell density. The N-terminus of the New1 prion domain
fragment was truncated by various lengths of amino acids in order
to determine whether the portion of the fragment less rich in Q/N
residues were important for lysine decarboxylase function of the
fusion polypeptide, and whether their removal could improve growth
of the host. The first 9, 18, 36, and 45 amino acids of the New1
prion domain fragment in pCIB222 were truncated (SEQ ID NO: 11, 12,
13, and 14) using the PCR primer 222-de-R and the respective PCR
primers 222-de-9-F, 222-de18-F, 222-de36-F, and 222-de45-F.
Example 11: Improvements in Cell Density by H. Alvei Overexpressing
Variants of the New1 Prion Domain Fused to CadA
[0143] E. coli BL21 was transformed with either pCIB222 or a
variant of pCIB222 with the first 9, 18, 27, 36, or 45 amino acids
of the New1 prion domain fragment truncated. Three single colonies
from each transformation were grown overnight at 37.degree. C. in 4
mL of LB medium with ampicillin (100 .mu.g/mL). The following day,
the absorbance of the overnight culture was measured at Abs600. The
OD of each sample is presented in Table 4, and the SDS-PAGE
analysis of each sample is shown in FIG. 2.
TABLE-US-00006 TABLE 4 OD of truncated variants of a fusion
polypeptide consisting of a prion domain fragment and a lysine
decarboxylase. Strain Plasmid Amino acids deleted OD (Abs.sub.600)
E. coli pCIB222 none 5.4 .+-. 0.2 pCIB222-de9 FPPKKFKDL 6.5 .+-.
0.4 pCIB222-de18 FPPKKFKDLNSFLDDQPK 6.1 .+-. 0.2 pCIB222-de36
FPPKKFKDLNSFLDDQPK 6.3 .+-. 0.1 DPNLVASPFGGYFKNPAA pCIB222-de45
FPPKKFKDLNSFLDDQPK 6.2 .+-. 0.1 DPNLVASPFGGYFKNPAA DAGSNNASK
[0144] Table 4 shows that the truncated versions of the New1 prion
domain fragment improved growth of the E. coli BL21 host compared
to the control with the entire prion domain fragment. There was no
significant difference in the final OD between the strains
producing different versions of the truncated enzymes. Even though
no difference could be seen between the truncated versions based on
the OD data, the SDS-PAGE analysis shows that significant increases
in total protein and the target enzyme are observed for the version
with 36 amino acids truncated. The versions of the enzymes with 9,
18, and 45 amino acids truncated show less total protein and target
enzyme compared to the control.
Example 12: Modification of the C-Terminal Amino Acids of the
Polynucleotide Encoding a Fragment of New1 Fused to CadA
[0145] It is common for synthetic proteins that fold slowly or
poorly to be degraded when overexpressed. Therefore, reducing
protein degradation in order to increase the time a polypeptide has
to fold would increase the amount of functional protein in a cell.
Previous work (Trepod C M & Mott J E, J Biotechnol 84, 273-284,
2000) reported that specific sequences of short polypeptides when
fused to the C-terminus of a protein can increases protein
stability and reduce intracellular protein degradation. These short
polypeptides are derived from the C-terminus of proteins such as
bovine somatotropin (BST), RpoC, .lamda.CI, Rho, RecA, Bla, and
TufA.
[0146] Three different short polypeptides derived from the genes
BST (C2), E. coli .lamda.CI (C4), and E. coli recA (C6) were fused
to the C-terminus of the New1 prion domain fragment in pCIB222
using the respective PCR primers 222-C2-F, 222-C2-R, 222-C4-F,
222-C4-R, 222-C6-F, and 222-C6-R. The BST fragment consists of the
amino acid sequence RRFGEASSAF (SEQ ID NO: 15 and 16), the
.lamda.CI fragment consists of the amino acid sequence ASQWPEETFG
(SEQ ID NO: 17 and 18), and the RecA fragment consists of the amino
acid sequence EGVAETNEDF (SEQ ID NO: 19 and 20).
Example 13: Improvements in Total Enzyme Activity by E. coli
Overexpressing C-Terminal Variants of the New1 Prion Domain Fused
to CadA
[0147] E. coli BL21 was transformed with either pCIB222 or a
variant of pCIB222 where the BST, .lamda.CI, or RecA fragment is
fused to the C-terminus of the New1 prion domain fragment. Three
single colonies from each transformation were grown overnight at
37.degree. C. in 4 mL of LB medium with ampicillin (100 .mu.g/mL).
The following day, 0.6 mL of each overnight culture was added to
0.4 mL of lysine-HCl and PLP to a final concentration of 160 g/L
and 0.1 mM, respectively. Each mixture was incubated at 37.degree.
C. for 2 hours. Cadaverine production from each sample was
quantified using NMR, and yield was calculated by dividing the
molar amount of cadaverine produced by the molar amount of lysine
added. The yield from each sample after 2 hours is presented in
Table 5.
TABLE-US-00007 TABLE 5 Cadaverine production by E. coli producing
C-terminal variants of a fusion polypeptide consisting of a prion
domain fragment and a lysine decarboxylase. Strain Plasmid Peptide
added Cadaverine Yield (%) E. coli pCIB222 none 32.2 .+-. 0.2
pCIB222-C2 RRFGEASSAF 34.8 .+-. 0.1 pCIB222-C4 ASQWPEETFG 41.5 .+-.
0.2 pCIB222-C6 EGVAETNEDF 37.5 .+-. 0.1
[0148] Table 5 shows that modification of the C-terminus of the
New1 prion domain fragment with an additional short polypeptide
derived from .lamda.CI improved cadaverine yield from lysine.
Modification of the prion domain fragment with an additional short
polypeptide derived from either BST or RecA showed a less
significant change in cadaverine yield. The increase in total
activity observed in the strain harboring the .lamda.CI C-terminal
fragment suggests an increase in the stability of the triple fusion
polypeptide that consists of CadA, the New1 prion domain fragment,
and the .lamda.CI polypeptide compared to the double fusion
polypeptide that lacks the .lamda.CI polypeptide.
Example 14: Effect of the Addition of Sorbitol on Lysine
Decarboxylase Activity by a Polypeptide Consisting of New1 Fused to
CadA
[0149] Protein chaperones can be overexpressed in a host in order
to improve the amount of functional protein produced in a cell,
especially in the case of a synthetic protein that does not fold
well or is not native to the host. For example, the common
chaperone protein systems used in E. coli are GroEL/GroES,
DnaK/DnaJ/GrpE, ClpB, and heat shock proteins/IbpA/IbpB (de Marco
et al., BMC Biotechnol 7:32, 2007). Chemical chaperones that can
increase the amount of soluble and functional protein produced have
also been demonstrated (Prasad, et al., Appl Environ Microbiol 77,
4603-4609, 2011).
[0150] To improve the folding of the synthetic polypeptide that
consists of the New1 prion domain fused to a lysine decarboxylase
CadA, sorbitol was added during protein production. E. coli BL21
and H. alvei were transformed with pCIB222. Three single colonies
from each transformation were grown overnight at 37.degree. C. in 4
mL of LB medium with ampicillin (100 .mu.g/mL), and enough sorbitol
to reach a final concentration of either 0, 0.2, or 0.8 M. The
following day, 0.6 mL of each overnight culture was added to 0.4 mL
of lysine-HCl and PLP to a final concentration of 160 g/L and 0.1
mM, respectively. Each mixture was incubated at 37.degree. C. for 2
hours. Cadaverine production from each sample was quantified using
NMR, and yield/OD was calculated by dividing the molar amount of
cadaverine produced by the molar amount of lysine added and
dividing again by the absorbance of the overnight culture at
Abs600. The yield/OD from each sample is presented in Table 6.
TABLE-US-00008 TABLE 6 Production of cadaverine by E. coli and H.
alvei strains grown with sorbitol and overproducing the fusion
polypeptide. Cadaverine Sorbitol OD Yield/OD Strain Plasmid (mM)
(Abs.sub.600) (%) E. coli pCIB222 0 6.2 .+-. 0.3 15.7 .+-. 0.3 0.2
6.0 .+-. 0.2 9.2 .+-. 0.2 0.8 4.0 .+-. 0.1 9.0 .+-. 0.2 H. alvei 0
5.4 .+-. 0.2 7.9 .+-. 0.2 0.2 5 .+-. 0.2 9.3 .+-. 0.2 0.8 4.7 .+-.
0.1 10.0 .+-. 0.3
[0151] As shown in Table 6, the addition of sorbitol affected
enzyme activity differently depending on the strain used. In both
E. coli and H. alvei, sorbitol negatively affected growth at
concentration of 0.8 M compared to 0 M, and the effect on growth is
less significant at 0.2 M. The addition of sorbitol increased the
yield/OD when H. alvei was the host, whereas the addition of
sorbitol decreased the yield/OD when E. coli was the host. Although
the addition of sorbitol decreases the OD in H. alvei, sorbitol
increased the lysine decarboxylase activity per cell and increased
total activity per culture volume.
Example 15: In Vitro Lysine Decarboxylase Activity and Kinetics of
a Polypeptide Consisting of New1 Fused to CadA at Different pHs
[0152] According to the literature, the activity of lysine
decarboxylase at pH 8 is significantly less than its activity at pH
6 due to a structural change from a high oligomer state to a low
oligomer state. The activity of lysine decarboxylase with and
without the New1 prion fragment fused to its C-terminus was
compared at pH 6 and pH 8, in order to determine whether the prion
fragment can increase the tolerance of the polypeptide to alkaline
conditions.
[0153] 100 mL samples of H. avlei transformed with either pCIB128
or pCIB222 were lysed with a french press. The lysed samples were
centrifuged, and the supernatant was separated from the pellet in
order to perform in vitro experiments. The reaction rate of each
lysed sample was measured using NMR by sampling the amount of
lysine converted in the presence of PLP into cadaverine every 1.6
minutes for a total of 20 minutes, and taking the slope of the
linear portion of the yield curve. The samples were diluted so that
the reaction rate per volume (U) of lysed sample was the same. The
kinetic constants Vmax and Km for lysine of each lysed samples was
measured using the same U at an initial pH of either 6 or pH 8. By
normalizing for U, the concentration of active enzyme in each
sample is the same. The results of the kinetic analysis of the two
samples are shown in Table 7.
TABLE-US-00009 TABLE 7 Kinetic analysis of the effect of pH on
lysed samples of H. avlei producing lysine decarboxylase with and
without the New1 prion domain fragment. pH CIB128 CIB222 6 Vmax
(mmol/min) 3.9 4.1 Km (mM) 27 25 8 Vmax (mmol/min) 2.6 3.7 Km (mM)
27 25
[0154] In accordance with the literature, wild-type lysine
decarboxylase (CIB128) lost a significant amount of activity at pH
8 compared to pH 6. The reduction in activity at pH 8 compared to
pH 6 was 33%. Surprisingly, the lysine decarboxylase fused to the
New1 prion domain (CIB222) only lost 10% of its activity when the
initial pH was 8 compared to 6. The Km of either wild-type or
modified lysine decarboxylase for lysine did not change even though
the initial pH changed. Since the amount of active enzyme added is
normalized by U, the ability for the modified enzyme to better
tolerate alkaline pH is likely due to its ability to maintain a
higher oligomer state compared to the wild-type enzyme.
Example 16: In Vitro Lysine Decarboxylase Activity and Kinetics of
a Polypeptide Consisting of New1 Fused to CadA at Different
Temperatures
[0155] Tolerance to high temperature is another beneficial trait of
a biological enzyme in a large-scale production system, especially
during the summer months in order to reduce the cost of cooling the
reactor. Most enzymes operate within a narrow temperature range,
and temperatures higher than that range tend to cause the enzymes
to denature or not exist in structural states necessary for
function. The lysine decarboxylase fused with a New1 prion domain
fragment showed increased tolerance to high pH. It is possible that
the increased structural stability provided by the prion domain
fragment is useful not only for tolerating high pH, but also high
temperature. The activities of lysine decarboxylase with and
without the New1 prion domain fragment were determined following
incubation for different periods of time at 37.degree. C.,
45.degree. C., and 55.degree. C., in order to determine whether the
enzymes are able to maintain the structural integrity necessary for
function at high temperatures.
[0156] 100 mL samples of H. avlei transformed with either pCIB128
or pCIB222 were lysed with a french press. The lysed samples were
centrifuged, and the supernatant was separated from the pellet in
order to perform in vitro experiments. The reaction rate of each
lysed sample was measured using NMR in the presence of PLP by
sampling the amount of lysine converted into cadaverine every 1.6
minutes for a total of 20 minutes, and taking the slope of the
linear portion of the yield curve. The samples were diluted so that
the reaction rate per volume (U) of lysed sample was the same.
Equal amounts of enzyme based on U were incubated at 37.degree. C.,
45.degree. C., and 55.degree. C. for 0, 1, 2, 4, and 20 hours.
After incubation at the specific temperature and time period, the
reaction rate of each enzyme sample was determined at 37.degree. C.
The effects of temperature and time on the two different enzymes
are shown in Table 8.
TABLE-US-00010 TABLE 8 Relative activity after incubation at
different temperatures for different periods of time of lysine
decarboxylase with or without the New1 prion domain fragment. Temp
Time (h) Strain (.degree. C.) 0 1 2 4 20 CIB128 37 100 100 98 81 70
45 100 95 92 55 37 55 100 8 6 0 0 CIB222 37 100 100 95 93 93 45 100
94 98 89 69 55 100 93 80 71 51
[0157] Table 8 shows the surprising discovery that not only does
the New1 prion domain fragment increase the tolerance of lysine
decarboxylase for alkaline pH, but it also increases the tolerance
of the enzyme for high temperature. The wild-type lysine
decarboxylase (CIB128) lost almost all of its activity after
incubation at 55.degree. C. for one hour. However, the lysine
decarboxylase fused with a fragment of the New1 prion domain
(CIB222) was able to maintain 93% of its activity. Furthermore,
when no detectable activity by the wild-type enzyme was observed
after 4 hours of incubation at 55.degree. C., the fusion
polypeptide still maintained 71% of its activity. Therefore, the
increased stability imparted on the acid decarboxylase by fusing it
with a prion domain fragment is not specific for tolerating a
single environmental stress, and can enable the new enzyme to
function across a wider range of operating conditions useful for
industrial production.
TABLE-US-00011 Table of plasmids used in Examples Host Protein(s)
Overexpressed Plasmid Strain CadA pCIB71 CadA, Cat pCIB128 CadA,
CadA pCIB146 CadA-New1 pCIB222 CadA-Sup35 pCIB223 New1-CadA pCIB241
Sup35-CadA pCIB242 CadA-New1 (.DELTA.9) pCIB222-de9 CadA-New1
(.DELTA.18) pCIB222-de18 CadA-New1 (.DELTA.36) pCIB222-de36
CadA-New1 (.DELTA.45) pCIB222-de45 CadA-New1-C2 pCIB222-C2
CadA-New1-C4 pCIB222-C4 CadA-New1-C6 pCIB222-C6 E. coli CadA-New1
pCIB222 CIB222-EC E. coli CadA-New1 (.DELTA.9) pCIB222-de9
CIB222-de9 E. coli CadA-New1 (.DELTA.18) pCIB222-de18 CIB222-de18
E. coli CadA-New1 (.DELTA.36) pCIB222-de36 CIB222-de36 E. coli
CadA-New1 (.DELTA.45) pCIB222-de45 CIB222-de45 E. coli CadA-New1-C2
pCIB222-C2 CIB222-C2 E. coli CadA-New1-C4 pCIB222-C4 CIB222-C4 E.
coli CadA-New1-C6 pCIB222-C6 CIB222-C6 H. avlei CadA, Cat pCIB128
CIB128 H. avlei CadA-New1 pCIB222 CIB222 H. avlei CadA-Sup35
pCIB223 CIB223 H. avlei New1-CadA pCIB241 CIB241 H. avlei
Sup35-CadA pCIB242 CIB242
TABLE-US-00012 Table of primer sequences used in Examples. Name
Sequence (5'-3') cadA-F
ggcgagctcacacaggaaacagaccatgaacgttattgcaatattgaatcac cadA-R
ggctctagaccacttcccttgtacgagc cadA-F2
atttcacacaggaaacagctatgaacgttattgcaatattgaat cadA-R2
agctgtttcctgtgtgaaat cat-HindIII-F
ggcaagcttgagaaaaaaatcactggatatacc cat-NdeI-R
ggccatatgtaagggcaccaataactgcc cadAt-XbaI-R
ggctctagatttgctttcttctttcaatacc cat-XbaI-F
ggctctagagagaaaaaaatcactggatatacc New1-XbaI-F
ggctctagaggttctggctctggttctccg New1-HindIII-R
ggcaagcttttactggtagccctgaccgttg Sup35-XbaI-F
ggctctagaggtagcggctctggctctga Sup35-HindIII-R
ggcaagcttttagccaccctgtgggttaaact cadA-XbaI-F
ggctctagaatttcacacaggaaacagct cadA-HindIII-R
ggcaagcttcacttcccttgtacgagcta rbs2-SacI-F
ggcgagctcatgaacgttattgcaatattgaatc rbs2-SacI-R
ggcgagctcctcctgtgtgaaattg New1-SacI-F ggcgagctcatgggttctggctctggttc
New1-XbaI-R ggctctagactggtagccctgaccgttg Sup35-SacI-F
ggcgagctcatgggtagcggctctggc Sup35-XbaI-R
ggctctagagccaccctgtgggttaaact 222-de-R tctagatttgctttcttctttcaatacc
222-de-9-F gaagaaagcaaatctagaaagttcaaagacctgaactctttc ggtg
222-de-18-F gaagaaagcaaatctagagacgaccagccgaaagacccgaac 222-de-36-F
gaagaaagcaaatctagaaaaaacccagcggcggacgcggg 222-de-45-F
gaagaaagcaaatctagaaacaacgcgtctaagaaatcttc 222-C2-F
tttcggcgaagcgagcagcgcgttctaaaagcttaagagacaggatg 222-C2-R
cgctgctcgcttcgccgaaacgacgctggtagccctgaccgttgtat 222-C4-F
ccagtggccggaagaaaccttcggctaaaagcttaagagacaggatg 222-C4-R
aggtttcttccggccactggctcgcctggtagccctgaccgttgtat 222-C6-F
cgtggcggaaaccaacgaagatttctaaaagcttaagagacaggatg 222-C6-R
cttcgttggtttccgccacgccttcctggtagccctgaccgttgtat
Illustrative Sequences:
TABLE-US-00013 [0158] Escherichia coli cadA nucleic acid sequence
SEQ ID NO: 1
ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATC
CGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGAC
CGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTT
GACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAA
CCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGA
CCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCT
AATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACT
AAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCAC
ATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTT
GGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGC
TGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAAC
GCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGT
ATGTACTCTGCTCCAGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCG
CTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGT
AACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATT
GCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTAC
CAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGA
TGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATT
TACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGA
AACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGT
TAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCA
CTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAAG
GCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCAAATTCCGTA
AAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTATGGCAGC
CGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACG
GCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGC
TGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGC
ATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTAT
AACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTG
CGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATG
CTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAA
CTGGCTCAGAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTAT
CGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCATTCCAGAAA
GAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAA
CGCCAATATGATCCTTCCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAAT
GATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGG
CGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGG
CCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA CadA polypeptide
sequence SEQ ID NO: 2
MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWD
KYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQT
TDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDI
SISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILI
DRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPV
HAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVI
YETQSTHKLLAAFSQASMIFIVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMK
GNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFK
NIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLF
SIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLI
VHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPP
GVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA DGRYTVKVLKEESKK New1
prion subunit polypeptide sequence SEQ ID NO: 3
FPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQG
GNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ Sup35 prion subunit
polypeptide sequence SEQ ID NO: 4
SDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQ
QGGYQQYNPQGGYQQDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGG New1 prion subunit
nucleic acid sequence SEQ ID NO: 5
TTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGACGACCAGCCGAAAGA
CCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAACCCAGCGGCGGACGC
GGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCAGCGTAACTGGAAAC
AGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACTCTAATTACAACAACT
ACAACAACTACAATAACTATAATAACTACAACAACTACAACAATTATAACAAATAC
AACGGTCAGGGCTACCAG Sup35 prion subunit nucleic acid sequence SEQ ID
NO: 6 TCTGACTCTAACCAAGGTAATAACCAGCAGAACTACCAACAATACTCTCAGAACGG
CAACCAGCAGCAGGGCAACAACCGCTATCAAGGCTACCAAGCGTACAACGCGCAGG
CACAGCCAGCAGGTGGCTACTACCAGAATTACCAGGGTTACTCTGGTTACCAGCAA
GGTGGTTATCAACAGTATAATCCGCAGGGCGGCTATCAGCAGGACGCAGGTTACCA
GCAACAATATAACCCTCAGGGCGGCTATCAGCAATACAACCCGCAAGGCGGTTATC
AACAACAGTTTAACCCACAGGGTGGC New1 prion subunitand linker polypeptide
sequence. The linker sequence is underlined SEQ ID NO: 7
GSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQR
NWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ Sup35 prion subunit
and linker polypeptide sequence SEQ ID NO: 8
GSGSGSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQG
YSGYQQGGYQQYNPQGGYQQDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGG New1 prion
subunit and linker nucleic acid sequence. The region encoding the
linker is underlined. SEQ ID NO: 9
GGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGAC
GACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAAC
CCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCA
GCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACT
CTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAAC
AATTATAACAAATACAACGGTCAGGGCTACCAG Sup35 prion subunit and linker
nucleic acid sequence. The region encoding the linker is
underlined. SEQ ID NO: 10
GGTAGCGGCTCTGGCTCTGACTCTAACCAAGGTAATAACCAGCAGAACTACCAACA
ATACTCTCAGAACGGCAACCAGCAGCAGGGCAACAACCGCTATCAAGGCTACCAAG
CGTACAACGCGCAGGCACAGCCAGCAGGTGGCTACTACCAGAATTACCAGGGTTAC
TCTGGTTACCAGCAAGGTGGTTATCAACAGTATAATCCGCAGGGCGGCTATCAGCAG
GACGCAGGTTACCAGCAACAATATAACCCTCAGGGCGGCTATCAGCAATACAACCC
GCAAGGCGGTTATCAACAACAGTTTAACCCACAGGGTGGC New1 prion subunit and
linker with 9 amino acid truncation polypeptide sequence. The
linker is underlined. SEQ ID NO: 11
GSGSGNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNY
QQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ New1 prion subunit and
linker with 18 amino acid truncation polypeptide sequence. The
linker is underlined. SEQ ID NO: 12
GSGSGDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSY
DSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ New1 prion subunit and linker with
36 amino acid truncation polypeptide sequence. The linker is
underlined. SEQ ID NO: 13
GSGSGDAGSNNASKKSSYQQQRNWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYN
NYNNYNKYNGQGYQ New1 prion subunit and linker with 45 amino acid
truncation polypeptide sequence. The linker is underlined. SEQ ID
NO: 14 GSGSGKSSYQQQRNWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYN
GQGYQ New1 prion domain fragment and linker with BST C-terminal
fragment polypeptide sequence. The linker is underlined. SEQ ID NO:
15 GSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQR
NWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQRRFGEASS AF New1
prion subunit and linker with BST C-terminal fragment nucleic acid
sequence. The region encoding the linker is underlined SEQ ID NO:
16 GGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGAC
GACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAAC
CCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCA
GCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACT
CTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAAC
AATTATAACAAATACAACGGTCAGGGCTACCAGCGTCGTTTCGGCGAAGCGAGCAG CGCGTTC
New1 prion subunit and linker with AEI C-terminal fragment
polypeptide sequence. The linker is underlined. SEQ ID NO: 17
GSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQR
NWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQASQWPEE TFG New1
prion domain fragment and linker with AEI C-terminal fragment
nucleic acid sequence. The region encoding the linker is
underlined. SEQ ID NO: 18
GGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGAC
GACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAAC
CCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCA
GCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACT
CTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAAC
AATTATAACAAATACAACGGTCAGGGCTACCAGGCGAGCCAGTGGCCGGAAGAAAC CTTCGGC
New1 prion domain fragment and linker with RecA C-terminal fragment
polypeptide sequence. The linker is underlined. SEQ ID NO: 19
GSGSGFPPKKFKDLNSFLDDQPKDPNLVASPFGGYFKNPAADAGSNNASKKSSYQQQR
NWKQGGNYQQGGYQSYDSNYNNYNNYNNYNNYNNYNNYNKYNGQGYQEGVAETN EDF New1
subunit and linker with RecA C-terminal fragment nucleic acid
sequence. The region encoding the linker is underlined. SEQ ID NO:
20 GGTTCTGGCTCTGGTTTTCCGCCGAAAAAGTTCAAAGACCTGAACTCTTTCCTGGAC
GACCAGCCGAAAGACCCGAACCTGGTTGCGTCTCCGTTCGGTGGCTACTTCAAAAAC
CCAGCGGCGGACGCGGGTTCTAACAACGCGTCTAAGAAATCTTCTTACCAGCAGCA
GCGTAACTGGAAACAGGGTGGCAACTATCAGCAAGGTGGTTACCAGTCTTACGACT
CTAATTACAACAACTACAACAACTACAATAACTATAATAACTACAACAACTACAAC
AATTATAACAAATACAACGGTCAGGGCTACCAGGAAGGCGTGGCGGAAACCAACGA AGATTTC
LdcC polypeptide sequence SEQ ID NO: 21
MNIIAIMGPHGVFYKDEPIKELESALVAQGFQIIWPQNSVDLLKFIEHNPRICGVIFDWDE
YSLDLCSDINQLNEYLPLYAFINTHSTMDVSVQDMRMALWFFEYALGQAEDIAIRMRQ
YTDEYLDNITPPFTKALFTYVKERKYTFCTPGHMGGTAYQKSPVGCLFYDFFGGNTLKA
DVSISVTELGSLLDHTGPHLEAEEYIARTFGAEQSYIVTNGTSTSNKIVGMYAAPSGSTLL
IDRNCHKSLAHLLMMNDVVPVWLKPTRNALGILGGIPRREFTRDSIEEKVAATTQAQWP
VHAVITNSTYDGLLYNTDWIKQTLDVPSIHFDSAWVPYTHFHPIYQGKSGMSGERVAGK
VIFETQSTHKMLAALSQASLIHIKGEYDEEAFNEAFMMHTTTSPSYPIVASVETAAAMLR
GNPGKRLINRSVERALHFRKEVQRLREESDGWFFDIWQPPQVDEAECWPVAPGEQWHG
FNDADADHMFLDPVKVTILTPGMDEQGNMSEEGIPAALVAKFLDERGIVVEKTGPYNLL
FLFSIGIDKTKAMGLLRGLTEFKRSYDLNLRIKNMLPDLYAEDPDFYRNMRIQDLAQGIH
KLIRKHDLPGLMLRAFDTLPEMIMTPHQAWQRQIKGEVETIALEQLVGRVSANMILPYP
PGVPLLMPGEMLTKESRTVLDFLLMLCSVGQHYPGFETDIHGAKQDEDGVYRVRVLKM AG AdiA
polypeptide sequence SEQ ID NO: 22
MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFS
YQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILED
TADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRF
YHDYYGENLFRTDMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGSN
RTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQP
ETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAW
YGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFS
RFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYK
EFTADGSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKD
IPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFS
MGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMF
AWLKENNPGARLNEAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPP
GIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA SpeA
polypeptide sequence SEQ ID NO: 23
MSDDMSMGLPSSAGEHGVLRSMQEVAMSSQEASKMLRTYNIAWWGNNYYDVNELGH
ISVCPDPDVPEARVDLAQLVKTREAQGQRLPALFCFPQILQHRLRSINAAFKRARESYGY
NGDYFLVYPIKVNQHRRVIESLIHSGEPLGLEAGSKAELMAVLAHAGMTRSVIVCNGYK
DREYIRLALIGEKMGHKVYLVIEKMSEIAIVLDEAERLNVVPRLGVRARLASQGSGKWQ
SSGGEKSKFGLAATQVLQLVETLREAGRLDSLQLLHFHLGSQMANIRDIATGVRESARF
YVELHKLGVNIQCFDVGGGLGVDYEGTRSQSDCSVNYGLNEYANNIIWAIGDACEENG
WLPHPTVITESGRAVTAHHTVLVSNIIGVERNEYTVPTAPAEDAPRALQSMWETWQEMHE
PGTRRSLREWLHDSQMDLHDIHIGYSSGIFSLQERAWAEQLYLSMCHEVQKQLDPQNR
AHRPIIDELQERMADKMYVNFSLFQSMPDAWGIDQLFPVLPLEGLDQVPERRAVLLDIT
CDSDGAIDHYIDGDGIATTMPMPEYDPENPPMLGFFMVGAYQEILGNMHNLFGDTEAV
DVFVFPDGSVEVELSDEGDTVADMLQYVQLDPKTLLTQFRDQVKKTDLDAELQQQFLE
EFEAGLYGYTYLEDE SpeC polypeptide sequence SEQ ID NO: 24
MKSMNIAASSELVSRLSSHRRVVALGDTDFTDVAAVVITAADSRSGILALLKRTGFHLP
VFLYSEHAVELPAGVTAVINGNEQQWLELESAACQYEENLLPPFYDTLTQYVEMGNSTF
ACPGHQHGAFFKKHPAGRHFYDFFGENVFRADMCNADVKLGDLLIHEGSAKDAQKFA
AKVFHADKTYFVLNGTSAANKVVTNALLTRGDLVLFDRNNHKSNHHGALIQAGATPV
YLEASRNPFGFIGGIDAHCFNEEYLRQQIRDVAPEKADLPRPYRLAIIQLGTYDGTVYNA
RQVIDTVGHLCDYILFDSAWVGYEQFIPMMADSSPLLLELNENDPGIFVTQSVHKQQA
GFSQTSQIHKKDNHIRGQARFCPHKRLNNAFMLHASTSPFYPLFAALDVNAKIHEGESG
RRLWAECVEIGIEARKAILARCKLFRPFIPPVVDGKLWQDYPTSVLASDRRFFSFEPGAK
WHGFEGYAADQYFVDPCKLLLTTPGIDAETGEYSDFGVPATILAHYLRENGIVPEKCDL
NSILFLLTPAESHEKLAQLVAMLAQFEQHIEDDSPLVEVLPSVYNKYPVRYRDYTLRQLC
QEMHDLYVSFDVKDLQKAMFRQQSFPSVVMNPQDAHSAYIRGDVELVRIRDAEGRIAA
EGALPYPPGVLCVVPGEVWGGAVQRYFLALEEGVNLLPGFSPELQGVYSETDADGVKR LYGYVLK
SpeF polypeptide sequence SEQ ID NO: 25
MSKLKIAVSDSCPDCFTTQRECIYINESRNIDVAAIVLSLNDVTCGKLDEIDATGYGIPVFI
ATENQERVPAEYLPRISGVFENCESRREFYGRQLETAASHYETQLRPPFFRALVDYVNQ
GNSAFDCPGHQGGEFFRRHPAGNQFVEYFGEALFRADLCNADVAMGDLLIHEGAPCIA
QQHAAKVFNADKTYFVLNGTSSSNKVVLNALLTPGDLVLFDRNNHKSNHHGALLQAG
ATPVYLETARNPYGFIGGIDAHCFEESYLRELIAEVAPQRAKEARPFRLAVIQLGTYDGTI
YNARQVVDKIGHLCDYILFDSAWVGYEQFIPMMADCSPLLLDLNENDPGILVTQSVHKQ
QAGFSQTSQIHKKDSHIKGQQRYVPHKRMNNAFMMHASTSPFYPLFAALNINAKMHEG
VSGRNMWMDCVVNGINARKLILDNCQHIRPFVPELVDGKPWQSYETAQIAVDLRFFQF
VPGEHWHSFEGYAENQYFVDPCKLLLTTPGIDARNGEYEAFGVPATILANFLRENGVVP
EKCDLNSILFLLTPAEDMAKLQQLVALLVRFEKLLESDAPLAEVLPSIYKQHEERYAGYT
LRQLCQEMHDLYARHNVKQLQKEMFRKEHFPRVSMNPQEANYAYLRGEVELVRLPDA
GRIAAEGALPYPPGVLCVVPGEIWGGAVLRYFSALEEGINLLPGFAPELQGVYIEEHDG
RKQVWCYVIKPRDAQSTLLKGEKL GadA polypeptide sequence SEQ ID NO: 26
MDQKLLTDFRSELLDSRFGAKAISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNL
ATFCQTWDDENVHKLMDLSINKNWIDKEEYPQSAAIDLRCVNMVADLWHAPAPKNGQ
AVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARY
WDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQ
ADTGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWR
DEEALPQELVFNVDYLGGQIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQV
AAYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPA
FTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT GadB
polypeptide sequence SEQ ID NO: 27
MDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNL
ATFCQTWDDENVHKLMDLSINKNWIDKEEYPQSAAIDLRCVNMVADLWHAPAPKNGQ
AVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARY
WDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQ
ADTGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWR
DEEALPQELVFNVDYLGGQIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQV
AAYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPA
FTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT
Sequence CWU 1
1
6812148DNAEscherichia coli 1atgaacgtta ttgcaatatt gaatcacatg
ggggtttatt ttaaagaaga acccatccgt 60gaacttcatc gcgcgcttga acgtctgaac
ttccagattg tttacccgaa cgaccgtgac 120gacttattaa aactgatcga
aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180aaatataatc
tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac
240gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg
tttacagatt 300agcttctttg aatatgcgct gggtgctgct gaagatattg
ctaataagat caagcagacc 360actgacgaat atatcaacac tattctgcct
ccgctgacta aagcactgtt taaatatgtt 420cgtgaaggta aatatacttt
ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480agcccggtag
gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt
540tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca
caaagaagca 600gaacagtata tcgctcgcgt ctttaacgca gaccgcagct
acatggtgac caacggtact 660tccactgcga acaaaattgt tggtatgtac
tctgctccag caggcagcac cattctgatt 720gaccgtaact gccacaaatc
gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780tatttccgcc
cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc
840cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg
gccggtacat 900gctgtaatta ccaactctac ctatgatggt ctgctgtaca
acaccgactt catcaagaaa 960acactggatg tgaaatccat ccactttgac
tccgcgtggg tgccttacac caacttctca 1020ccgatttacg aaggtaaatg
cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080gaaacccagt
ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt
1140aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac
caccacttct 1200ccgcactacg gtatcgtggc gtccactgaa accgctgcgg
cgatgatgaa aggcaatgca 1260ggtaagcgtc tgatcaacgg ttctattgaa
cgtgcgatca aattccgtaa agagatcaaa 1320cgtctgagaa cggaatctga
tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380acgactgaat
gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat
1440aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg
gatggaaaaa 1500gacggcacca tgagcgactt tggtattccg gccagcatcg
tggcgaaata cctcgacgaa 1560catggcatcg ttgttgagaa aaccggtccg
tataacctgc tgttcctgtt cagcatcggt 1620atcgataaga ccaaagcact
gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680gacctgaacc
tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc
1740tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat
tgttcaccac 1800aatctgccgg atctgatgta tcgcgcattt gaagtgctgc
cgacgatggt aatgactccg 1860tatgctgcat tccagaaaga gctgcacggt
atgaccgaag aagtttacct cgacgaaatg 1920gtaggtcgta ttaacgccaa
tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980ccgggtgaaa
tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt
2040gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata
ccgtcaggct 2100gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa
21482715PRTEscherichia coli 2Met Asn Val Ile Ala Ile Leu Asn His
Met Gly Val Tyr Phe Lys Glu1 5 10 15Glu Pro Ile Arg Glu Leu His Arg
Ala Leu Glu Arg Leu Asn Phe Gln 20 25 30Ile Val Tyr Pro Asn Asp Arg
Asp Asp Leu Leu Lys Leu Ile Glu Asn 35 40 45Asn Ala Arg Leu Cys Gly
Val Ile Phe Asp Trp Asp Lys Tyr Asn Leu 50 55 60Glu Leu Cys Glu Glu
Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr65 70 75 80Ala Phe Ala
Asn Thr Tyr Ser Thr Leu Asp Val Ser Leu Asn Asp Leu 85 90 95Arg Leu
Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp 100 105
110Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile
115 120 125Leu Pro Pro Leu Thr Lys Ala Leu Phe Lys Tyr Val Arg Glu
Gly Lys 130 135 140Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr
Ala Phe Gln Lys145 150 155 160Ser Pro Val Gly Ser Leu Phe Tyr Asp
Phe Phe Gly Pro Asn Thr Met 165 170 175Lys Ser Asp Ile Ser Ile Ser
Val Ser Glu Leu Gly Ser Leu Leu Asp 180 185 190His Ser Gly Pro His
Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe 195 200 205Asn Ala Asp
Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn 210 215 220Lys
Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile225 230
235 240Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser
Asp 245 250 255Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr
Gly Ile Leu 260 265 270Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala
Thr Ile Ala Lys Arg 275 280 285Val Lys Glu Thr Pro Asn Ala Thr Trp
Pro Val His Ala Val Ile Thr 290 295 300Asn Ser Thr Tyr Asp Gly Leu
Leu Tyr Asn Thr Asp Phe Ile Lys Lys305 310 315 320Thr Leu Asp Val
Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr 325 330 335Thr Asn
Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly 340 345
350Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly
Asp Val 370 375 380Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His
Thr Thr Thr Ser385 390 395 400Pro His Tyr Gly Ile Val Ala Ser Thr
Glu Thr Ala Ala Ala Met Met 405 410 415Lys Gly Asn Ala Gly Lys Arg
Leu Ile Asn Gly Ser Ile Glu Arg Ala 420 425 430Ile Lys Phe Arg Lys
Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly 435 440 445Trp Phe Phe
Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys 450 455 460Trp
Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp465 470
475 480Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr
Pro 485 490 495Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile
Pro Ala Ser 500 505 510Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile
Val Val Glu Lys Thr 515 520 525Gly Pro Tyr Asn Leu Leu Phe Leu Phe
Ser Ile Gly Ile Asp Lys Thr 530 535 540Lys Ala Leu Ser Leu Leu Arg
Ala Leu Thr Asp Phe Lys Arg Ala Phe545 550 555 560Asp Leu Asn Leu
Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu 565 570 575Asp Pro
Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn 580 585
590Ile His Lys Leu Ile Val His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala
Ala Phe 610 615 620Gln Lys Glu Leu His Gly Met Thr Glu Glu Val Tyr
Leu Asp Glu Met625 630 635 640Val Gly Arg Ile Asn Ala Asn Met Ile
Leu Pro Tyr Pro Pro Gly Val 645 650 655Pro Leu Val Met Pro Gly Glu
Met Ile Thr Glu Glu Ser Arg Pro Val 660 665 670Leu Glu Phe Leu Gln
Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly 675 680 685Phe Glu Thr
Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr 690 695 700Thr
Val Lys Val Leu Lys Glu Glu Ser Lys Lys705 710
7153100PRTSaccharomyces cerevisiae 3Phe Pro Pro Lys Lys Phe Lys Asp
Leu Asn Ser Phe Leu Asp Asp Gln1 5 10 15Pro Lys Asp Pro Asn Leu Val
Ala Ser Pro Phe Gly Gly Tyr Phe Lys 20 25 30Asn Pro Ala Ala Asp Ala
Gly Ser Asn Asn Ala Ser Lys Lys Ser Ser 35 40 45Tyr Gln Gln Gln Arg
Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln Gly 50 55 60Gly Tyr Gln Ser
Tyr Asp Ser Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn65 70 75 80Asn Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys Tyr Asn Gly 85 90 95Gln
Gly Tyr Gln 1004102PRTSaccharomyces cerevisiae 4Ser Asp Ser Asn Gln
Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr Ser1 5 10 15Gln Asn Gly Asn
Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr Gln 20 25 30Ala Tyr Asn
Ala Gln Ala Gln Pro Ala Gly Gly Tyr Tyr Gln Asn Tyr 35 40 45Gln Gly
Tyr Ser Gly Tyr Gln Gln Gly Gly Tyr Gln Gln Tyr Asn Pro 50 55 60Gln
Gly Gly Tyr Gln Gln Asp Ala Gly Tyr Gln Gln Gln Tyr Asn Pro65 70 75
80Gln Gly Gly Tyr Gln Gln Tyr Asn Pro Gln Gly Gly Tyr Gln Gln Gln
85 90 95Phe Asn Pro Gln Gly Gly 1005300DNAArtificial SequenceNew1
prion subunit nucleic acid sequence.Codon optimized. 5tttccgccga
aaaagttcaa agacctgaac tctttcctgg acgaccagcc gaaagacccg 60aacctggttg
cgtctccgtt cggtggctac ttcaaaaacc cagcggcgga cgcgggttct
120aacaacgcgt ctaagaaatc ttcttaccag cagcagcgta actggaaaca
gggtggcaac 180tatcagcaag gtggttacca gtcttacgac tctaattaca
acaactacaa caactacaat 240aactataata actacaacaa ctacaacaat
tataacaaat acaacggtca gggctaccag 3006306DNAArtificial SequenceSup35
prion subunit nucleic acid sequence.Codon optimized 6tctgactcta
accaaggtaa taaccagcag aactaccaac aatactctca gaacggcaac 60cagcagcagg
gcaacaaccg ctatcaaggc taccaagcgt acaacgcgca ggcacagcca
120gcaggtggct actaccagaa ttaccagggt tactctggtt accagcaagg
tggttatcaa 180cagtataatc cgcagggcgg ctatcagcag gacgcaggtt
accagcaaca atataaccct 240cagggcggct atcagcaata caacccgcaa
ggcggttatc aacaacagtt taacccacag 300ggtggc 3067105PRTArtificial
SequenceNew1 prion subunit and linker polypeptide sequence. The
linker sequence is GSGSG(in the front of the sequence). 7Gly Ser
Gly Ser Gly Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser1 5 10 15Phe
Leu Asp Asp Gln Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe 20 25
30Gly Gly Tyr Phe Lys Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala
35 40 45Ser Lys Lys Ser Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly
Gly 50 55 60Asn Tyr Gln Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr
Asn Asn65 70 75 80Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn
Tyr Asn Asn Tyr 85 90 95Asn Lys Tyr Asn Gly Gln Gly Tyr Gln 100
1058107PRTArtificial SequenceSup35 prion subunit and linker
polypeptide sequence.The linker polypeptide sequence is GSGSG(in
the front of the sequence) 8Gly Ser Gly Ser Gly Ser Asp Ser Asn Gln
Gly Asn Asn Gln Gln Asn1 5 10 15Tyr Gln Gln Tyr Ser Gln Asn Gly Asn
Gln Gln Gln Gly Asn Asn Arg 20 25 30Tyr Gln Gly Tyr Gln Ala Tyr Asn
Ala Gln Ala Gln Pro Ala Gly Gly 35 40 45Tyr Tyr Gln Asn Tyr Gln Gly
Tyr Ser Gly Tyr Gln Gln Gly Gly Tyr 50 55 60Gln Gln Tyr Asn Pro Gln
Gly Gly Tyr Gln Gln Asp Ala Gly Tyr Gln65 70 75 80Gln Gln Tyr Asn
Pro Gln Gly Gly Tyr Gln Gln Tyr Asn Pro Gln Gly 85 90 95Gly Tyr Gln
Gln Gln Phe Asn Pro Gln Gly Gly 100 1059315DNAArtificial
SequenceNew1 prion subunit and linker nucleic acid sequence.The
region encoding the linker is GGTTCTGGCTCTGGT(in the front of
sequence). 9ggttctggct ctggttttcc gccgaaaaag ttcaaagacc tgaactcttt
cctggacgac 60cagccgaaag acccgaacct ggttgcgtct ccgttcggtg gctacttcaa
aaacccagcg 120gcggacgcgg gttctaacaa cgcgtctaag aaatcttctt
accagcagca gcgtaactgg 180aaacagggtg gcaactatca gcaaggtggt
taccagtctt acgactctaa ttacaacaac 240tacaacaact acaataacta
taataactac aacaactaca acaattataa caaatacaac 300ggtcagggct accag
31510321DNAArtificial SequenceSup35 prion subunit and linker
nucleic acid sequence.The region encoding the linker is
GGTAGCGGCTCTGGC(in the front of the sequence). 10ggtagcggct
ctggctctga ctctaaccaa ggtaataacc agcagaacta ccaacaatac 60tctcagaacg
gcaaccagca gcagggcaac aaccgctatc aaggctacca agcgtacaac
120gcgcaggcac agccagcagg tggctactac cagaattacc agggttactc
tggttaccag 180caaggtggtt atcaacagta taatccgcag ggcggctatc
agcaggacgc aggttaccag 240caacaatata accctcaggg cggctatcag
caatacaacc cgcaaggcgg ttatcaacaa 300cagtttaacc cacagggtgg c
3211196PRTArtificial SequenceNew1 prion subunit and linker with 9
amino acid truncation polypeptide sequence.The linker is GSGSG(in
the front of the sequence). 11Gly Ser Gly Ser Gly Asn Ser Phe Leu
Asp Asp Gln Pro Lys Asp Pro1 5 10 15Asn Leu Val Ala Ser Pro Phe Gly
Gly Tyr Phe Lys Asn Pro Ala Ala 20 25 30Asp Ala Gly Ser Asn Asn Ala
Ser Lys Lys Ser Ser Tyr Gln Gln Gln 35 40 45Arg Asn Trp Lys Gln Gly
Gly Asn Tyr Gln Gln Gly Gly Tyr Gln Ser 50 55 60Tyr Asp Ser Asn Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn65 70 75 80Tyr Asn Asn
Tyr Asn Asn Tyr Asn Lys Tyr Asn Gly Gln Gly Tyr Gln 85 90
951287PRTArtificial SequenceNew1 prion subunit and linker with 18
amino acid truncation polypeptide sequence.The linker is GSGSG(in
the front of the sequence). 12Gly Ser Gly Ser Gly Asp Pro Asn Leu
Val Ala Ser Pro Phe Gly Gly1 5 10 15Tyr Phe Lys Asn Pro Ala Ala Asp
Ala Gly Ser Asn Asn Ala Ser Lys 20 25 30Lys Ser Ser Tyr Gln Gln Gln
Arg Asn Trp Lys Gln Gly Gly Asn Tyr 35 40 45Gln Gln Gly Gly Tyr Gln
Ser Tyr Asp Ser Asn Tyr Asn Asn Tyr Asn 50 55 60Asn Tyr Asn Asn Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys65 70 75 80Tyr Asn Gly
Gln Gly Tyr Gln 851369PRTArtificial SequenceNew1 prion subunit and
linker with 36 amino acid truncation polypeptide sequence.The
linker is GSGSG(in the front of the sequence). 13Gly Ser Gly Ser
Gly Asp Ala Gly Ser Asn Asn Ala Ser Lys Lys Ser1 5 10 15Ser Tyr Gln
Gln Gln Arg Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln 20 25 30Gly Gly
Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn Tyr Asn Asn Tyr 35 40 45Asn
Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Lys Tyr Asn 50 55
60Gly Gln Gly Tyr Gln651460PRTArtificial SequenceNew1 prion subunit
and linker with 45 amino acid truncation polypeptide sequence.The
linker is GSGSG(in the front of the sequence). 14Gly Ser Gly Ser
Gly Lys Ser Ser Tyr Gln Gln Gln Arg Asn Trp Lys1 5 10 15Gln Gly Gly
Asn Tyr Gln Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn 20 25 30Tyr Asn
Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr 35 40 45Asn
Asn Tyr Asn Lys Tyr Asn Gly Gln Gly Tyr Gln 50 55
6015115PRTArtificial SequenceNew1 prion domain fragment and linker
with BST C-terminal fragment polypeptide sequence.The linker is
GSGSG(in the front of the sequence). 15Gly Ser Gly Ser Gly Phe Pro
Pro Lys Lys Phe Lys Asp Leu Asn Ser1 5 10 15Phe Leu Asp Asp Gln Pro
Lys Asp Pro Asn Leu Val Ala Ser Pro Phe 20 25 30Gly Gly Tyr Phe Lys
Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala 35 40 45Ser Lys Lys Ser
Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly 50 55 60Asn Tyr Gln
Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn65 70 75 80Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr 85 90
95Asn Lys Tyr Asn Gly Gln Gly Tyr Gln Arg Arg Phe Gly Glu Ala Ser
100 105 110Ser Ala Phe 11516345DNAArtificial SequenceNew1 prion
subunit and linker with BST C-terminal fragment nucleic acid
sequence.The region encoding the linker is GGTTCTGGCTCTGGT(in the
front of the sequence). 16ggttctggct ctggttttcc gccgaaaaag
ttcaaagacc tgaactcttt cctggacgac 60cagccgaaag acccgaacct ggttgcgtct
ccgttcggtg gctacttcaa aaacccagcg 120gcggacgcgg gttctaacaa
cgcgtctaag aaatcttctt accagcagca gcgtaactgg 180aaacagggtg
gcaactatca gcaaggtggt taccagtctt acgactctaa ttacaacaac
240tacaacaact acaataacta taataactac aacaactaca acaattataa
caaatacaac 300ggtcagggct accagcgtcg tttcggcgaa gcgagcagcg cgttc
34517115PRTArtificial SequenceNew1 prion subunit and linker with
??CI C-terminal fragment polypeptide sequence.The linker is
GSGSG(in the front of the sequence). 17Gly Ser Gly Ser Gly Phe Pro
Pro Lys Lys Phe Lys Asp Leu Asn Ser1 5 10 15Phe Leu Asp Asp Gln Pro
Lys Asp Pro Asn Leu Val Ala Ser Pro Phe 20 25 30Gly Gly Tyr Phe Lys
Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala 35 40 45Ser Lys Lys Ser
Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly 50 55 60Asn Tyr Gln
Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn65 70 75 80Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr 85 90
95Asn Lys Tyr Asn Gly Gln Gly Tyr Gln Ala Ser Gln Trp Pro Glu Glu
100 105 110Thr Phe Gly 11518345DNAArtificial SequenceNew1 prion
domain fragment and linker with ??CI C-terminal fragment nucleic
acid sequence.The region encoding the linker is GGTTCTGGCTCTGGT(in
the front of the sequence). 18ggttctggct ctggttttcc gccgaaaaag
ttcaaagacc tgaactcttt cctggacgac 60cagccgaaag acccgaacct ggttgcgtct
ccgttcggtg gctacttcaa aaacccagcg 120gcggacgcgg gttctaacaa
cgcgtctaag aaatcttctt accagcagca gcgtaactgg 180aaacagggtg
gcaactatca gcaaggtggt taccagtctt acgactctaa ttacaacaac
240tacaacaact acaataacta taataactac aacaactaca acaattataa
caaatacaac 300ggtcagggct accaggcgag ccagtggccg gaagaaacct tcggc
34519115PRTArtificial SequenceNew1 prion domain fragment and linker
with RecA C-terminal fragment polypeptide sequence.The linker is
GSGSG(in the front of the sequence). 19Gly Ser Gly Ser Gly Phe Pro
Pro Lys Lys Phe Lys Asp Leu Asn Ser1 5 10 15Phe Leu Asp Asp Gln Pro
Lys Asp Pro Asn Leu Val Ala Ser Pro Phe 20 25 30Gly Gly Tyr Phe Lys
Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala 35 40 45Ser Lys Lys Ser
Ser Tyr Gln Gln Gln Arg Asn Trp Lys Gln Gly Gly 50 55 60Asn Tyr Gln
Gln Gly Gly Tyr Gln Ser Tyr Asp Ser Asn Tyr Asn Asn65 70 75 80Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr 85 90
95Asn Lys Tyr Asn Gly Gln Gly Tyr Gln Glu Gly Val Ala Glu Thr Asn
100 105 110Glu Asp Phe 11520345DNAArtificial SequenceNew1 subunit
and linker with RecA C-terminal fragment nucleic acid sequence.The
region encoding the linker is GGTTCTGGCTCTGGT(in the front of the
sequence). 20ggttctggct ctggttttcc gccgaaaaag ttcaaagacc tgaactcttt
cctggacgac 60cagccgaaag acccgaacct ggttgcgtct ccgttcggtg gctacttcaa
aaacccagcg 120gcggacgcgg gttctaacaa cgcgtctaag aaatcttctt
accagcagca gcgtaactgg 180aaacagggtg gcaactatca gcaaggtggt
taccagtctt acgactctaa ttacaacaac 240tacaacaact acaataacta
taataactac aacaactaca acaattataa caaatacaac 300ggtcagggct
accaggaagg cgtggcggaa accaacgaag atttc 34521713PRTEscherichia coli
21Met Asn Ile Ile Ala Ile Met Gly Pro His Gly Val Phe Tyr Lys Asp1
5 10 15Glu Pro Ile Lys Glu Leu Glu Ser Ala Leu Val Ala Gln Gly Phe
Gln 20 25 30Ile Ile Trp Pro Gln Asn Ser Val Asp Leu Leu Lys Phe Ile
Glu His 35 40 45Asn Pro Arg Ile Cys Gly Val Ile Phe Asp Trp Asp Glu
Tyr Ser Leu 50 55 60Asp Leu Cys Ser Asp Ile Asn Gln Leu Asn Glu Tyr
Leu Pro Leu Tyr65 70 75 80Ala Phe Ile Asn Thr His Ser Thr Met Asp
Val Ser Val Gln Asp Met 85 90 95Arg Met Ala Leu Trp Phe Phe Glu Tyr
Ala Leu Gly Gln Ala Glu Asp 100 105 110Ile Ala Ile Arg Met Arg Gln
Tyr Thr Asp Glu Tyr Leu Asp Asn Ile 115 120 125Thr Pro Pro Phe Thr
Lys Ala Leu Phe Thr Tyr Val Lys Glu Arg Lys 130 135 140Tyr Thr Phe
Cys Thr Pro Gly His Met Gly Gly Thr Ala Tyr Gln Lys145 150 155
160Ser Pro Val Gly Cys Leu Phe Tyr Asp Phe Phe Gly Gly Asn Thr Leu
165 170 175Lys Ala Asp Val Ser Ile Ser Val Thr Glu Leu Gly Ser Leu
Leu Asp 180 185 190His Thr Gly Pro His Leu Glu Ala Glu Glu Tyr Ile
Ala Arg Thr Phe 195 200 205Gly Ala Glu Gln Ser Tyr Ile Val Thr Asn
Gly Thr Ser Thr Ser Asn 210 215 220Lys Ile Val Gly Met Tyr Ala Ala
Pro Ser Gly Ser Thr Leu Leu Ile225 230 235 240Asp Arg Asn Cys His
Lys Ser Leu Ala His Leu Leu Met Met Asn Asp 245 250 255Val Val Pro
Val Trp Leu Lys Pro Thr Arg Asn Ala Leu Gly Ile Leu 260 265 270Gly
Gly Ile Pro Arg Arg Glu Phe Thr Arg Asp Ser Ile Glu Glu Lys 275 280
285Val Ala Ala Thr Thr Gln Ala Gln Trp Pro Val His Ala Val Ile Thr
290 295 300Asn Ser Thr Tyr Asp Gly Leu Leu Tyr Asn Thr Asp Trp Ile
Lys Gln305 310 315 320Thr Leu Asp Val Pro Ser Ile His Phe Asp Ser
Ala Trp Val Pro Tyr 325 330 335Thr His Phe His Pro Ile Tyr Gln Gly
Lys Ser Gly Met Ser Gly Glu 340 345 350Arg Val Ala Gly Lys Val Ile
Phe Glu Thr Gln Ser Thr His Lys Met 355 360 365Leu Ala Ala Leu Ser
Gln Ala Ser Leu Ile His Ile Lys Gly Glu Tyr 370 375 380Asp Glu Glu
Ala Phe Asn Glu Ala Phe Met Met His Thr Thr Thr Ser385 390 395
400Pro Ser Tyr Pro Ile Val Ala Ser Val Glu Thr Ala Ala Ala Met Leu
405 410 415Arg Gly Asn Pro Gly Lys Arg Leu Ile Asn Arg Ser Val Glu
Arg Ala 420 425 430Leu His Phe Arg Lys Glu Val Gln Arg Leu Arg Glu
Glu Ser Asp Gly 435 440 445Trp Phe Phe Asp Ile Trp Gln Pro Pro Gln
Val Asp Glu Ala Glu Cys 450 455 460Trp Pro Val Ala Pro Gly Glu Gln
Trp His Gly Phe Asn Asp Ala Asp465 470 475 480Ala Asp His Met Phe
Leu Asp Pro Val Lys Val Thr Ile Leu Thr Pro 485 490 495Gly Met Asp
Glu Gln Gly Asn Met Ser Glu Glu Gly Ile Pro Ala Ala 500 505 510Leu
Val Ala Lys Phe Leu Asp Glu Arg Gly Ile Val Val Glu Lys Thr 515 520
525Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540Lys Ala Met Gly Leu Leu Arg Gly Leu Thr Glu Phe Lys Arg
Ser Tyr545 550 555 560Asp Leu Asn Leu Arg Ile Lys Asn Met Leu Pro
Asp Leu Tyr Ala Glu 565 570 575Asp Pro Asp Phe Tyr Arg Asn Met Arg
Ile Gln Asp Leu Ala Gln Gly 580 585 590Ile His Lys Leu Ile Arg Lys
His Asp Leu Pro Gly Leu Met Leu Arg 595 600 605Ala Phe Asp Thr Leu
Pro Glu Met Ile Met Thr Pro His Gln Ala Trp 610 615 620Gln Arg Gln
Ile Lys Gly Glu Val Glu Thr Ile Ala Leu Glu Gln Leu625 630 635
640Val Gly Arg Val Ser Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655Pro Leu Leu Met Pro Gly Glu Met Leu Thr Lys Glu Ser Arg
Thr Val 660 665 670Leu Asp Phe Leu Leu Met Leu Cys Ser Val Gly Gln
His Tyr Pro Gly 675 680 685Phe Glu Thr Asp Ile His Gly Ala Lys Gln
Asp Glu Asp Gly Val Tyr 690 695 700Arg Val Arg Val Leu Lys Met Ala
Gly705 71022755PRTEscherichia coli 22Met Lys Val Leu Ile Val Glu
Ser Glu Phe Leu His Gln Asp Thr Trp1 5 10 15Val Gly Asn Ala Val Glu
Arg Leu Ala Asp Ala Leu Ser Gln Gln Asn 20 25 30Val Thr Val Ile Lys
Ser Thr Ser Phe Asp Asp Gly Phe Ala Ile Leu 35 40 45Ser Ser Asn Glu
Ala Ile Asp Cys Leu Met Phe Ser Tyr Gln Met Glu 50 55 60His Pro Asp
Glu His Gln Asn Val Arg Gln Leu Ile Gly Lys Leu His65 70 75 80Glu
Arg Gln Gln Asn Val Pro Val Phe Leu Leu Gly Asp Arg Glu Lys 85 90
95Ala Leu Ala Ala Met Asp Arg Asp Leu Leu Glu Leu Val Asp Glu Phe
100 105 110Ala Trp Ile Leu Glu Asp Thr Ala Asp Phe Ile Ala Gly Arg
Ala Val 115 120 125Ala Ala Met Thr Arg Tyr Arg Gln Gln Leu Leu Pro
Pro Leu Phe Ser 130 135 140Ala Leu Met Lys Tyr Ser Asp Ile His Glu
Tyr Ser Trp Ala Ala Pro145 150 155 160Gly His Gln Gly Gly Val Gly
Phe Thr Lys Thr Pro Ala Gly Arg Phe 165 170 175Tyr His Asp Tyr Tyr
Gly Glu Asn Leu Phe Arg Thr Asp Met Gly Ile 180 185 190Glu Arg Thr
Ser Leu Gly Ser Leu Leu Asp His Thr Gly Ala Phe Gly 195 200 205Glu
Ser Glu Lys Tyr Ala Ala Arg Val Phe Gly Ala Asp Arg Ser Trp 210 215
220Ser Val Val Val Gly Thr Ser Gly Ser Asn Arg Thr Ile Met Gln
Ala225 230 235 240Cys Met Thr Asp Asn Asp Val Val Val Val Asp Arg
Asn Cys His Lys 245 250 255Ser Ile Glu Gln Gly Leu Met Leu Thr Gly
Ala Lys Pro Val Tyr Met 260 265 270Val Pro Ser Arg Asn Arg Tyr Gly
Ile Ile Gly Pro Ile Tyr Pro Gln 275 280 285Glu Met Gln Pro Glu Thr
Leu Gln Lys Lys Ile Ser Glu Ser Pro Leu 290 295 300Thr Lys Asp Lys
Ala Gly Gln Lys Pro Ser Tyr Cys Val Val Thr Asn305 310 315 320Cys
Thr Tyr Asp Gly Val Cys Tyr Asn Ala Lys Glu Ala Gln Asp Leu 325 330
335Leu Glu Lys Thr Ser Asp Arg Leu His Phe Asp Glu Ala Trp Tyr Gly
340 345 350Tyr Ala Arg Phe Asn Pro Ile Tyr Ala Asp His Tyr Ala Met
Arg Gly 355 360 365Glu Pro Gly Asp His Asn Gly Pro Thr Val Phe Ala
Thr His Ser Thr 370 375 380His Lys Leu Leu Asn Ala Leu Ser Gln Ala
Ser Tyr Ile His Val Arg385 390 395 400Glu Gly Arg Gly Ala Ile Asn
Phe Ser Arg Phe Asn Gln Ala Tyr Met 405 410 415Met His Ala Thr Thr
Ser Pro Leu Tyr Ala Ile Cys Ala Ser Asn Asp 420 425 430Val Ala Val
Ser Met Met Asp Gly Asn Ser Gly Leu Ser Leu Thr Gln 435 440 445Glu
Val Ile Asp Glu Ala Val Asp Phe Arg Gln Ala Met Ala Arg Leu 450 455
460Tyr Lys Glu Phe Thr Ala Asp Gly Ser Trp Phe Phe Lys Pro Trp
Asn465 470 475 480Lys Glu Val Val Thr Asp Pro Gln Thr Gly Lys Thr
Tyr Asp Phe Ala 485 490 495Asp Ala Pro Thr Lys Leu Leu Thr Thr Val
Gln Asp Cys Trp Val Met 500 505 510His Pro Gly Glu Ser Trp His Gly
Phe Lys Asp Ile Pro Asp Asn Trp 515 520 525Ser Met Leu Asp Pro Ile
Lys Val Ser Ile Leu Ala Pro Gly Met Gly 530 535 540Glu Asp Gly Glu
Leu Glu Glu Thr Gly Val Pro Ala Ala Leu Val Thr545 550 555 560Ala
Trp Leu Gly Arg His Gly Ile Val Pro Thr Arg Thr Thr Asp Phe 565 570
575Gln Ile Met Phe Leu Phe Ser Met Gly Val Thr Arg Gly Lys Trp Gly
580 585 590Thr Leu Val Asn Thr Leu Cys Ser Phe Lys Arg His Tyr Asp
Ala Asn 595 600 605Thr Pro Leu Ala Gln Val Met Pro Glu Leu Val Glu
Gln Tyr Pro Asp 610 615 620Thr Tyr Ala Asn Met Gly Ile His Asp Leu
Gly Asp Thr Met Phe Ala625 630 635 640Trp Leu Lys Glu Asn Asn Pro
Gly Ala Arg Leu Asn Glu Ala Tyr Ser 645 650 655Gly Leu Pro Val Ala
Glu Val Thr Pro Arg Glu Ala Tyr Asn Ala Ile 660 665 670Val Asp Asn
Asn Val Glu Leu Val Ser Ile Glu Asn Leu Pro Gly Arg 675 680 685Ile
Ala Ala Asn Ser Val Ile Pro Tyr Pro Pro Gly Ile Pro Met Leu 690 695
700Leu Ser Gly Glu Asn Phe Gly Asp Lys Asn Ser Pro Gln Val Ser
Tyr705 710 715 720Leu Arg Ser Leu Gln Ser Trp Asp His His Phe Pro
Gly Phe Glu His 725 730 735Glu Thr Glu Gly Thr Glu Ile Ile Asp Gly
Ile Tyr His Val Met Cys 740 745 750Val Lys Ala
75523658PRTEscherichia coli 23Met Ser Asp Asp Met Ser Met Gly Leu
Pro Ser Ser Ala Gly Glu His1 5 10 15Gly Val Leu Arg Ser Met Gln Glu
Val Ala Met Ser Ser Gln Glu Ala 20 25 30Ser Lys Met Leu Arg Thr Tyr
Asn Ile Ala Trp Trp Gly Asn Asn Tyr 35 40 45Tyr Asp Val Asn Glu Leu
Gly His Ile Ser Val Cys Pro Asp Pro Asp 50 55 60Val Pro Glu Ala Arg
Val Asp Leu Ala Gln Leu Val Lys Thr Arg Glu65 70 75 80Ala Gln Gly
Gln Arg Leu Pro Ala Leu Phe Cys Phe Pro Gln Ile Leu 85 90 95Gln His
Arg Leu Arg Ser Ile Asn Ala Ala Phe Lys Arg Ala Arg Glu 100 105
110Ser Tyr Gly Tyr Asn Gly Asp Tyr Phe Leu Val Tyr Pro Ile Lys Val
115 120 125Asn Gln His Arg Arg Val Ile Glu Ser Leu Ile His Ser Gly
Glu Pro 130 135 140Leu Gly Leu Glu Ala Gly Ser Lys Ala Glu Leu Met
Ala Val Leu Ala145 150 155 160His Ala Gly Met Thr Arg Ser Val Ile
Val Cys Asn Gly Tyr Lys Asp 165 170 175Arg Glu Tyr Ile Arg Leu Ala
Leu Ile Gly Glu Lys Met Gly His Lys 180 185 190Val Tyr Leu Val Ile
Glu Lys Met Ser Glu Ile Ala Ile Val Leu Asp 195 200 205Glu Ala Glu
Arg Leu Asn Val Val Pro Arg Leu Gly Val Arg Ala Arg 210 215 220Leu
Ala Ser Gln Gly Ser Gly Lys Trp Gln Ser Ser Gly Gly Glu Lys225 230
235 240Ser Lys Phe Gly Leu Ala Ala Thr Gln Val Leu Gln Leu Val Glu
Thr 245 250 255Leu Arg Glu Ala Gly Arg Leu Asp Ser Leu Gln Leu Leu
His Phe His 260 265 270Leu Gly Ser Gln Met Ala Asn Ile Arg Asp Ile
Ala Thr Gly Val Arg 275 280 285Glu Ser Ala Arg Phe Tyr Val Glu Leu
His Lys Leu Gly Val Asn Ile 290 295 300Gln Cys Phe Asp Val Gly Gly
Gly Leu Gly Val Asp Tyr Glu Gly Thr305 310 315 320Arg Ser Gln Ser
Asp Cys Ser Val Asn Tyr Gly Leu Asn Glu Tyr Ala 325 330 335Asn Asn
Ile Ile Trp Ala Ile Gly Asp Ala Cys Glu Glu Asn Gly Leu 340 345
350Pro His Pro Thr Val Ile Thr Glu Ser Gly Arg Ala Val Thr Ala His
355 360 365His Thr Val Leu Val Ser Asn Ile Ile Gly Val Glu Arg Asn
Glu Tyr 370 375 380Thr Val Pro Thr Ala Pro Ala Glu Asp Ala Pro Arg
Ala Leu Gln Ser385 390 395 400Met Trp Glu Thr Trp Gln Glu Met His
Glu Pro Gly Thr Arg Arg Ser 405 410 415Leu Arg Glu Trp Leu His Asp
Ser Gln Met Asp Leu His Asp Ile His 420 425 430Ile Gly Tyr Ser Ser
Gly Ile Phe Ser Leu Gln Glu Arg Ala Trp Ala 435 440 445Glu Gln Leu
Tyr Leu Ser Met Cys His Glu Val Gln Lys Gln Leu Asp 450 455 460Pro
Gln Asn Arg Ala His Arg Pro Ile Ile Asp Glu Leu Gln Glu Arg465 470
475 480Met Ala Asp Lys Met Tyr Val Asn Phe Ser Leu Phe Gln Ser Met
Pro 485 490 495Asp Ala Trp Gly Ile Asp Gln Leu Phe Pro Val Leu Pro
Leu Glu Gly 500 505 510Leu Asp Gln Val Pro Glu Arg Arg Ala Val Leu
Leu Asp Ile Thr Cys 515 520 525Asp Ser Asp Gly Ala Ile Asp His Tyr
Ile Asp Gly Asp Gly Ile Ala 530 535 540Thr Thr Met Pro Met
Pro Glu Tyr Asp Pro Glu Asn Pro Pro Met Leu545 550 555 560Gly Phe
Phe Met Val Gly Ala Tyr Gln Glu Ile Leu Gly Asn Met His 565 570
575Asn Leu Phe Gly Asp Thr Glu Ala Val Asp Val Phe Val Phe Pro Asp
580 585 590Gly Ser Val Glu Val Glu Leu Ser Asp Glu Gly Asp Thr Val
Ala Asp 595 600 605Met Leu Gln Tyr Val Gln Leu Asp Pro Lys Thr Leu
Leu Thr Gln Phe 610 615 620Arg Asp Gln Val Lys Lys Thr Asp Leu Asp
Ala Glu Leu Gln Gln Gln625 630 635 640Phe Leu Glu Glu Phe Glu Ala
Gly Leu Tyr Gly Tyr Thr Tyr Leu Glu 645 650 655Asp
Glu24711PRTEscherichia coli 24Met Lys Ser Met Asn Ile Ala Ala Ser
Ser Glu Leu Val Ser Arg Leu1 5 10 15Ser Ser His Arg Arg Val Val Ala
Leu Gly Asp Thr Asp Phe Thr Asp 20 25 30Val Ala Ala Val Val Ile Thr
Ala Ala Asp Ser Arg Ser Gly Ile Leu 35 40 45Ala Leu Leu Lys Arg Thr
Gly Phe His Leu Pro Val Phe Leu Tyr Ser 50 55 60Glu His Ala Val Glu
Leu Pro Ala Gly Val Thr Ala Val Ile Asn Gly65 70 75 80Asn Glu Gln
Gln Trp Leu Glu Leu Glu Ser Ala Ala Cys Gln Tyr Glu 85 90 95Glu Asn
Leu Leu Pro Pro Phe Tyr Asp Thr Leu Thr Gln Tyr Val Glu 100 105
110Met Gly Asn Ser Thr Phe Ala Cys Pro Gly His Gln His Gly Ala Phe
115 120 125Phe Lys Lys His Pro Ala Gly Arg His Phe Tyr Asp Phe Phe
Gly Glu 130 135 140Asn Val Phe Arg Ala Asp Met Cys Asn Ala Asp Val
Lys Leu Gly Asp145 150 155 160Leu Leu Ile His Glu Gly Ser Ala Lys
Asp Ala Gln Lys Phe Ala Ala 165 170 175Lys Val Phe His Ala Asp Lys
Thr Tyr Phe Val Leu Asn Gly Thr Ser 180 185 190Ala Ala Asn Lys Val
Val Thr Asn Ala Leu Leu Thr Arg Gly Asp Leu 195 200 205Val Leu Phe
Asp Arg Asn Asn His Lys Ser Asn His His Gly Ala Leu 210 215 220Ile
Gln Ala Gly Ala Thr Pro Val Tyr Leu Glu Ala Ser Arg Asn Pro225 230
235 240Phe Gly Phe Ile Gly Gly Ile Asp Ala His Cys Phe Asn Glu Glu
Tyr 245 250 255Leu Arg Gln Gln Ile Arg Asp Val Ala Pro Glu Lys Ala
Asp Leu Pro 260 265 270Arg Pro Tyr Arg Leu Ala Ile Ile Gln Leu Gly
Thr Tyr Asp Gly Thr 275 280 285Val Tyr Asn Ala Arg Gln Val Ile Asp
Thr Val Gly His Leu Cys Asp 290 295 300Tyr Ile Leu Phe Asp Ser Ala
Trp Val Gly Tyr Glu Gln Phe Ile Pro305 310 315 320Met Met Ala Asp
Ser Ser Pro Leu Leu Leu Glu Leu Asn Glu Asn Asp 325 330 335Pro Gly
Ile Phe Val Thr Gln Ser Val His Lys Gln Gln Ala Gly Phe 340 345
350Ser Gln Thr Ser Gln Ile His Lys Lys Asp Asn His Ile Arg Gly Gln
355 360 365Ala Arg Phe Cys Pro His Lys Arg Leu Asn Asn Ala Phe Met
Leu His 370 375 380Ala Ser Thr Ser Pro Phe Tyr Pro Leu Phe Ala Ala
Leu Asp Val Asn385 390 395 400Ala Lys Ile His Glu Gly Glu Ser Gly
Arg Arg Leu Trp Ala Glu Cys 405 410 415Val Glu Ile Gly Ile Glu Ala
Arg Lys Ala Ile Leu Ala Arg Cys Lys 420 425 430Leu Phe Arg Pro Phe
Ile Pro Pro Val Val Asp Gly Lys Leu Trp Gln 435 440 445Asp Tyr Pro
Thr Ser Val Leu Ala Ser Asp Arg Arg Phe Phe Ser Phe 450 455 460Glu
Pro Gly Ala Lys Trp His Gly Phe Glu Gly Tyr Ala Ala Asp Gln465 470
475 480Tyr Phe Val Asp Pro Cys Lys Leu Leu Leu Thr Thr Pro Gly Ile
Asp 485 490 495Ala Glu Thr Gly Glu Tyr Ser Asp Phe Gly Val Pro Ala
Thr Ile Leu 500 505 510Ala His Tyr Leu Arg Glu Asn Gly Ile Val Pro
Glu Lys Cys Asp Leu 515 520 525Asn Ser Ile Leu Phe Leu Leu Thr Pro
Ala Glu Ser His Glu Lys Leu 530 535 540Ala Gln Leu Val Ala Met Leu
Ala Gln Phe Glu Gln His Ile Glu Asp545 550 555 560Asp Ser Pro Leu
Val Glu Val Leu Pro Ser Val Tyr Asn Lys Tyr Pro 565 570 575Val Arg
Tyr Arg Asp Tyr Thr Leu Arg Gln Leu Cys Gln Glu Met His 580 585
590Asp Leu Tyr Val Ser Phe Asp Val Lys Asp Leu Gln Lys Ala Met Phe
595 600 605Arg Gln Gln Ser Phe Pro Ser Val Val Met Asn Pro Gln Asp
Ala His 610 615 620Ser Ala Tyr Ile Arg Gly Asp Val Glu Leu Val Arg
Ile Arg Asp Ala625 630 635 640Glu Gly Arg Ile Ala Ala Glu Gly Ala
Leu Pro Tyr Pro Pro Gly Val 645 650 655Leu Cys Val Val Pro Gly Glu
Val Trp Gly Gly Ala Val Gln Arg Tyr 660 665 670Phe Leu Ala Leu Glu
Glu Gly Val Asn Leu Leu Pro Gly Phe Ser Pro 675 680 685Glu Leu Gln
Gly Val Tyr Ser Glu Thr Asp Ala Asp Gly Val Lys Arg 690 695 700Leu
Tyr Gly Tyr Val Leu Lys705 71025732PRTEscherichia coli 25Met Ser
Lys Leu Lys Ile Ala Val Ser Asp Ser Cys Pro Asp Cys Phe1 5 10 15Thr
Thr Gln Arg Glu Cys Ile Tyr Ile Asn Glu Ser Arg Asn Ile Asp 20 25
30Val Ala Ala Ile Val Leu Ser Leu Asn Asp Val Thr Cys Gly Lys Leu
35 40 45Asp Glu Ile Asp Ala Thr Gly Tyr Gly Ile Pro Val Phe Ile Ala
Thr 50 55 60Glu Asn Gln Glu Arg Val Pro Ala Glu Tyr Leu Pro Arg Ile
Ser Gly65 70 75 80Val Phe Glu Asn Cys Glu Ser Arg Arg Glu Phe Tyr
Gly Arg Gln Leu 85 90 95Glu Thr Ala Ala Ser His Tyr Glu Thr Gln Leu
Arg Pro Pro Phe Phe 100 105 110Arg Ala Leu Val Asp Tyr Val Asn Gln
Gly Asn Ser Ala Phe Asp Cys 115 120 125Pro Gly His Gln Gly Gly Glu
Phe Phe Arg Arg His Pro Ala Gly Asn 130 135 140Gln Phe Val Glu Tyr
Phe Gly Glu Ala Leu Phe Arg Ala Asp Leu Cys145 150 155 160Asn Ala
Asp Val Ala Met Gly Asp Leu Leu Ile His Glu Gly Ala Pro 165 170
175Cys Ile Ala Gln Gln His Ala Ala Lys Val Phe Asn Ala Asp Lys Thr
180 185 190Tyr Phe Val Leu Asn Gly Thr Ser Ser Ser Asn Lys Val Val
Leu Asn 195 200 205Ala Leu Leu Thr Pro Gly Asp Leu Val Leu Phe Asp
Arg Asn Asn His 210 215 220Lys Ser Asn His His Gly Ala Leu Leu Gln
Ala Gly Ala Thr Pro Val225 230 235 240Tyr Leu Glu Thr Ala Arg Asn
Pro Tyr Gly Phe Ile Gly Gly Ile Asp 245 250 255Ala His Cys Phe Glu
Glu Ser Tyr Leu Arg Glu Leu Ile Ala Glu Val 260 265 270Ala Pro Gln
Arg Ala Lys Glu Ala Arg Pro Phe Arg Leu Ala Val Ile 275 280 285Gln
Leu Gly Thr Tyr Asp Gly Thr Ile Tyr Asn Ala Arg Gln Val Val 290 295
300Asp Lys Ile Gly His Leu Cys Asp Tyr Ile Leu Phe Asp Ser Ala
Trp305 310 315 320Val Gly Tyr Glu Gln Phe Ile Pro Met Met Ala Asp
Cys Ser Pro Leu 325 330 335Leu Leu Asp Leu Asn Glu Asn Asp Pro Gly
Ile Leu Val Thr Gln Ser 340 345 350Val His Lys Gln Gln Ala Gly Phe
Ser Gln Thr Ser Gln Ile His Lys 355 360 365Lys Asp Ser His Ile Lys
Gly Gln Gln Arg Tyr Val Pro His Lys Arg 370 375 380Met Asn Asn Ala
Phe Met Met His Ala Ser Thr Ser Pro Phe Tyr Pro385 390 395 400Leu
Phe Ala Ala Leu Asn Ile Asn Ala Lys Met His Glu Gly Val Ser 405 410
415Gly Arg Asn Met Trp Met Asp Cys Val Val Asn Gly Ile Asn Ala Arg
420 425 430Lys Leu Ile Leu Asp Asn Cys Gln His Ile Arg Pro Phe Val
Pro Glu 435 440 445Leu Val Asp Gly Lys Pro Trp Gln Ser Tyr Glu Thr
Ala Gln Ile Ala 450 455 460Val Asp Leu Arg Phe Phe Gln Phe Val Pro
Gly Glu His Trp His Ser465 470 475 480Phe Glu Gly Tyr Ala Glu Asn
Gln Tyr Phe Val Asp Pro Cys Lys Leu 485 490 495Leu Leu Thr Thr Pro
Gly Ile Asp Ala Arg Asn Gly Glu Tyr Glu Ala 500 505 510Phe Gly Val
Pro Ala Thr Ile Leu Ala Asn Phe Leu Arg Glu Asn Gly 515 520 525Val
Val Pro Glu Lys Cys Asp Leu Asn Ser Ile Leu Phe Leu Leu Thr 530 535
540Pro Ala Glu Asp Met Ala Lys Leu Gln Gln Leu Val Ala Leu Leu
Val545 550 555 560Arg Phe Glu Lys Leu Leu Glu Ser Asp Ala Pro Leu
Ala Glu Val Leu 565 570 575Pro Ser Ile Tyr Lys Gln His Glu Glu Arg
Tyr Ala Gly Tyr Thr Leu 580 585 590Arg Gln Leu Cys Gln Glu Met His
Asp Leu Tyr Ala Arg His Asn Val 595 600 605Lys Gln Leu Gln Lys Glu
Met Phe Arg Lys Glu His Phe Pro Arg Val 610 615 620Ser Met Asn Pro
Gln Glu Ala Asn Tyr Ala Tyr Leu Arg Gly Glu Val625 630 635 640Glu
Leu Val Arg Leu Pro Asp Ala Glu Gly Arg Ile Ala Ala Glu Gly 645 650
655Ala Leu Pro Tyr Pro Pro Gly Val Leu Cys Val Val Pro Gly Glu Ile
660 665 670Trp Gly Gly Ala Val Leu Arg Tyr Phe Ser Ala Leu Glu Glu
Gly Ile 675 680 685Asn Leu Leu Pro Gly Phe Ala Pro Glu Leu Gln Gly
Val Tyr Ile Glu 690 695 700Glu His Asp Gly Arg Lys Gln Val Trp Cys
Tyr Val Ile Lys Pro Arg705 710 715 720Asp Ala Gln Ser Thr Leu Leu
Lys Gly Glu Lys Leu 725 73026466PRTEscherichia coli 26Met Asp Gln
Lys Leu Leu Thr Asp Phe Arg Ser Glu Leu Leu Asp Ser1 5 10 15Arg Phe
Gly Ala Lys Ala Ile Ser Thr Ile Ala Glu Ser Lys Arg Phe 20 25 30Pro
Leu His Glu Met Arg Asp Asp Val Ala Phe Gln Ile Ile Asn Asp 35 40
45Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu Ala Thr Phe Cys
50 55 60Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met Asp Leu Ser
Ile65 70 75 80Asn Lys Asn Trp Ile Asp Lys Glu Glu Tyr Pro Gln Ser
Ala Ala Ile 85 90 95Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp
His Ala Pro Ala 100 105 110Pro Lys Asn Gly Gln Ala Val Gly Thr Asn
Thr Ile Gly Ser Ser Glu 115 120 125Ala Cys Met Leu Gly Gly Met Ala
Met Lys Trp Arg Trp Arg Lys Arg 130 135 140Met Glu Ala Ala Gly Lys
Pro Thr Asp Lys Pro Asn Leu Val Cys Gly145 150 155 160Pro Val Gln
Ile Cys Trp His Lys Phe Ala Arg Tyr Trp Asp Val Glu 165 170 175Leu
Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe Met Asp Pro Lys 180 185
190Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly Val Val Pro Thr
195 200 205Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro Gln Pro
Leu His 210 215 220Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile
Asp Ile Asp Met225 230 235 240His Ile Asp Ala Ala Ser Gly Gly Phe
Leu Ala Pro Phe Val Ala Pro 245 250 255Asp Ile Val Trp Asp Phe Arg
Leu Pro Arg Val Lys Ser Ile Ser Ala 260 265 270Ser Gly His Lys Phe
Gly Leu Ala Pro Leu Gly Cys Gly Trp Val Ile 275 280 285Trp Arg Asp
Glu Glu Ala Leu Pro Gln Glu Leu Val Phe Asn Val Asp 290 295 300Tyr
Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn Phe Ser Arg Pro305 310
315 320Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu Arg Leu Gly
Arg 325 330 335Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln Val
Ala Ala Tyr 340 345 350Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr
Glu Phe Ile Cys Thr 355 360 365Gly Arg Pro Asp Glu Gly Ile Pro Ala
Val Cys Phe Lys Leu Lys Asp 370 375 380Gly Glu Asp Pro Gly Tyr Thr
Leu Tyr Asp Leu Ser Glu Arg Leu Arg385 390 395 400Leu Arg Gly Trp
Gln Val Pro Ala Phe Thr Leu Gly Gly Glu Ala Thr 405 410 415Asp Ile
Val Val Met Arg Ile Met Cys Arg Arg Gly Phe Glu Met Asp 420 425
430Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser Leu Lys Tyr Leu
435 440 445Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln Asn Ser
Phe Lys 450 455 460His Thr46527466PRTEscherichia coli 27Met Asp Lys
Lys Gln Val Thr Asp Leu Arg Ser Glu Leu Leu Asp Ser1 5 10 15Arg Phe
Gly Ala Lys Ser Ile Ser Thr Ile Ala Glu Ser Lys Arg Phe 20 25 30Pro
Leu His Glu Met Arg Asp Asp Val Ala Phe Gln Ile Ile Asn Asp 35 40
45Glu Leu Tyr Leu Asp Gly Asn Ala Arg Gln Asn Leu Ala Thr Phe Cys
50 55 60Gln Thr Trp Asp Asp Glu Asn Val His Lys Leu Met Asp Leu Ser
Ile65 70 75 80Asn Lys Asn Trp Ile Asp Lys Glu Glu Tyr Pro Gln Ser
Ala Ala Ile 85 90 95Asp Leu Arg Cys Val Asn Met Val Ala Asp Leu Trp
His Ala Pro Ala 100 105 110Pro Lys Asn Gly Gln Ala Val Gly Thr Asn
Thr Ile Gly Ser Ser Glu 115 120 125Ala Cys Met Leu Gly Gly Met Ala
Met Lys Trp Arg Trp Arg Lys Arg 130 135 140Met Glu Ala Ala Gly Lys
Pro Thr Asp Lys Pro Asn Leu Val Cys Gly145 150 155 160Pro Val Gln
Ile Cys Trp His Lys Phe Ala Arg Tyr Trp Asp Val Glu 165 170 175Leu
Arg Glu Ile Pro Met Arg Pro Gly Gln Leu Phe Met Asp Pro Lys 180 185
190Arg Met Ile Glu Ala Cys Asp Glu Asn Thr Ile Gly Val Val Pro Thr
195 200 205Phe Gly Val Thr Tyr Thr Gly Asn Tyr Glu Phe Pro Gln Pro
Leu His 210 215 220Asp Ala Leu Asp Lys Phe Gln Ala Asp Thr Gly Ile
Asp Ile Asp Met225 230 235 240His Ile Asp Ala Ala Ser Gly Gly Phe
Leu Ala Pro Phe Val Ala Pro 245 250 255Asp Ile Val Trp Asp Phe Arg
Leu Pro Arg Val Lys Ser Ile Ser Ala 260 265 270Ser Gly His Lys Phe
Gly Leu Ala Pro Leu Gly Cys Gly Trp Val Ile 275 280 285Trp Arg Asp
Glu Glu Ala Leu Pro Gln Glu Leu Val Phe Asn Val Asp 290 295 300Tyr
Leu Gly Gly Gln Ile Gly Thr Phe Ala Ile Asn Phe Ser Arg Pro305 310
315 320Ala Gly Gln Val Ile Ala Gln Tyr Tyr Glu Phe Leu Arg Leu Gly
Arg 325 330 335Glu Gly Tyr Thr Lys Val Gln Asn Ala Ser Tyr Gln Val
Ala Ala Tyr 340 345 350Leu Ala Asp Glu Ile Ala Lys Leu Gly Pro Tyr
Glu Phe Ile Cys Thr 355 360 365Gly Arg Pro Asp Glu Gly Ile Pro Ala
Val Cys Phe Lys Leu Lys Asp 370 375 380Gly Glu Asp Pro Gly Tyr Thr
Leu Tyr Asp Leu Ser Glu Arg Leu Arg385 390 395 400Leu Arg Gly Trp
Gln Val Pro Ala Phe Thr Leu Gly Gly Glu Ala Thr 405 410 415Asp Ile
Val Val Met Arg Ile Met Cys Arg Arg Gly Phe Glu Met Asp 420 425
430Phe Ala Glu Leu Leu Leu Glu Asp Tyr Lys Ala Ser Leu Lys Tyr Leu
435
440 445Ser Asp His Pro Lys Leu Gln Gly Ile Ala Gln Gln Asn Ser Phe
Lys 450 455 460His Thr4652819PRTSaccharomyces cerevisiae 28Gln Gln
Gln Arg Asn Trp Lys Gln Gly Gly Asn Tyr Gln Gln Tyr Gln1 5 10 15Ser
Tyr Asn2930PRTSaccharomyces cerevisiae 29Ser Asn Tyr Asn Asn Tyr
Asn Asn Tyr Asn Asn Tyr Asn Asn Tyr Asn1 5 10 15Asn Tyr Asn Asn Tyr
Asn Lys Tyr Asn Gly Gln Gly Tyr Gln 20 25 30308PRTSaccharomyces
cerevisiae 30Pro Gln Gly Gly Tyr Gln Gln Asn1 5319PRTSaccharomyces
cerevisiae 31Phe Pro Pro Lys Lys Phe Lys Asp Leu1
53218PRTSaccharomyces cerevisiae 32Phe Pro Pro Lys Lys Phe Lys Asp
Leu Asn Ser Phe Leu Asp Asp Gln1 5 10 15Pro Lys3336PRTSaccharomyces
cerevisiae 33Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser Phe Leu
Asp Asp Gln1 5 10 15Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe Gly
Gly Tyr Phe Lys 20 25 30Asn Pro Ala Ala 353445PRTSaccharomyces
cerevisiae 34Phe Pro Pro Lys Lys Phe Lys Asp Leu Asn Ser Phe Leu
Asp Asp Gln1 5 10 15Pro Lys Asp Pro Asn Leu Val Ala Ser Pro Phe Gly
Gly Tyr Phe Lys 20 25 30Asn Pro Ala Ala Asp Ala Gly Ser Asn Asn Ala
Ser Lys 35 40 453510PRTBovine adenovirus type 1 35Arg Arg Phe Gly
Glu Ala Ser Ser Ala Phe1 5 103610PRTEscherichia coli 36Ala Ser Gln
Trp Pro Glu Glu Thr Phe Gly1 5 103710PRTEscherichia coli 37Glu Gly
Val Ala Glu Thr Asn Glu Asp Phe1 5 103852DNAArtificial
SequencePrimer cadA-F 38ggcgagctca cacaggaaac agaccatgaa cgttattgca
atattgaatc ac 523928DNAArtificial SequencePrimer cadA-R
39ggctctagac cacttccctt gtacgagc 284044DNAArtificial SequencePrimer
cadA-F2 40atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat
444120DNAArtificial SequencePrimer cadA-R2 41agctgtttcc tgtgtgaaat
204233DNAArtificial SequencePrimer cat-HindIII-F 42ggcaagcttg
agaaaaaaat cactggatat acc 334329DNAArtificial SequencePrimer
cat-NdeI-R 43ggccatatgt aagggcacca ataactgcc 294431DNAArtificial
SequencePrimer cadAt-XbaI-R 44ggctctagat ttgctttctt ctttcaatac c
314533DNAArtificial SequencePrimer cat-XbaI-F 45ggctctagag
agaaaaaaat cactggatat acc 334630DNAArtificial SequencePrimer
New1-XbaI-F 46ggctctagag gttctggctc tggttctccg 304731DNAArtificial
SequencePrimer New1-HindIII-R 47ggcaagcttt tactggtagc cctgaccgtt g
314829DNAArtificial SequencePrimer Sup35-XbaI-F 48ggctctagag
gtagcggctc tggctctga 294932DNAArtificial SequencePrimer
Sup35-HindIII-R 49ggcaagcttt tagccaccct gtgggttaaa ct
325029DNAArtificial SequencePrimer cadA-XbaI-F 50ggctctagaa
tttcacacag gaaacagct 295129DNAArtificial SequencePrimer
cadA-HindIII-R 51ggcaagcttc acttcccttg tacgagcta
295234DNAArtificial SequencePrimer rbs2-SacI-F 52ggcgagctca
tgaacgttat tgcaatattg aatc 345325DNAArtificial SequencePrimer
rbs2-SacI-R 53ggcgagctcc tcctgtgtga aattg 255429DNAArtificial
SequencePrimer New1-SacI-F 54ggcgagctca tgggttctgg ctctggttc
295528DNAArtificial SequencePrimer New1-XbaI-R 55ggctctagac
tggtagccct gaccgttg 285627DNAArtificial SequencePrimer Sup35-SacI-F
56ggcgagctca tgggtagcgg ctctggc 275729DNAArtificial SequencePrimer
Sup35-XbaI-R 57ggctctagag ccaccctgtg ggttaaact 295828DNAArtificial
SequencePrimer 222-de-R 58tctagatttg ctttcttctt tcaatacc
285946DNAArtificial SequencePrimer 222-de-9-F 59gaagaaagca
aatctagaaa gttcaaagac ctgaactctt tcggtg 466042DNAArtificial
SequencePrimer 222-de-18-F 60gaagaaagca aatctagaga cgaccagccg
aaagacccga ac 426141DNAArtificial SequencePrimer 222-de-36-F
61gaagaaagca aatctagaaa aaacccagcg gcggacgcgg g 416241DNAArtificial
SequencePrimer 222-de-45-F 62gaagaaagca aatctagaaa caacgcgtct
aagaaatctt c 416347DNAArtificial SequencePrimer 222-C2-F
63tttcggcgaa gcgagcagcg cgttctaaaa gcttaagaga caggatg
476447DNAArtificial SequencePrimer 222-C2-R 64cgctgctcgc ttcgccgaaa
cgacgctggt agccctgacc gttgtat 476547DNAArtificial SequencePrimer
222-C4-F 65ccagtggccg gaagaaacct tcggctaaaa gcttaagaga caggatg
476647DNAArtificial SequencePrimer 222-C4-R 66aggtttcttc cggccactgg
ctcgcctggt agccctgacc gttgtat 476747DNAArtificial SequencePrimer
222-C6-F 67cgtggcggaa accaacgaag atttctaaaa gcttaagaga caggatg
476847DNAArtificial SequencePrimer 222-C6-R 68cttcgttggt ttccgccacg
ccttcctggt agccctgacc gttgtat 47
* * * * *