U.S. patent application number 17/509544 was filed with the patent office on 2022-07-14 for constitutive yeast llp promotor-based expression systems.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Novartis AG. Invention is credited to Clemens Achmuller, Franz Hartner, Thomas Specht, Ferdinand Zepeck.
Application Number | 20220220161 17/509544 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220220161 |
Kind Code |
A1 |
Achmuller; Clemens ; et
al. |
July 14, 2022 |
Constitutive Yeast LLP Promotor-Based Expression Systems
Abstract
The present invention belongs to the field of biotechnology,
recombinant protein production, molecular biology, microbiology and
microbial genetics. It provides a modified eukaryotic cell that is
modified to the effect that the modified eukaryotic cell is not
able to provide an SSN6-like related protein or an SSN6-like
protein that exerts its wildtype function and/or wildtype activity,
the amount of SSN6-like related protein or of SSN6-like protein
being present in the modified eukaryotic cell differs from the
amount of SSN6-like related protein or of SSN6-like protein being
present in its wildtype form, and/or essentially no SSN6-like
related protein or SSN6-like protein is present in the modified
cell. Additionally, the present invention provides a polynucleotide
sequence comprising a modified ssn6-like related gene or modified
ssn6-like gene, and a vector comprising said polynucleoptide.
Additionally provided is an expression vector comprising a promoter
that is repressed in the presence of SSN6-like protein or SSN6-like
related protein, and a host cell comprising said vectors. The
present invention further refers to a method for determining the
purity of a composition by using the modified eukaryotic cell, to a
method of expressing gene(s) of interest, and eukaryotic cells
comprising modified ssn6-like related gene or ssn6-like gene.
Inventors: |
Achmuller; Clemens; (Kundl,
AT) ; Zepeck; Ferdinand; (Kundl, AT) ;
Hartner; Franz; (Kundl, AT) ; Specht; Thomas;
(Kundl, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Appl. No.: |
17/509544 |
Filed: |
October 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15554552 |
Aug 30, 2017 |
11203620 |
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PCT/EP2016/054478 |
Mar 2, 2016 |
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17509544 |
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International
Class: |
C07K 14/39 20060101
C07K014/39; C12N 15/81 20060101 C12N015/81; C07K 14/395 20060101
C07K014/395 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 3, 2015 |
EP |
15157284.9 |
Claims
1-27. (canceled)
28. A polynucleotide sequence comprising a modified ssn6-like gene,
wherein either (A) or (B) applies: (A): the ssn6-like gene is
defined as comprising SEQ ID NO: 1, the nucleotide sequence of the
ssn6-like gene is modified by introduction of a point mutation, or
a partial deletion, wherein, if said polynucleotide sequence is (a)
introduced into a suitable expression system, and tried to be
expressed, essentially no SSN6-like protein is expressed, or (b)
expressed in a suitable expression system, SSN6-like protein is
expressed that does not exert its wildtype function and/or wildtype
activity, and/or (c) expressed in a suitable expression system, an
amount of SSN6-like protein is expressed that differs from the
amount of wildtype SSN6-like protein; (B): the polynucleotide
sequence comprises SEQ ID NO: 7.
29. The polynucleotide sequence according to claim 28, wherein the
polynucleotide comprises modifications of SEQ ID NO: 1.
30. A The polynucleotide according to claim 28, wherein the
polynucleotide is a vector.
31. An expression vector comprising a promoter, wherein said
promoter is characterized in that it is repressed in the presence
of SSN6-like protein if said expression vector is introduced into a
suitable expression system, wherein the ssn6-like protein is
defined as comprising the amino acid sequence as depicted in SEQ ID
NO: 8, and wherein said promoter is an LLP promoter comprising SEQ
ID NO: 133 or modified versions thereof, said modified versions
being characterized in that they still exhibit the promoter
function, wherein an LLP protein, which is encoded by the
nucleotide sequence depicted in SEQ ID NO: 16, is not encoded by
the polynucleotide sequence of this expression vector.
32-33. (canceled)
34. The expression vector of claim 31, which further comprises one
or more gene(s) of interest, wherein this one or more gene(s) of
interest is under control of said LLP promoter.
35. (canceled)
36. A host cell, comprising the vector according to claim 30.
37-45. (canceled)
46. The polynucleotide sequence according to claim 28, wherein, if
said polynucleotide sequence is expressed in a suitable expression
system, or is introduced in a suitable expression system and tried
to be expressed, the amount of SSN6-like protein is reduced, or
there is no SSN6-like protein at all, compared to the amount of
SSN6-like protein that is expressed when the corresponding wildtype
polynucleotide sequence is expressed under comparable or the same
conditions.
47. The polynucleotide sequence according to claim 28, comprising
SEQ ID NO: 7.
48. The expression vector of claim 34, wherein the one or more
gene(s) of interest is (are) selected from the group consisting of
genes encoding enzymes, antibodies or fragments thereof, hormones,
structural proteins, and protein-antigens being present in
vaccines.
49. A host cell, comprising an expression vector according to claim
31.
Description
FIELD OF THE INVENTION
[0001] The present invention belongs to the field of biotechnology,
recombinant protein production, molecular biology, microbiology and
microbial genetics. Specifically, the present invention relates to
a modified eukaryotic cell, or expression system, respectively,
comprising a modified gene, a polynucleotide sequence comprising
said modified gene, an expression vector comprising said
polynucleotide sequence, a host cell comprising said expression
vector, as well as to the use of said components.
[0002] Further, the present invention refers to methods using said
components.
DESCRIPTION OF THE BACKGROUND ART
[0003] Expression systems for the expression of genes of interest
(GOI) and also for the production of proteins of interest are
widely known and of great importance in the field of molecular
biology, microbiology and microbial genetics.
[0004] Until now, there are several expression systems known that
allow the expression of certain desired genes and proteins, such as
cell-based systems and cell-free systems. Cell-based systems
comprise bacterial systems and eukaryotic systems, and cell-free
systems are based for instance on the use of purified RNA
polymerase, ribosomes, tRNA and ribonucleotides or are based on the
use of systems containing cell-free protein synthesis systems such
as for example rabbit reticulocyte lysates. The choice of the
respectively used expression system depends on various factors such
as the type of protein (eukaryotic or prokaryotic source of the
protein) that is to be expressed, or whether post-translational
modifications are necessary in order to ensure proper function
and/or structure.
[0005] For instance, in general, if eukaryotic proteins are to be
expressed, typically a eukaryotic expression system is selected.
The reason therefore is that most eukaryotic proteins are modified
for instance by phospohorylation, acylation, methylation,
ubiquitylation or glycosylation. Bacteria are known to be no ideal
host organisms for such modified proteins, as bacteria are
prokaryotes which are not equipped with the full enzymatic
machinery to accomplish the post-translational modifications or
molecular folding that are required for many proteins to function
properly. Thus, if eukaryotic proteins are to be expressed,
typically expression systems using eukaryotic cells are used. Such
expression systems are for instance based on yeast, fungi or insect
cells. Expression systems in yeast use either inducible or
non-inducible promoters for expression. Inducible promoters for
methylotrophic yeast such as Pichia pastoris (P. Pastoris) often
require the use of methanol as inducing substance.
[0006] Despite the known expression systems, there is still a need
for an improved expression system, in particular for a eukaryotic
expression system. Improvements are for instance desired with
regard to the handling of the expression system, costs, safety
aspects (e.g. avoiding the use of highly inflammable inducers such
as methanol), complexity of the system, the time needed for
expressing the proteins, or the yield of the expressed
proteins.
SUMMARY OF THE INVENTION
[0007] The present invention provides the following aspects,
subject-matters and preferred embodiments, which respectively taken
alone or in combination, contribute to solving the object of the
present invention:
[0008] (1) A modified eukaryotic cell, which is modified compared
to its wildtype cell at least in that it comprises [0009] a
modified ssn6-like gene or a modified ssn6-like related gene,
and/or [0010] a modified expression level of SSN6-like protein or
of SSN6-like related protein, or [0011] in that [0012] an ssn6-like
gene or an ssn6-like related gene is deleted,
[0013] respectively to the effect that [0014] the modified
eukaryotic cell is not able to provide an SSN6-like protein or an
SSN6-like related protein that exerts its wildtype function and/or
wildtype activity, [0015] the amount of SSN6-like protein or of
SSN6-like related protein being present in the modified eukaryotic
cell differs from the amount of SSN6-like protein or of SSN6-like
related protein being present in its wildtype form, and/or [0016]
no SSN6-like protein or SSN6-like related protein is present in the
modified cell,
[0017] wherein said modified eukaryotic cell, compared to its
wildtype cell, exhibits different SSN6-like protein or SSN6-like
related-protein activity and/or function, preferably with respect
to the proteins' ability in regulating gene expression.
[0018] In a preferred embodiment, said modified eukaryotic cell
exhibits different SSN6-like protein or SSN6-like related protein
activity and/or function with respect to the proteins' activity
and/or function in regulating the expression of genes that are
under control of the lectin-like protein (LLP) promoter.
[0019] (2) The modified eukaryotic cell according to item (1),
wherein said modified eukaryotic cell exhibits reduced SSN6-like
protein or SSN6-like related-protein activity and/or reduced
function, or no SSN6-like protein or SSN6-like related-protein
activity and/or function at all, preferably with regard to the
proteins' activity and/or function in regulating gene expression,
preferably in regulating the expression of genes that are under
control of the LLP promoter.
[0020] (3) The modified eukaryotic cell according to item (1) or
(2), wherein the ssn6-like gene and/or the ssn6-like related gene
is/are modified in a regulatory sequence, such as promoter(s),
enhancer(s), terminator(s), silencer(s), IRES-sequence(s),
ribosome-binding site(s), and sequence(s) stabilizing or
destabilizing the mRNA by secondary structure(s), and/or in a
coding sequence.
[0021] (4) The modified eukryotic cell according to any of items
(1) to (3), wherein the modified expression level of SSN6-like
protein or of SSN6-like related protein is obtained by modifying
the coding sequence of the ssn6-like gene and/or of the ssn6-like
related gene.
[0022] (5) The modified eukaryotic cell according to any of items
(1) to (4), wherein the total activity, function, preferably gene
regulatory function, and/or amount of SSN6-like protein or
SSN6-like related protein of the modified eukaryotic cell is at
most 20%, preferably at most 15%, more preferably at most 10%, even
more preferably at most 5% of the total activity, function and/or
amount of SSN6-like protein or SSN6-like related protein of its
wildtype form, and most preferably there is no activity and/or no
function of the SSN6-like protein or SSN6-like related protein of
the modified eukaryotic cell at all.
[0023] (6) The modified eukaryotic cell according to any of items
(1) to (5), wherein the ssn6-like gene or the ssn6-like related
gene and/or the expression level of the SSN6-like protein or of the
SSN6-like related protein is modified by [0024] introduction of one
or more point mutations, e.g. substitution, insertion or deletion
of a single or more nucleotides in the polynucleotide sequence of
the ssn6-like gene or of the ssn6-like related gene, [0025] partial
or complete deletion of the polynucleotide sequence of the
ssn6-like gene or of the ssn6-like related gene, and/or [0026]
partial or complete replacement of the polynucleotide sequence of
the ssn6-like gene or of the ssn6-like related gene by a different,
e.g. heterologous, nucleotide sequence,
[0027] wherein the polynucleotide sequence of the ssn6-like gene or
of the ssn6-like related gene comprises coding and regulatory
polynucleotide sequences, and wherein said regulatory
polynucleotide sequences of the ssn6-like gene or of the ssn6-like
related gene comprise promoters, enhancers, terminators, silencers,
IRES-sequences, ribosome-binding sites, or sequences stabilizing or
destabilizing the mRNA by secondary structures.
[0028] (7) The modified eukaryotic cell according to any of the
preceding items, wherein said cell is a fungal cell, preferably a
yeast cell, more preferably selected from the group consisting of
Saccharomyces species (e.g., Saccharomyces cerevisiae),
Kluyveromyces species (e.g., Kluyveromyces lactis), Torulaspora
species, Yarrowia species (e.g., Yarrowia lipolitica),
Schizosaccharomyces species (e.g., Schizosaccharomyces pombe),
Pichia species (e.g., Pichia pastoris or Pichia methanolica),
Hansenula species (e.g., Hansenula polymorpha), Torulopsis species,
Komagataella species, Candida species (e.g., Candida boidinii), and
Karwinskia species, even more preferably the eukaryotic cell is a
Pichia cell, most preferably a Pichia pastoris cell according to
current classification.
[0029] (8) The modified eukaryotic cell according to item (7),
wherein the cell can be any Pichia pastoris cell, preferably a cell
of a strain selected from the group consisting of NRRL Y-11430,
CBS704, GS115, and KM71.
[0030] (9) The modified eukaryotic cell according to any of the
preceding items, wherein said modified eukaryotic cell comprises a
polynucleotide sequence which represents a modification of SEQ ID
NO: 1, preferably the modified eukaryotic cell comprises SEQ ID NO:
7.
[0031] SEQ ID NO: 1 represents the coding region of the ssn6-like
gene.
[0032] SEQ ID NO: 7 represents modified ssn6-like nucleotide
sequence.
[0033] (10) The modified eukaryotic cell according to any of the
proceeding items, wherein said wildtype cell contains a SSN6-like
or SSN6-like related protein, which protein comprises one or both
of the consensus amino acid sequences depicted in SEQ ID NO: 63 and
64.
[0034] In other words, the eukaryotic cell, prior to its
modification resulting in said modified eukaryotic cell, contained
an SSN6-like or SSN6-like related protein, which protein comprised
one or both of the amino acid sequences depicted in SEQ ID NO: 63
and 64.
[0035] (11) A polynucleotide sequence comprising, preferably
consisting of, a modified ssn6-like gene or nucleotide sequence,
e.g. as depicted in SEQ ID NO: 7, or a modified ssn6-like related
gene,
[0036] wherein, if said polynucleotide sequence
[0037] (a) is introduced into a suitable expression system, and
tried to be expressed, essentially no SSN6-like protein or
SSN6-like related protein is expressed, or
[0038] (b) is expressed in a suitable expression system, SSN6-like
protein or SSN6-like related protein is expressed that does not
exert its wildtype function and/or wildtype activity, preferably
with respect to its activity and/or function in regulating gene
expression, more preferably in regulating the expression of genes
that are under control of the LLP promoter, and/or
[0039] (c) is expressed in a suitable expression system, an amount
of SSN6-like protein or SSN6-like related protein is expressed that
differs from the amount of wildtype SSN6-like protein or of
SSN6-like related protein.
[0040] In order to assess any of (a) to (c), the corresponding
wildtype polynucleotide sequence is tried to be expressed under
comparable or the same conditions, under which the modified
ssn6-like gene or modified ssn6-like related gene is tried to be
expressed.
[0041] (12) The polynucleotide sequence according to item (11),
wherein, if said polynucleotide sequence is introduced into a
suitable expression system, and tried to be expressed, or is
expressed in a suitable expression system, the amount of SSN6-like
protein or SSN6-like related protein is reduced, or there is
essentially no, preferably no, SSN6-like protein or SSN6-like
related protein at all present, compared to the amount of SSN6-like
protein or SSN6-like related protein that is expressed when the
corresponding wildtype polynucleotide sequence is expressed under
comparable or the same conditions.
[0042] (13) The polynucleotide sequence according to item (11) or
(12), wherein a regulatory sequence of the ssn6-like gene or of the
ssn6-like related gene is selected from the group comprising
promoters, enhancers, terminators, silencers, IRES-sequences,
ribosome-binding sites, and sequences stabilizing or destabilizing
the mRNA by secondary structures, preferably said regulatory
sequence is selected from the group consisting of promoters,
enhancers, and terminators.
[0043] (14) The polynucleotide sequence according to any of items
(11) to (13), wherein the ssn6-like gene or the ssn6-like related
gene has been modified as defined in item (6).
[0044] (15) The polynucleotide sequence according to any of items
(11) to (14), wherein the polynucleotide sequence comprises,
preferably consists of, modifications of SEQ ID NO: 1, preferably
the polynucleotide sequence comprises, more preferably consists of,
SEQ ID NO: 7.
[0045] (16) A nucleic acid sequence comprising the polynucleotide
sequence according to any of items (11) to (15).
[0046] (17) A vector comprising the polynucleotide sequence/nucleic
acid sequence according to any of items (11) to (16).
[0047] (18) A host cell comprising the vector according to item
(17), or a polynucleotide according to any of items (11) to
(13).
[0048] (19) The host cell according to item (18), wherein said host
cell is a bacterium, preferably Escherichia coli.
[0049] (20) A polypeptide encoded by the polynucleotide sequence
according to any of items (11) to (15).
[0050] (21) An expression vector comprising a promoter, wherein
said promoter is characterized in that it is repressed in the
presence of SSN6-like related protein or SSN6-like protein if said
expression vector is introduced into a suitable expression system,
e.g. into a eukaryotic cell as defined in item (7) or (8).
[0051] (22) The expression vector according to item (21), which
further comprises one or more gene(s) of interest.
[0052] (23) The expression vector according to item (22), wherein
the gene(s) of interest is/are under control of the promoter
defined in item (21).
[0053] (24) The expression vector according to any of items (21) to
(23), wherein said promoter is an LLP promoter, preferably an LLP
promoter comprising, preferably consisting of, SEQ ID NO: 2 or 12,
or consisting of a sequence having a length of 1000, 775, 675, 605,
576, 512, 472, 415, 404, 372, 305, 285, 235, 165, or 100
nucleotides counted in each case from the 3'-end of SEQ ID NO: 12,
or modified versions thereof, said modified versions being
characterized in that they still exhibit the promoter function.
[0054] (25) The expression vector according to any of items (21) to
(24), wherein, if the promoter is an LLP promoter as defined in
item (24), LLP protein is not encoded by the polynucleotide
sequence of this expression vector, and preferably the gene of
interest is selected from the group consisting of genes encoding
enzymes, antibodies or fragments thereof, hormones, structural
proteins (such as albumin), and protein-antigens being suitable for
vaccines.
[0055] (26) The expression vector according to any of items (21) to
(24), wherein, if the promoter is an LLP promoter as defined in
item (24), LLP protein is encoded by the polynucleotide sequence of
this expression vector, and preferably the gene of interest is
selected from the group consisting of genes encoding enzymes,
antibodies or fragments thereof, hormones, structural proteins
(such as albumin), and protein-antigens being suitable for
vaccines.
[0056] (27) The expression vector according to any of items (21) to
(26), wherein said expression vector further comprises features
selected from any one of the following: [0057] (i) a selection
marker; [0058] (ii) a purification marker; [0059] (iii) a signal
sequence, preferably an alpha-factor secreting signal sequence,
more preferably the MFalpha pre-pro signal sequence, even more
preferably a signal sequence comprising or consisting of,
preferably consisting of, SEQ ID NO: 14 or SEQ ID NO: 21; [0060]
(iv) an origin of replication; and/or [0061] (v) a nucleotide
sequence for targeted and/or random integration into the genome of
a host cell.
[0062] In a preferred embodiment, the signal sequence (iii) of the
expression vector according to item (27) comprises or consists,
preferably consists of, SEQ ID NO: 14.
[0063] (28) The expression vector according to any of items (21) to
(27), wherein said vector contains more than one promoter as
defined in item (21), and/or wherein said vector contains more than
one LLP promotor according to item (24), preferably 2, 3, 4, 5, or
6 LLP promoters according to item (24) and preferably different LLP
promoters, e.g. different length LLP-promoters, according to item
(24) which result in different expression rates of the genes of
interest under control of said LLP promoter.
[0064] (29) The expression vector according to any of items (21) to
(27), wherein said vector contains, besides one or more LLP
promoters, in addition one or more other promoters different from a
LLP promoter, which other promoters result in expression rates
different to the expression rates of the LLP promoters.
[0065] (30) Use of an expression vector according to item (28) or
(29) for the expression of a multimeric protein, said multimeric
protein consisting of two or more individual protein chains, which
individual protein chains are connected to each other by one or
more disulfide bridges and/or which individual protein chains form
a multimeric protein by other forms of protein chain-protein chain
interactions.
[0066] (31) Use according to item (30) wherein said multimeric
protein is expressed by transforming a cell with two or more
individual vectors, wherein each vector is containing one or more
promoter(s) as defined in item (21) and/or in item (24), or wherein
at least one vector contains a LLP promoter and at least one vector
contains other promoter different from a LLP promoter.
[0067] (32) A host cell, comprising the vector according to item
(17) or any of items (21) to (29).
[0068] (33) The host cell according to item (32), wherein said host
cell is a bacterium, preferably Escherichia coli, or the modified
eukaryotic cell according to any of items (1) to (9).
[0069] (34) The modified eukaryotic cell according to any of items
(1) to (10), comprising the expression vector according to any of
items (21) to (29).
[0070] (35) An expression system, comprising [0071] a) the modified
eukaryotic cell as defined in any of items (1) to (10); [0072] b)
the expression vector as defined in any of items (21) to (29),
wherein said expression vector can also be present in linearized
form and/or at least parts of the vector being integrated into the
genome of the modified eukaryotic cell.
[0073] (36) The modified eukaryotic cell according to any of items
(1) to (10), further comprising a promoter as defined in item (21)
or (24).
[0074] (37) The modified eukaryotic cell according to item (36),
further comprising (a) gene(s) of interest being under control of
the promoter as defined in item (21) or (24).
[0075] (38) The modified eukaryotic cell according to item (37),
wherein the gene(s) of interest is (are) as defined in item
(25).
[0076] (39) The modified eukaryotic cell according to item (37) or
(38), wherein the LLP gene is not expressed in addition to the
gene(s) of interest, or wherein the LLP gene is expressed in
addition to the gene(s) of interest.
[0077] (40) The modified eukaryotic cell according to item (39),
[0078] wherein the LLP promoter or the LLP-like promoter controls
(a) gene(s) of interest which is (are) different from LLP, and in
addition another copy of said promoter controls the LLP-gene (e.g.
Option 2 in FIG. 3D); or [0079] wherein the LLP promoter or the
LLP-like promoter only controls (a) gene(s) of interest which is
(are) different from LLP and no LLP-gene is present (e.g. Option 1
in FIG. 3C).
[0080] (41) Use of [0081] (A) the modified eukaryotic cell, [0082]
(B) the polynucleotide sequence, [0083] (C) the expression vector,
[0084] (D) the host cell, or [0085] (E) an expression system
[0086] according to any of items (1) to (40)
[0087] in a method of expressing, preferably overexpressing,
gene(s) of interest.
[0088] (42) Method for determining the purity of a composition
comprising the expression product of a gene of interest, comprising
the following steps: [0089] (a) expressing gene(s) of interest by
using a modified eukaryotic cell according to any of items (1) to
(10) and by using an expression vector according to item (25),
wherein [0090] (a1) the modified eukaryotic cell comprises a gene
encoding LLP protein under control of an LLP promoter or an
LLP-like promoter, and wherein [0091] (a2) the expression vector
comprises one or more gene(s) of interest under control of an LLP
promoter or an LLP-like promoter, wherein said gene(s) of interest
does (do) not encode LLP, [0092] thereby obtaining a composition
comprising the expression product of the gene(s) of interest, i.e.
the protein(s) of interest, and the LLP protein; [0093] (b)
determining the amount of the expression product of the gene(s) of
interest, i.e. the amount of the protein(s) of interest, and the
amount of LLP protein being present in the composition obtained in
step (a), wherein the amount of LLP protein compared to the amount
of expression product of the gene(s) of interest, i.e. of the
protein(s) of interest, is indicative for the purity of the
composition obtained in step (a); and, optionally, [0094] (c)
subjecting the composition of step (a) to one or more downstream
purification step(s), followed by step (b) for determining the
amount of the the protein(s) of interest, and the amount of LLP
protein being present in the composition obtained after having
carried out said downstream purification step, i.e. monitoring
host-cell-protein depletion (purity of gene of interest protein in
the course of its purification).
[0095] (43) A method of expressing one or more gene(s) of interest
in a eukaryotic cell comprising an ssn6-like gene or an ssn6-like
related gene, wherein the translation of the mRNA transcript of the
ssn6-like gene or the ssn6-like related gene is prevented by
hybridizing a complementary sequence or a partial sequence thereof
to the mRNA transcript.
[0096] (44) The method of item (43), wherein the partial sequence
of the complementary sequence is a siRNA, anti-sense RNA, a
ribozyme, or triplex RNA or DNA.
[0097] (45) A method of expressing one or more gene(s) of interest
in a eukaryotic cell comprising an SSN6-like related expression
cassette or SSN6-like expression cassette, wherein the SSN6-like
related protein or the SSN6-like protein in said eukaryotic cell is
modified or inhibited in its function and/or activity.
[0098] (46) The method according to item (45), wherein the
SSN6-like related protein or SSN6-like protein of said eukaryotic
cell is modified or inhibited in its function and/or activity in
regulating the LLP promoter, preferably the SSN6-like related
protein or SSN6-like protein exhibits reduced SSN6-like protein or
SSN6-like related-protein activity and/or function, or no SSN6-like
protein or SSN6-like related-protein activity and/or function at
all.
[0099] (47) A eukaryotic cell comprising [0100] a modified
ssn6-like gene or a modified ssn6-like related gene, wherein said
gene has inserted a foreign nucleotide sequence, to the effect that
[0101] (i) the eukaryotic cell is not able to provide an SSN6-like
protein or an SSN6-like related protein that exerts its wildtype
function and/or wildtype activity, preferably with respect to its
activity and/or function in regulating the LLP promoter or LLP-like
promoter, [0102] (ii) the amount of SSN6-like protein or of
SSN6-like related protein being present in the eukaryotic cell
differs from the amount of SSN6-like protein or of SSN6-like
related protein being present in its wildtype form, preferably the
amount of the SSN6-like protein or of the SSN6-like related protein
is reduced, and/or [0103] (iii) no SSN6-like protein or SSN6-like
related protein is present in the eukaryotic cell,
[0104] and [0105] a gene of interest that replaces a part or all of
the coding region of the gene encoding the LLP protein, and that is
under control of the LLP promoter, to the effect that essentially
no LLP protein is present in the eukaryotic cell,
[0106] preferably wherein said eukaryotic cell exhibits reduced
SSN6-like protein or SSN6-like related-protein activity and/or
function, or no SSN6-like protein or SSN6-like related-protein
activity and/or function at all.
[0107] (48) A eukaryotic cell comprising [0108] a modified
ssn6-like gene or a modified ssn6-like related gene, wherein said
gene has inserted a foreign nucleotide sequence, to the effect that
[0109] (i) the eukaryotic cell is not able to provide an SSN6-like
protein or an SSN6-like related protein that exerts its wildtype
function and/or wildtype activity, preferably with respect to its
activity and/or function in regulating the LLP promoter, [0110]
(ii) the amount of SSN6-like protein or of SSN6-like related
protein being present in the eukaryotic cell differs from the
amount of SSN6-like protein or of SSN6-like related protein being
present in its wildtype form, preferably the amount of the
SSN6-like protein or of the SSN6-like related protein is reduced,
and/or [0111] (iii) no SSN6-like protein or SSN6-like related
protein is present in the eukaryotic cell, [0112] a gene of
interest under control of the LLP promoter, and [0113] an llp
gene,
[0114] preferably wherein said eukaryotic cell exhibits reduced
SSN6-like protein or SSN6-like related-protein activity and/or
function, or no SSN6-like protein or SSN6-like related-protein
activity and/or function at all.
[0115] (49) Nucleotide sequence comprising at it's 5'-end a
nucleotide sequence which codes for the peptide sequence of the
LLP-signal sequence and which is depicted in SEQ ID NO: 3, and
further comprising a nucleotide sequence which is coding for a
protein different to the native LLP-protein sequence.
[0116] (50) Use of a nucleotide sequence according to item (45) for
the manufacture of a non-LLP-protein in a yeast cell, which yeast
cell secretes said non-LLP-protein into the cell culture medium,
due to the secretion-promoting activity of said peptide sequence of
the LLP-signal sequence which is depicted in SEQ ID NO: 3.
[0117] (51) Use of a promotor comprising the LLP-promoter sequence
according to SEQ ID NO: 12 (1000 bp of LLP-promoter), or according
to SEQ ID NO: 2 (605 bp of LLP-promoter), or according to SEQ ID
NO: 129 (576 bp of LLP-promoter), or according to SEQ ID NO: 130
(512 bp of LLP-promoter), or according to SEQ ID NO: 131 (472 bp of
LLP-promoter), or according to SEQ ID NO: 132 (404 bp of
LLP-promoter), or according to SEQ ID NO: 133 (372 bp of
LLP-promoter), or according to SEQ ID NO: 134 (305 bp of
LLP-promoter), in a vector for expression of a gene of interest,
wherein said LLP-promoter sequence has at least 30%, preferably
40%, preferably 50%, preferably 60%, preferably 70%, preferably,
preferably 80%, preferably 90%, preferably 95%, preferably 97%,
preferably 99%, preferably 100% sequence identity with SEQ ID NO:
12.
[0118] (52) Vector comprising the LLP-promoter sequence as defined
in item (51).
[0119] (53) Host cell comprising a vector according to item (52) or
comprising one or more of the LLP-promoter sequences according to
item (51).
[0120] (54) Nucleic acid comprising one or more of the LLP-promoter
sequences according to item (51). In a preferred embodiment, the
nucleic acid according to item (54) is used as a promoter.
Definitions of Terms as Used within the Meaning of the Present
Invention
[0121] Within the meaning of the present invention, the SSN6-like
protein is a protein that comprises, preferably consists of, the
amino acid sequence as depicted in SEQ ID NO: 8.
[0122] The term "ssn6-like gene" denotes a gene encoding the
SSN6-like protein.
[0123] The coding region of the ssn6-like gene of P. pastoris is
depicted in SEQ ID NO: 1.
[0124] Genes or proteins that resemble the ssn6-like gene or
SSN6-like protein in respect of function, activity and sequence, or
only in respect of function and activity, are denoted herein as
"ssn6-like related genes" or "ssn6-like related proteins".
[0125] Within the meaning of the present invention, the term "gene"
denotes a certain nucleic acid sequence that encodes a polypeptide
or an RNA chain that has a function in the organism. The nucleic
acid sequence comprises regulatory regions (herein also denoted as
regulatory polynucleotide sequences or regulatory sequences),
transcribed regions (such as regions that code for proteins, but
also regions that are transcribed but do not code for proteins,
such as introns) and/or other functional sequence regions.
[0126] In general, regulatory regions are primarily regions
flanking the 3'- and 5-region of the coding region of the gene, but
certain regulatory regions such as Trans-regulatory elements, might
be located quite distant within the same chromosome or might even
be located on a different chromosome. In particular, within the
meaning of the present invention, the regulatory regions of the
ssn6-like (or ssn6-like related) gene comprise the flanking region
being located up to 200 nucleotides up- and downstream of the 3'-
and 5'-end of the ssn6-like coding region. Examples of regulatory
polynucleotide sequences are promoters, enhancers, terminators,
silencers, IRES-sequences, ribosome-binding sites, and sequences
stabilizing or destabilizing the mRNA by secondary structures.
Within the meaning of the present invention, the term "regulatory
polynucleotide sequence" denotes polynucleotide sequences that
modify the expression of genes, for example the ssn6-like gene
and/or of the ssn6 gene, and/or a gene of interest and/or the llp
gene.
[0127] "Protein-antigens that are suitbale for vaccines" are
antigens that are able to elicit a proper, desired immune response
upon vaccination. For instance, such antigens are neuraminidase
(NA) or haemagglutinin (HA) of influenza virus. Further antigens
that are suitbale for vaccination purpose are known to a person
skilled in the art. Methods for testing the suitability of proteins
for eliciting a proper immune response are known to a skilled
person.
[0128] A "polynucleotide" or "nucleic acid" sequence includes DNA
(desoxyribonucleic acid) or RNA (ribonucleic acid), in single
stranded or double stranded form or otherwise.
[0129] In general, the term "ssn6-like gene" denotes a gene
encoding the SSN6-like protein. The ssn6-like gene belongs to an
evolutionary conserved family of proteins (see FIG. 9 A), and it is
known that certain yeasts, flies, worms and mammals contain
proteins that resemble SSN6-like proteins in sequence and
function/activity. These proteins are denoted herein as "SSN6-like
related proteins", and the genes that encode these proteins are
denoted herein as "ssn6-like related genes". Examples of yeasts
that contain proteins that belong to the SSN6-like related protein
family are Pichia pastoris (P. Pastoris), Saccharomyces cerevisiae
(S. cerevisiae) and Candida albicans (C. albicans).
[0130] Within the meaning of the present invention, the term
"ssn6-like-related protein" for example denotes the respective
proteins whose NCBI (National Center for Biotechnology Information,
USA) GenBank accession numbers are noted on the left hand site of
FIG. 9A. FIG. 9A only shows an internal part of these sequences
aligned to the corresponding sequence part of the P. Pastoris
ssn6-like sequence (NCBI GenBank accession number CCA36593.1). In
detail these proteins include XP_004181958.1 (SEQ ID NO: 24),
KDQ17717.1 (SEQ ID NO: 25), CCK71477.1 (SEQ ID NO: 26), EDK38165.2
(SEQ ID NO: 27), XP_003688172.1 (SEQ ID NO: 28), XP_003667908.1
(SEQ ID NO: 29), EGA59684.1 (SEQ ID NO: 30), EDN64727.1 (SEQ ID NO:
31), AAA34545.1 (SEQ ID NO: 32), NP_009670.3 (SEQ ID NO: 33),
XP_001731010.1 (SEQ ID NO: 34), CCU98386.1 (SEQ ID NO: 35),
XP_646078.1 (SEQ ID NO: 36), XP_003288629.1 (SEQ ID NO: 37),
CCF50299.1 (SEQ ID NO: 38), XP_761648.1 (SEQ ID NO: 39),
XP_007880878.1 (SEQ ID NO: 40), EPB82504.1 (SEQ ID NO: 41),
CDK26448.1 (SEQ ID NO: 42), ESW97404.1 (SEQ ID NO: 43), CCH42354.1
(SEQ ID NO: 44), CDR41214.1 (SEQ ID NO: 45), XP_002489776.1 (SEQ ID
NO: 46), XP_002770760.1 (SEQ ID NO: 47), EDK37317.2 (SEQ ID NO:
48), XP_001485744.1 (SEQ ID NO: 49), XP_002619527.1 (SEQ ID NO:
50), XP_004200097.1 (SEQ ID NO: 51), XP_004199242.1 (SEQ ID NO:
52), XP_002419644.1 (SEQ ID NO: 53), BAF31137.1 (SEQ ID NO: 54),
XP_719833.1 (SEQ ID NO: 55), EMG49052.1 (SEQ ID NO: 56),
XP_002551300.1 (SEQ ID NO: 57), XP_001526425.1 (SEQ ID NO: 58),
CCE42279.1 (SEQ ID NO: 59), XP_003868368.1 (SEQ ID NO: 60),
XP_001387682.2 (SEQ ID NO: 61), XP_007376632.1 (SEQ ID NO: 62) as
found in the NCBI data base under
http://www.ncbi.nlm.nih.gov/nucleotide/.
[0131] Within the meaning of the present invention, the term
"SSN6-like protein" denotes the protein comprising (preferably
consisting of) the amino acid sequence as depicted in the sequence
depicted in FIG. 2A (SEQ ID NO: 8).
[0132] Furthermore within the meaning of the present invention, the
term "SSN6-like protein" denotes a protein comprising consensus
amino acid sequences depicted in FIG. 9 B or 9 C, namely:
TABLE-US-00001 FIG. 9 B: Consensus sequence 1 (SEQ ID NO: 63)
W(CGL)(SLTA)(IMV)G(VINSTK)LY(YFLA)(QNSRKE)
(INLM)(GNSKR)Q(NFYL)(HRETPAK)D(AST)(LI) (DGTNASE)(AV)(YF) and/or
FIG. 9 C: Consensus sequence 2 (SEQ ID NO: 64)
W(YFLED)(NDG)L(GLAS)(TSIQC)(LIV)YE(TSARKQ)
(CS)(NHSDEH)(DNK-RGF)(Q-H)(ILTHVAS)
(TSNQERIAMG)D(ASV)(LIASC)(DHNE)(SA)(YC) (EAKRQMTLNDS)(RQK)
[0133] The both consensus sequences are written in single letter
code and groups of amino acids in brackets. Groups of amino acids
in brackets means that either amino acid in the brackets can be
present at that position within the consensus sequence. For example
the consensus sequence in FIG. 9 B starts with W at position 1, and
at position 2 there can be present either C or G or L, at position
3 there can be present either S or L or T or A, etc.
[0134] The corresponding amino acid position within the context of
the P. pastoris SSN6-like protein is given below the consensus
sequences of FIGS. 9 B and 9 C, e.g. the Tryptophane residue (W) in
position 1 of the consensus sequence in FIG. 9 B corresponds to the
amino acid position 352 of the P. pastoris SSN6-like protein
sequence.
[0135] In case there is written a "-" (dash), for example at
position 407 of consensus sequence in FIG. 9 C, (Q-H), this means
that on this position there can be either a Q, no amino acid, or a
H.
[0136] Further within the meaning of the present invention, the
term "ssn6-like related gene" or "SSN6-like related protein"
denotes a gene or protein that resembles the ssn6-like gene or
SSN6-like protein in respect of function, activity and sequence, or
only in respect of function and activity.
[0137] The coding region of the ssn6-like gene of P. Pastoris is
depicted in FIG. 1A (SEQ ID NO: 1).
[0138] The amino acid sequence of the SSN6-like protein of P.
Pastoris is depicted in FIG. 1 J (SEQ ID NO: 13).
[0139] The term "llp gene" denotes a gene encoding LLP protein. SEQ
ID NO: 16 depicts the nucleotide sequence of the coding region of
the llp gene from P. Pastoris, including the signal sequence.
[0140] "Resembling with respect to function" or "resembling with
respect to activity" defines that the ssn6-like related gene
product, i.e. the SSN6-like related protein that is encoded by the
"ssn6-like related gene", exhibits essentially the same function
and activity as the SSN6-like protein of P. Pastoris (depicted in
SEQ ID NO: 13) with regard to the LLP-promoter ("Lectin-like
protein with similarity to Flo1p"), when said function and activity
is compared in a suitable system when applying comparable,
preferably corresponding, conditions. The function and activity
that is of interest in the present invention, and thus has to be
compared or assessed, respectively, is the ability of the SSN6-like
protein and SSN6-like related protein, respectively, to regulate,
preferably repress (reduce), and even more preferably prevent, the
expression of genes that are under control of the LLP-promoter. For
instance, in a wildtype situation, typically the gene that is under
control of the LLP promoter is a gene encoding LLP protein. In this
case, in order to determine whether the SSN6-like related protein
exhibits essentially the same function and/or activity as the
SSN6-like protein, for instance the amount of LLP protein being
present is compared, i.e. the amount of LLP protein in an
expression system wherein an ssn6-like related gene (and, thus, an
SSN6-like related protein) is present, compared to an expression
system wherein an SSN6-like gene (and, thus, an SSN6-like protein)
is present. If it is shown that the candidate ssn6-like related
gene and the ssn6-like gene (and its gene products, (candidate)
SSN6-like related protein and SSN6-like protein, respectively (also
referred to herein as "SSN6-like/SSN6-like related protein" or
"SSN6-like related/SSN6-like protein") exhibit essentially the same
function/activity with regard to the LLP-promoter (and with regard
to the gene that is under control of said promoter, such as the llp
gene), e.g. with regard to the determined amount of LLP protein,
then the candidate ssn6-like related gene is a ssn6-like related
gene within the meaning of the present invention.
[0141] A promoter is defined as a DNA regulatory region capable of
binding an RNA polymerase in a cell and that initiates
transcription of a particular gene to which it operably links.
Typically, promoters are located near the transcription start sites
of the respective gene(s) that is under control of said promoter,
on the same strand. FIG. 1 B (SEQ ID NO: 2) and FIG. 1 I (SEQ ID
NO: 12) show nucleotide sequences of the LLP promoter of P.
Pastoris (FIG. 1 B=first 605 nucleotides 5' from ATG start codon,
FIG. 1 I=fist 1000 nucleotides 5' from ATG start codon).
[0142] Whenever reference is made herein to an LLP promoter, an
"LLP-like promoter" is also comprised by this expression. Within
the meaning of the present invention, an LLP-like promoter is a
promoter that exhibits essentially the same function/activity as
the LLP promoter, i.e. is still able to essentially exert the LLP
promoter's wildtype function/activity.
[0143] In order to determine whether a candidate ssn6-like-related
gene and SSN6-like-related protein, respectively, exhibits
essentially the same function and activity as the ssn6-like-gene
and SSN6-like protein, respectively, with respect to the
LLP-promoter, any suitable method that is known to a person skilled
in the art can be used. Examples of such methods are measuring the
amount of LLP protein in the supernatant of a cell culture for
example by Enzyme Linked Inmmunosorbent Assay (ELISA), or by
western blotting, or measuring the level of LLP mRNA by northern
blotting or by quantitative Polymerase Chain Reaction (qPCR) or
reverse transcriptase qPCR, or measuring the activity of the
LLP-promoter or LLP-like promoter ("LLP/LLP-like promoter") for
example by using luciferase reporter gene assays or by using
LLP-promoter-green fluorescent protein (GFP) constructs, etc. All
these methods are well known to a person skilled in the art and
represent routine work. A textbook comprising protocols for routine
methods is for instance Sambrook et al., "Molecular Cloning: A
Laboratory Manual", 4.sup.th Edition, Cold Spring Harbor Laboratory
Press, (2012), referred to herein as Sambrook et al. Within the
meaning of the present invention, a candidate ssn6-like-related
gene is considered as exhibiting essentially the same
function/activity as the ssn6-like-gene, if its effect (and the
effect of its gene product, respectively) on the LLP protein
expression is essentially the same.
[0144] Within the meaning of the present invention, the expression
"essentially the same" defines a deviation of up to 20%, preferably
of up to 10%, more preferably of up to 7% and even more preferably
of up to 3% of a given value.
[0145] Accordingly, the expression "essentially no" defines that
there is an amount/activity of the respective matter (such as
protein) present (or left) that corresponds to at most 20%,
preferably at most 10%, more preferably at most 7%, and even more
preferably at most 3% of the respective wildtype amount/activity.
"Essentially no" also includes the absence of the respective
matter/amount/activity, preferably meaning below the detection
limit of the methods described in this application for that
matter/amount/activity.
[0146] Within the meaning of the present invention the term
"reduced", e.g. "reduced" activity, "reduced" function, or
"reduced" amount denotes that the total activity, function,
preferably gene regulatory function, or amount of a matter (e.g.
SSN6-like protein or SSN6-like related protein of the modified
eukaryotic cell; or LLP promoter) is at most 20%, preferably at
most 15%, more preferably at most 10%, even more preferably at most
5% of the total activity, function and/or amount of the wildtype
form of this matter (e.g. SSN6-like protein or SSN6-like related
protein; or LLP promoter).
[0147] It is possible that nucleotide sequences and the proteins
they encode, respectively, which exhibit essentially the same
function and activity as ssn6-like gene and SSN6-like protein,
respectively, exhibit a comparably low degree of sequence identity
such as 50%, 40% or 30% or even lower, such as 25%, 20%, 19%, 18%,
17%, 16%, 15%, 12%, 10%, or 5%. Thus, ssn6-like-related
genes/SSN6-like-related proteins within the meaning of the present
invention are genes/proteins that--first of all--exhibit
essentially the same function/activity as the ssn6-like
gene/SSN6-like protein with regard to the LLP protein (in wild type
situation). Hence, in a preferred embodiment of the present
invention, the term "ssn6-like-related gene" denotes genes that
resemble the ssn6-like gene as defined herein in respect of
function/activity, i.e. the ability of the SSN6-like-related
protein being encoded by the ssn6-like-related gene to regulate the
expression of genes that are under control of the LLP-promoter, and
LLP-promoter sequence, respectively.
[0148] It is also possible that an SSN6-like related protein
comprises the consensus amino acid sequences depicted in FIG. 9 B
and/or FIG. 9C.
[0149] In a further preferred embodiment of the present invention,
the "ssn6-like related gene" resembles the ssn6-like gene as
defined herein not only in respect of function/activity, i.e. the
ability of the SSN6-like related protein being encoded by the
ssn6-like related gene to regulate the expression of genes that are
under control of the LLP-promoter or modified LLP promoter, and
LLP-promoter sequence, respectively, but additionally originates
from a microorganism being selected from the group consisting of
Komagataella pastoris CBS 7435 (Synonym/other names: Pichia
pastoris, Pichia pastoris CBS 7435), Komagataella pastoris GS115
(Synonym/other names: Pichia pastoris, Pichia pastoris GS115),
Scheffersomyces stipitis CBS 6054 (Synonym/other names: Pichia
stipitis, Pichia stipitis CBS 6054), Millerozyma farinosa CBS 7064
(other name: Pichia farinosa CBS 7064), Candida parapsilosis,
Candida orthopsilosis Co 90-125, Debaryomyces hansenii CBS767,
Spathaspora passalidarum NRRL Y-27907, Candida albicans, Candida
albicans, Candida albicans SC5314, Candida maltosa Xu316, Candida
tropicalis MYA-3404 (other name: Candida tropicalis T1),
Lodderomyces elongisporus NRRL YB-4239 (other name: Saccharomyces
elongisporus), Clavispora lusitaniae ATCC 4272 (genebank anamorph:
Candida lusitaniae ATCC 42720), Meyerozyma guilliermondii ATCC 6260
(genebank anamorph: Pichia guilliermondii ATCC 6260),
Wickerhamomyces ciferrii, Ogataea parapolymorpha DL-1 (synonym and
other names: Hansenula polymorpha, Hansenula polymorpha DL-1,
Ogataea angusta DL-1, Ogataea parapolymorpha ATCC 26012, Ogataea
parapolymorpha DL-1, Pichia angusta DL-1), Cyberlindnera fabianii
(synonyms and other names: Hansenula fabianii, Pichia fabianii, . .
. ) Kuraishia capsulata CBS 1993, Dictyostelium discoideum AX4
(belongs to social amoebae), Tetrapisispora phaffii CBS 4417
(synonym: Fabospora phaffii, Dictyostelium purpureum (belongs to
social amoebae), Pseudozyma flocculosa PF-1, Malassezia globosa CBS
7966, Botryobasidium botryosum FD-172 SS1 (basidiomycete),
Naumovozyma dairenensis CBS 421 (synonyme: Saccharomyces
dairenensis), Tetrapisispora blattae CBS 6284, Mucor circinelloides
f. circinelloides 1006PhL (Early diverging fungal lineage),
Malassezia sympodialis ATCC 42132, Kazachstania naganishii CBS 8797
(Saccharomyces naganishii), Saccharomyces cerevisiae YJM789,
Saccharomyces cerevisiae FostersB, Saccharomyces cerevisiae,
Saccharomyces cerevisiae S288c, Ustilago hordei (Corn smut fungus,
basidiomycete), Meyerozyma guilliermondii ATCC 6260 (synonym/other
names: Candida guilliermondii, Pichia guilliermondii ATCC 6260),
and Ustilago maydis 521 (Corn smut fungus, basidiomycete).
[0150] In a preferred embodiment, the "modified ssn6-like gene" and
"modified ssn6-like related gene", and the respective proteins
these genes encode, do not correspond to/are not derived from
Saccharomyces cerevisiae ssn6 or Tup1 nucleotide or amino acid
sequences, especially do not correspond to the following
sequences:
[0151] SEQ ID NO: 135 (nucleotide sequence encoding SSN6)
[0152] SEQ ID NO: 136 (nucleotide sequence encoding Tup1)
[0153] SEQ ID NO: 137 (amino acid sequence of SSN6)
[0154] SEQ ID NO: 138, (amino acid sequence of Tup1).
[0155] In a further preferred embodiment, the modified eukaryotic
cell comprises modified ssn6-like genes or ssn6-like related genes,
but no other modified gene(s) that regulate(s) the expression of a
gene that is under control of a promoter that is repressed in the
presence of SSN6-like related protein or SSN6-like protein.
[0156] "Sequence identity" or "% identity" refers to the percentage
of residue matches between at least two polypeptide or
polynucleotide sequences aligned using a standardized algorithm.
Such an algorithm may insert, in a standardized and reproducible
way, gaps in the sequences being compared in order to optimize
alignment between two sequences, and therefore achieve a more
meaningful comparison of the two sequences. For purposes of the
present invention, the sequence identity between two amino acid
sequences or nucleotide is determined using the NCBI BLAST program
version 2.2.29 (Jan. 6, 2014) (Altschul et al., Nucleic Acids Res.
(1997) 25:3389-3402). Sequence identity of two amino acid sequences
can be determined with blastp set at the following parameters:
Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost:
Existence=11, Extension=1; Filter=low complexity activated; Filter
String: L; Compositional adjustments: Conditional compositional
score matrix adjustment. For purposes of the present invention, the
sequence identity between two nucleotide sequences is determined
using the NCBI BLAST program version 2.2.29 (Jan. 6, 2014) with
blastn set at the following exemplary parameters: Word Size: 11;
Expect value: 10; Gap costs: Existence=5, Extension=2; Filter=low
complexity activated; Match/Mismatch Scores: 2,-3; Filter String:
L; m.
[0157] Nucleic acid sequences alternatively can be characterized by
their degree of complementarity. As used herein, the term
"complementary" refers to the ability of purine and pyrimidine
nucleotide sequences to associate through hydrogen bonding to form
double-stranded nucleic acid molecules. Guanine and cytosine,
adenine and thymine, and adenine and uracil are complementary and
can associate through hydrogen bonding resulting in the formation
of doublestranded nucleic acid molecules when two nucleic acid
molecules have "complementary" sequences. The complementary DNA
sequences are referred to as a "complement." In accordance with the
invention "highly stringent conditions" means hybridization at
65.degree. C. in 5.times.SSPE and 50% formamide, and washing at
65.degree. C. in 0.5.times.SSPE. Conditions for high stringency
hybridization are described in Sambrook et al., "Molecular Cloning:
A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory
Press, (2001), incorporated herein by reference. In some
illustrative aspects, hybridization can occur along the full-length
of the isolated nucleic acid, or along part of its length, or to a
fragment thereof.
[0158] The following list indicates examples of organisms that
comprise ssn6-like related genes: Komagataella pastoris CBS 7435
(Synonym/other names: Pichia pastoris, Pichia pastoris CBS 7435),
Komagataella pastoris GS115 (Synonym/other names: Pichia pastoris,
Pichia pastoris GS115), Scheffersomyces stipitis CBS 6054
(Synonym/other names: Pichia stipitis, Pichia stipitis CBS 6054),
Millerozyma farinosa CBS 7064 (other name: Pichia farinosa CBS
7064), Candida parapsilosis, Candida orthopsilosis Co 90-125,
Debaryomyces hansenii CBS767, Spathaspora passalidarum NRRL
Y-27907, Candida albicans, Candida albicans, Candida albicans
SC5314, Candida maltosa Xu316, Candida tropicalis MYA-3404 (other
name: Candida tropicalis T1), Lodderomyces elongisporus NRRL
YB-4239 (other name: Saccharomyces elongisporus), Clavispora
lusitaniae ATCC 4272 (genebank anamorph: Candida lusitaniae ATCC
42720), Meyerozyma guilliermondii ATCC 6260 (genebank anamorph:
Pichia guilliermondii ATCC 6260), Wickerhamomyces ciferrii, Ogataea
parapolymorpha DL-1 (synonym and other names: Hansenula polymorpha,
Hansenula polymorpha DL-1, Ogataea angusta DL-1, Ogataea
parapolymorpha ATCC 26012, Ogataea parapolymorpha DL-1, Pichia
angusta DL-1), Cyberlindnera fabianii (synonyms and other names:
Hansenula fabianii, Pichia fabianii, . . . ), Kuraishia capsulata
CBS 1993, Dictyostelium discoideum AX4 (belongs to social amoebae),
Tetrapisispora phaffii CBS 4417 (synonym: Fabospora phaffii,
Dictyostelium purpureum (belongs to social amoebae), Pseudozyma
flocculosa PF-1, Malassezia globosa CBS 7966, Botryobasidium
botryosum FD-172 SS1 (asdmVcete), Naumovozyma dairenensis CBS 421
(synonyme: Saccharomyces dairenensis), Tetrapisispora blattae CBS
6284, Mucor circinelloides f. circinelloides 1006PhL (Early
diverging fungal lineage), Malassezia sympodialis ATCC 42132,
Kazachstania naganishii CBS 8797 (Saccharomyces naganishii),
Saccharomyces cerevisiae YJM789, Saccharomyces cerevisiae FostersB,
Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Ustilago
hordei (Corn smut fungus, basidiomycete), Meyerozyma guilliermondii
ATCC 6260 (synonym/other names: Candida guilliermondii, Pichia
guilliermondii ATCC 6260), Ustilago maydis 521, (Corn smut fungus,
basidiomycete).
[0159] Within the meaning of the present invention, the term
"wildtype" denotes that a certain matter is present in its natural
occurring state, and thus exerts its natural function and/or
activity. A "certain matter" can for instance be an organism such
as a microorganism, a cell, or, preferably, a certain protein such
as the SSN6 protein or the SSN6-like protein. The wildtype state,
or wildtype form, respectively, of a certain matter is
distinguishable from mutant forms of said matter, as for instance
structural mutant forms can result from modifications that are
carried out artificially, such as by in vitro, in vivo or ex vivo
modifications or manipulations. Modifications that can be carried
out artificially are described elsewhere herein. An organism that
comprises modifications is "modified" compared to its wildtype form
with regard to the modified structure.
[0160] Within the meaning of the present invention, the term
"function" defines the physiological role of a certain matter, such
as the SSN6-like protein or SSN6-like related protein, and the term
"activity" defines to which extent said certain matter exerts its
function. An example of a physiological role of a certain matter is
the regulating, preferably repressing, role of SSN6-like protein or
SSN6-like related protein, on the activity of the LLP-promoter, and
hence, on the expression of genes that are under control of said
promoter.
[0161] Within the meaning of the present invention, the term
"wildtype function" denotes the function a certain structure or
matter (such as an organism, a protein, or a gene) exerts in its
natural state. Accordingly, the term "wildtype activity" denotes
the activity a certain structure exerts in its natural state.
[0162] Within the meaning of the present invention, an organism can
be modified only with regard to a single specific structural and/or
functional feature, or with regard to multiple structural and/or
functional features. Specific structural and/or functional features
that are modified are features that modulate (i.e. influence,
either positively or negatively, preferably negatively) the
expression of the ssn6 gene or ssn6-like gene.
[0163] An example of a specific feature that can be modified is for
instance the nucleotide sequence of ssn6-like gene or ssn6-like
related gene, with this gene comprising a coding sequence and a
regulatory sequence that influences the expression of said genes as
disclosed elsewhere herein.
[0164] Regulatory sequences are sequences that control the
expression of certain genes. Within the meaning of the present
invention, said regulatory sequences are for example sequences that
control the expression of the ssn6-like gene, the ssn6-like related
gene or the LLP-gene, such as promoters, enhancers, terminators,
silencers, IRES-sequences, ribosome-binding sites, and sequences
stabilizing or destabilizing the mRNA by secondary structures.
[0165] Within the meaning of the present invention, the term
"heterologous nucleotide sequence" is a polynucleotide that does
not naturally occur in the cell, e.g. because the nucleotide
sequence of the polynucleotide does not naturally occur in
eukaryotic cells, such as eukaryotic cells as defined elsewhere
herein.
[0166] Within the meaning of the present invention, the names
"Komagataella pastoris" and "Pichia pastoris" are synonymous.
[0167] The names "NRRL Y-11430, CBS7435" are synonymously used in
the present invention. The genome of CBS7435 comprises chromosomes
1 to 4 and a mitochondrium. The respective nucleotide sequences
have the following GenBank Accession Numbers: Chromosome 1:
FR839628.1; Chromosome 2: FR839629.1; Chromosome 3: FR839630.1;
Chromosome 4: FR839631.1; Mitochondrium: FR839632.1 (Publication:
High-quality genome sequence of Pichia pastoris CBS7435, Kuberl A,
Schneider J, Thallinger G G, Anderl I, Wibberg D, Hajek T, Jaenicke
S, Brinkrolf K, Goesmann A, Szczepanowski R, Puhler A, Schwab H,
Glieder A, Pichler H, J Biotechnol, volume 154 issue 4, pages
312-320 year 2011).
[0168] A "vector" is a replicon, such as plasmid, phage, bacterial
artificial chromosome (BAC) or cosmid, into which another DNA
segment (e.g. a foreign gene) may be inserted so as to bring about
the replication of said inserted DNA segment, resulting in
expression of said inserted sequence. Vectors may comprise a
promoter and one or more control elements (e.g., enhancer elements)
that are homologous or heterologous to said inserted DNA segment
but are recognized and used by the host cell. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions
as a unit of DNA replication within a cell. A replicon can be an
autonomous unit. This means that it is capable of replication under
its own control. Within the meaning of the present invention, the
vector comprises a promoter, wherein said promoter is characterized
in that it is repressed in the presence of SSN6-like related
protein or SSN6-like protein if said vector is introduced into a
suitable host cell. In a preferred embodiment of the present
invention, the vector comprises a promoter for the coding sequence
of the LLP-protein and/or for the coding sequence of a protein of
interest, wherein said promoter is characterized in that it is
repressed in the presence of SSN6-like related protein or SSN6-like
protein if said vector is introduced into a suitable host cell.
[0169] A common type of vector is a "plasmid", which generally is a
self-contained molecule of double-stranded DNA, usually of
bacterial origin, that can readily accept additional (foreign) DNA
and which can be readily introduced into a suitable host cell. In
general, vectors enable the introduction of nucleotide sequences
into a host cell, so as to transform the host and, optionally, to
promote expression and/or replication of the introduced
sequence.
[0170] A vector often contains coding DNA and regulatory sequences
such as promoter DNA and has one or more restriction sites suitable
for inserting additional, e.g. foreign such as heterologous, DNA.
Promoter DNA and coding DNA may be from the same gene or from
different genes, and may be from the same or different organisms.
Vectors, such as recombinant cloning vectors, will often include
one or more replication systems for cloning or expression, one or
more markers for selection in the host, e.g. antibiotic resistance,
and one or more expression cassettes. Vector, or vector constructs,
respectively, may be produced using conventional molecular biology
and recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, 4.sup.th Edition (2012) Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 2012");
DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover
ed. 1985); F. M. Ausubel et al. (eds.), Current Protocols in
Molecular Biology, John Wiley & Sons, Inc. (1994).
[0171] Generally, the specific structural elements of a vector
depend on the intended use of said vector. For instance, in order
to propagate a vector in a host cell, it may contain one or more
"origins of replication" sites (often also termed "ori"), which is
a specific nucleic acid sequence at which replication is initiated.
Accordingly, the term "origin of replication" or "ori" refers to a
nucleic acid sequence that initiates nucleic acid replication.
[0172] "Ori T" refers to an "origin of transfer" that permits the
transfer of the vector from one bacterial cell to another.
[0173] Dependent on in which organism/cell the (expression) vector
is intended for being used, a person skilled in the art knows which
markers have to be present in the vector. If, for instance, the
vector is intended for being used in yeast, amongst others,
commonly used yeast markers are present, such as
beta-galactosidase, Zeocin, Geneticin, URA3, HIS3, LEU2, TRP1 and
LYS2. If the vector is intended for being used in bacteria such as
E. coli, amongst others an on (see above) and/or selectable markers
such as genes conferring antibiotic resistance can be present.
[0174] Suitable selectable markers depend on the respective system
that is used and are known to a person skilled in the art.
Transformed microorganisms, that is, those containing recombinant
molecules such as a vector (expression vector) or plasmid, may be
selected with a variety of positive and/or negative selection
methods or markers. For instance, a positive selection marker can
be a gene that allows growth in the absence of an essential
nutrient, such as an amino acid. A variety of suitable
positive/negative selection pairs are available and known in the
art. For example, various amino acid analogs known in the art could
be used as a negative selection, while growth on minimal media
(relative to the amino acid analog) could be used as a positive
selection. Visually detectable markers are also suitable for being
uses in the present invention, and may be positively and negatively
selected and/or screened using technologies such as fluorescence
activated cell sorting (FACS) or microfluidics. Examples of
detectable markers include various enzymes, prosthetic groups,
fluorescent markers, luminescent markers, bioluminescent markers,
and the like. Examples of suitable fluorescent proteins include,
but are not limited to, yellow fluorescent protein (YFP), green
fluorescence protein (GFP), cyan fluorescence protein (CFP),
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichiorotriazinylamine fluorescein, dansyl chloride, phycoerythrin
and the like. Examples of suitable bioluminescent markers include,
but are not limited to, luciferase (e.g., bacterial, firefly, click
beetle and the like), luciferin, aequorin and the like. Examples of
suitable enzyme systems having visually detectable signals include,
but are not limited to, galactosidases, glucorinidases,
phosphatases, peroxidases, cholinesterases and the like. In other
aspects, the positive selection marker is a gene that confers
resistance to a compound, which would be lethal to the cell in the
absence of the gene. For example, a cell expressing an antibiotic
resistance gene would survive in the presence of an antibiotic,
while a cell lacking the gene would not. For instance, the presence
of a tetracycline resistance gene could be positively selected for
in the presence of tetracycline, and negatively selected against in
the presence of fusaric acid. Suitable antibiotic resistance genes
include, but are not limited to, genes such as
ampicillin-resistance gene, neomycin-resistance gene,
blasticidin-resistance gene, hygromycin-resistance gene,
puromycin-resistance gene, chloramphenicol-resistance gene,
apramycin-resistance gene, geneticin-resistance gene,
zeocin-resistance gene, and the like. In certain aspects, the
negative selection marker is a gene that is lethal to the target
cell in the presence of a particular substrate. For example, the
thyA gene is lethal in the presence of trimethoprim. Accordingly,
cells that grow in the presence trimethoprim do not express the
thyA gene.
[0175] In order to purify the recombinant proteins, the respective
(expression) vectors used often comprise suitable purification
sequences (purification markers). For instance, a vector may
comprise a C-terminal c-myc epitope and polyhistidine sequence for
detection and purification of the recombinant protein(s).
[0176] As described elsewhere herein, a vector can be used for
introducing a nucleic acid sequence into a host cell, or into the
host cell's genome, such as the genome of a yeast such as P.
pastoris, or of a bacterium, such as E. coli. The introduction of
(heterologous) nucleic acid into a host cell's nucleic acid is
denoted as "recombination". Such a recombination process can be
targeted, i.e. take place at a defined, desired site of the genome
(nucleic acid sequence) it is to be introduced. In this case, the
recombination is denoted as "homologous recombination" (or
"targeted recombination"/"targeted integration"). Also, the
recombination process can be random ("random recombination"/"random
integration"). In this case, the recombination is
non-homologous.
[0177] The term "homologous recombination" refers to a type of
genetic recombination, a process of physical rearrangement
occurring between two different strands of DNA molecules.
Homologous recombination involves the alignment of identical or
similar sequences, a crossover between the aligned homologous DNA
strands of the two molecules, and breaking and repair of the DNA to
produce an exchange of material between the strands. Homologous
recombination is distinguished from other types of recombination.
For example, "site specific recombination", as exemplified by
invertible elements, resolvases, and some phage integration events
are examples of non-homologous recombination. Though in many cases
identical or similar sequences are required at the two recombining
sites, the sequences are short, distinguishing them from the longer
stretches (hundreds of base pairs) used in homologous
recombination. (J Rubnitz and S Subramani. 1984, Mol Cell Biol. 4:
2253-2258).
[0178] If the vector aligns in a non-homologous region of the
target nucleic acid, the recombination is random.
[0179] Within the meaning of the present invention, in connection
with homologous recombination, two DNA sequences are "substantially
homologous" if they are able to mediate a homologous recombination.
This may for instance the case when at least about 80%, preferably
at least about 90% or 95%, more preferably at least about 97% or
98% of the nucleotides match over a defined length of the DNA
sequences, as determined by sequence comparison algorithms known to
a person skilled in the art and described elsewhere in this
application. It is also possible that two DNA sequences exhibit
100% match of the nucleotides over a defined length. In this case,
the two DNA sequences are homologous to each other. Within the
meaning of the present invention, a DNA sequence in the vector,
e.g. the expression vector, is substantially homologous, or
homologous, to a desired target integration site in the host
cell.
[0180] Within the meaning of the present invention, both
integration/recombination events can occur, i.e. the homologous
recombination and the random recombination. For instance, if it is
desired to replace a certain gene (or coding sequence) by the gene
of interest, then a homologous recombination will be carried out.
Within the meaning of the present invention, it may be desired to
replace a gene that is under the control of a promoter that is
repressable by SSN6-like related or SSN6-like protein, such as a
nucleic acid sequence encoding LLP protein ("Option 1" in FIG. 3
C).
[0181] Within the meaning of the present invention it is however
also possible that it may be desired to not replace a gene that is
under the control of a promoter that is repressable by SSN6-like
related or SSN6-like protein, such as a nucleic acid sequence
encoding LLP protein, but to express said gene (such as the gene
encoding LLP), and to additionally express the gene of interest
("Option 2" in FIG. 3 D). In such a case, a random (heterologous)
recombination is carried out.
[0182] Within the meaning of the present invention, the term "gene
of interest" (GOI) refers to a gene that is intended for being
expressed in a host cell. In the present invention, genes of
interest are genes selected for example from the group encoding
enzymes, antibodies or fragments thereof, hormones, structural
proteins (e.g. albumin) and protein-antigens present in
vaccines.
[0183] Within the meaning of the present invention, the term
"expression cassette" refers to a part of vector DNA that is for
instance used for cloning and/or transformation. An expression
cassette comprises one or more genes and sequences controlling
their expression. An expression cassette can be inserted in a
nucleotide sequence such as a genome for instance by means of
homologous recombination or by heterologous recombination.
[0184] Within the meaning of the present invention, the term "host
cell" denotes any cell that, under suitable conditions, is capable
of propagating or expressing a vector such as an expression vector.
For instance, in the present invention an expression vector can
comprise a promoter, wherein said promoter is characterized in that
it is repressed in the presence of SSN6-like protein or SSN6-like
related protein.
[0185] The host cell that comprises a vector can be any suitable
host cell, such as a bacterial cell, preferably an E. coli cell. It
is also possible that the vector, such as the expression vector, is
present in a eukaryotic cell. In this case, the host cell is said
eukaryotic cell. If, as described elsewhere herein, said eukaryotic
cell is modified with regard to the ssn6-like gene, ssn6-like
related gene, SSN6-like protein or SSN6-like related protein, the
eukaryotic cell is denoted as "modified eukaryotic cell". In a
preferred embodiment of the present invention, the eukaryotic cell
is a fungal cell, preferably a yeast cell, more preferably selected
from the group consisting of Saccharomyces, Kluyveromyces, Candida,
Hansenula, Pichia, Komagataella and Torulopsis, even more
preferably the eukaryotic cell is a Pichia cell, most preferably a
Pichia pastoris cell.
[0186] If the expression vector is intended for being present in a
eukaryotic cell, which may be modified or not, it is preferred that
the expression vector comprises features selected from any one of
the following: a selection marker; a purification marker; a signal
sequence, preferably the yeast alpha-factor secreting signal
sequence, the yeast KILM1 signal peptide, the yeast PH01 signal
peptide, or the yeast SUC2 signal peptide, more preferably an
alpha-factor secreting signal sequence, even more preferably the
MFalpha pre-pro signal sequence; an origin of replication; and/or a
nucleotide sequence for targeted (option 1, of FIG. 3 C) and/or
random integration (option 2, FIG. 3 D) into the genome of a host
cell.
[0187] Within the meaning of the present invention, the eukaryotic
cells that are used are modified compared to their wildtype cell.
This means that the modified eukaryotic cell (at least) comprises a
modified ssn6-like related gene or a modified ssn6-like gene;
and/or a modified, preferably reduced, expression level of
SSN6-like related protein or of SSN6-like protein, or that an
ssn6-like related gene or an ssn6-like gene is deleted resulting in
the complete lack of said SSN6-like related protein or complete
lack of said SSN6-like protein.
[0188] The effect of the respective modifications is that the
modified eukaryotic cell is not able to provide an SSN6-like
related protein or an SSN6-like protein that exerts its wildtype
function and/or wildtype activity, preferably with respect to
regulating the LLP promoter; and/or the amount of SSN6-like related
protein or of SSN6-like protein being present in the modified
eukaryotic cell differs (preferably in that it is reduced) from the
amount of SSN6-like related protein or of SSN6-like protein being
present in its wildtype form; and/or no SSN6-like related protein
or SSN6-like protein is present in the modified cell. This, in
turn, has the effect that said modified eukaryotic cell, compared
to its wildtype cell, exhibits different (preferably reduced)
SSN6-like related protein or SSN6-like protein activity and/or
function, preferably with respect to its activity and/or function
in regulating the LLP promoter.
[0189] As described elsewhere herein, if the modified eukaryotic
cell is not able to provide a proper, sufficiently working
SSN6-like related protein or SSN6-like protein (e.g. in terms of
function, activity, and/or amount), as a result the regulatory
sequences that are controlled by said protein, such as promoters,
like the LLP promoter, are not affected in their function (e.g. are
not repressed) any more. This, in turn, results in an
overexpression of the protein that is under control of said
regulatory polynucleotide sequence.
[0190] In other words, proper, sufficiently working SSN6-like
related protein or SSN6-like protein represses the LLP promoter,
which means that the LLP promoter exhibits reduced function, when
compared to the function of the LLP promoter that is not repressed
by said proteins. As to the term "reduced", reference is made to
the description elsewhere herein.
[0191] Within the meaning of the present invention, the term
"overexpression" defines that the expression of a protein (encoded
for instance by the gene of interest) in a cell such as a
(modified) eukaryotic cell is at levels greater than normal in a
wildtype cell. For instance, in the present invention the modified
eukaryotic cell can be used in order to overexpress a gene of
interest.
[0192] Within the meaning of the present invention, the term
"modification" denotes any suitable amendment that is known to a
person skilled in the art that can be applied to a nucleotide
sequence or gene, respectively, that results in one or more of the
following effects (if the modified nucleotide sequence is expressed
in a suitable expression system): the SSN6-like or SSN6-like
related protein does not exert its wildtype function and/or
wildtype activity; the amount of SSN6-like or SSN6-like related
protein being present is lower as compared to non-modified cells;
and/or no SSN6-like or SSN6-like related protein is present at all.
In an embodiment of the present invention, the nucleotide sequence
of the ssn66-like or ssn66-like related gene, respectively, is
modified by introduction of a point mutation, e.g. substitution,
insertion or deletion of a single or more nucleotides; by partial
or complete deletion; and/or by replacement by a different, e.g.
heterologous, nucleotide sequence.
[0193] It is also possible that the expression level of the
affected SSN6-like or SSN6-like related protein is modified, for
instance by introduction of a point mutation, e.g. substitution,
insertion or deletion of a single or more nucleotides in regulatory
polynucleotide sequences of the affected ssn6-like or ssn6-like
related nucleotide sequence or ssn6-like or ssn6-like related gene,
respectively; by partial or complete deletion of regulatory
polynucleotide sequences of the affected ssn6-like or ssn6-like
related nucleotide sequence or ssn6-like or ssn6-like related gene,
respectively; and/or by replacement of the regulatory
polynucleotide sequences of the affected ssn6-like or ssn6-like
related nucleotide sequence or ssn6 or ssn6-like gene,
respectively, by different, e.g. heterologous, nucleotide
sequences. The regulatory polynucleotide sequences are selected
from the group as defined elsewhere herein.
[0194] In order to assess whether a modification in the meaning of
the present invention has been carried out on a nucleotide
sequence, suitable sequence alignments can be carried out: For
instance, the sequence of the potentially modified polynucleotide
sequence can be aligned with the respective wildtype counterpart.
The resulting alignment indicates whether a modification has been
carried out, and if so, which kind of modification.
[0195] Within the meaning of the present invention, the term
"coding sequence" denotes a nucleotide sequence (e.g. heterologous
or homologous nucleotide sequence or heterologous or homologous
polynucleotide, respectively), such as a nucleotide sequence being
comprised in a gene of interest, that encodes an expression
product, such as an RNA or polypeptide, that, when expressed,
results in production of the product (e.g. polypeptide (for
instance a heterologous polypeptide), such as enzymes, antibodies
or fragments thereof, hormones, or protein-antigens present in
vaccines).
[0196] Here, the term "a promoter that is repressed" defines that
the promoter is not able to exert its full, natural function and
its full, natural activity. An example of such a promoter that is
repressed in the presence of SSN6-like or SSN-like related protein
is the LLP promoter: It is not yet known which mechanism is
underlying said repression, however it is speculated that the
repression could be based on alteration of the local chromatin
structure, or on interaction with the general transcription
machinery (Smith et al., TIBS 25, July 2000, 325-330).
[0197] Within the meaning of the present invention, as a result of
the repression of the promoter, the expression of a gene that is
under control of said promoter is influenced to the effect that the
respective encoded protein is not expressed; and that the amount of
said expressed protein differs from the wildtype amount of said
protein.
[0198] Within the meaning of the present invention a coding
sequence (e.g. of a heterologous polynucleotide) is "under control
of" (or the like) a transcriptional or translational control
sequence (regulatory polynucleotide sequence) when the regulatory
polynucleotide sequence directs RNA, preferably mRNA, which then
may be spliced (if it contains introns) and, optionally, translated
into a protein encoded by the coding sequence. In an embodiment of
the present invention, a gene (such as a gene of interest) is under
control of the LLP promoter. This means that as long as the
promoter works as in a wildtype situation (for instance, is
repressed by SSN6-like protein or SSN6-like related protein), a
gene that is under control of said promoter is not expressed or
expressed at low levels when the cells are cultivated in standard
conditions. As soon as the LLP promoter is de-repressed due to the
presence of a modified SSN6-like related protein or SSN6-like
protein or different cultivation conditions, a gene that is under
control of this promoter is expressed at high to very high levels
(compared to a situation where the promoter is not repressed).
Based on the ratio underlying the present invention, it is likely
that a SSN6-like protein or a SSN6-like related protein might be
blocked not only be mutation of the ssn6-like gene or the ssn6-like
related gene, but also by inhibitors of ssn6-like gene or protein
or by inhibitors of ssn6-like related gene or protein, which also
could result in increased activity of the LLP-promoter.
[0199] The term "functional" defines that the respective matter
exhibits its natural function.
[0200] The term "selecting" refers to the identification and
isolation of a recipient cell that contains the vector of interest.
Transformed microorganisms, that is, those containing recombinant
molecules such as a vector or plasmid, may be selected with a
variety of positive and/or negative selection methods or markers.
Details according to such selection methods and markers are
described elsewhere in this application. Negative selection markers
include, but are not limited to, genes such as thyA, sacB, gnd,
gapC, zwJ, talA, taiB, ppc, gdhA, pgi, Jbp, pykA, cit, acs, edd,
icdA, groEL, secA and the like.
[0201] In general, the term "expression system" denotes a system
that is specifically designed for the production of a gene product
of choice, also referred to herein as protein of interest (POI). In
the present invention, the expression system comprises a modified
ssn6-like related gene or a modified ssn6-like gene, or an
ssn6-like related gene or an ssn6-like gene and a modified
regulatory polynucleotide sequence that is involved in the
regulation of the expression of said genes, to the effect that said
expression system is not able to express an SSN6-like related
protein or SSN6-like protein, that SSN6-like related protein or
SSN6-like protein is expressed that does not exert its wildtype
function and/or wildtype activity, and/or that an amount of
SSN6-like related protein or SSN6-like protein is expressed that
differs, preferably is lower, when compared to the amount of
SSN6-like related protein or of SSN6-like protein that is expressed
by said expression system when the corresponding wildtype
polynucleotide sequence is expressed by such an expression system
under the same conditions.
[0202] The expression system of the present invention is a
eukaryotic expression system, i.e. an expression system that
comprises a eukaryotic cell. In particular, within the meaning of
the present invention, said eukaryotic cell is modified as
described elsewhere herein.
DETAILED DESCRIPTION OF THE INVENTION
[0203] The present invention is now described in more detail by
preferred embodiments and examples, which are however presented for
illustrative purpose only and shall not be understood as limiting
the scope of the present invention in any way.
[0204] Although expression systems for expressing proteins are
widely known in prior art, these expression systems often exhibit
disadvantages, for instance with regard to the yield of the
expression product, or with regard to the expression system
itself.
[0205] The present invention is based on the surprising general
finding that an improved constitutive eukaryotic expression system
can be generated by modifying an ssn6-like related gene or
ssn6-like gene ("ssn6-like related/ssn6-like gene") that is present
in a eukaryotic cell, and/or by modifying the expression level of
SSN6-like related protein or SSN6-like protein; or by deleting the
ssn6-like related gene or ssn6-like gene, respectively to the
effect that the resulting modified eukaryotic cell is not able to
provide an SSN6-like related protein or an SSN6-like protein that
exerts its wildtype function and/or wildtype activity in particular
with regard to regulatory sequences such as a promoter that is
regulated by said proteins (such as the LLP promoter); the amount
of SSN6-like related protein or of SSN6-like protein being present
in the modified eukaryotic cell differs (preferably is lower) from
the amount of SSN6-like related protein or of SSN6-like protein
being present in its wildtype form, to the effect that said
proteins are not able to exert their wildtype function and/or
activity in particular with regard to regulatory sequences such as
a promoter that is regulated by said proteins; and/or no SSN6-like
related protein or SSN6-like protein ("SSN6-like related/SSN6-like
protein") is present in the modified cell, again to the effect that
said proteins are not able to exert their wildtype function and/or
activity in particular with regard to regulatory sequences such as
a promoter that is regulated by said proteins. Thus, by preventing
the SSN6-like related protein or SSN6-like protein from exerting
its natural (wildtype) function, which function comprises the
repressive direct and/or indirect interactions with regulatory
sequences such as promoters (e.g. the LLP promoter), the expression
of a gene that is under control of said regulatory sequences (e.g.
LLP promoter) in a suitable expression system is, at least to a
certain degree, not repressed any more. In other other words, the
respective gene being under control of said promoter is
overexpressed compared to its wildtype expression.
[0206] By applying the principle underlying the present invention,
a eukaryotic expression system is provided, which is for instance
improved with regard to expression product yield compared to the
yield that is obtained when applying prior art expression systems.
Moreover, the inventive expression system is improved with regard
to safety aspects: Contrary to certain prior art eukaryotic
expression systems, the present eukaryotic expression system is not
based on the commonly used AOX (alcohol oxidase) promoters, which
are tightly regulated by methanol. The presence of flammable and
toxic methanol represents a major security risk, and also imposes a
risk on alcohol-sensitive expression products or host cells.
Especially in large scale fermentation of recombinant proteins the
use of methanol is a serious safety and environmental risk.
Alternative eukaryotic expression systems that use promoters that
are not regulated by methanol are for instance based on the
constitutive GAP (glyceraldehyde-3-phosphate dehydrogenase)
promoter. However, the resulting yield for instance when applying a
GAP-based expression system leaves much to be desired.
[0207] Within the meaning of the present invention it has been
surprisingly found that the modification of an ssn6-like related
gene or of an ssn6-like gene and/or the modification of the
expression level of SSN6-like related protein or SSN6-like protein
that is present in a eukaryotic cell to the effect that the
resulting modified eukaryotic cell is not able to exert the
wildtype function of SSN6-like related protein or SSN6-like protein
with regard to a regulatory sequence that is regulated by said
SSN6-like related protein or SSN6-like protein, such as a promoter
(e.g. the LLP promoter), results in an enhanced expression of the
gene that is under control of said regulatory sequence.
[0208] Thus, the present invention provides a modified eukaryotic
cell, which is modified compared to its wildtype cell at least in
that it comprises [0209] a modified ssn6-like related gene or a
modified ssn6-like gene, and/or [0210] a modified expression level
of SSN6-like related protein or of SSN6-like protein, or [0211] in
that [0212] an ssn6-like related gene or an ssn6-like gene is
deleted,
[0213] respectively to the effect that [0214] the modified
eukaryotic cell is not able to provide an SSN6-like related protein
or an SSN6-like protein that exerts its wildtype function and/or
wildtype activity, [0215] the amount of SSN6-like related protein
or of SSN6-like protein being present in the modified eukaryotic
cell differs from the amount of SSN6-like related protein or of
SSN6-like protein being present in its wildtype form, and/or [0216]
no SSN6-like related protein or SSN6-like protein is present in the
modified cell.
[0217] In accordance with the ratio underlying the present
invention, the resulting modified eukaryotic cell, when compared to
its wildtype cell, exhibits different SSN6-like related protein or
SSN6-like protein activity and/or function, preferably with respect
to its activity and/or function in regulating the expression of a
gene.
[0218] Whenever the term "wildtype function" is used in connection
with SSN6-like related protein or SSN6-like protein, or the
respective genes encoding said proteins, the function with regard
to a regulatory sequence that is regulated by SSN6-like related
protein or SSN6-like protein, such as a promoter (preferably the
LLP promoter), is referred to.
[0219] As it is known to a person skilled in the art, the
expression of a gene is, amongst others, regulated by a complex
interplay of various factors that contribute to the regulation of
the expression of (a) certain gene(s), such as promoters,
enhancers, terminators, silencers, transcription factors,
IRES-sequences, ribosome-binding sites, and sequences stabilizing
or destabilizing the mRNA by secondary structures or enhancers.
[0220] Thus, in a preferred embodiment, the resulting modified
eukaryotic cell exhibits different SSN6-like related protein or
SSN6-like protein activity and/or function with regard to any
promoter that is regulated by said proteins, preferably the LLP
promoter. Hence, it is a preferred embodiment of the present
invention that the modified eukaryotic cell, when compared to its
wildtype cell, exhibits different SSN6-like related protein or
SSN6-like protein activity and/or function with regard to
regulating the expression of genes that are under control of the
LLP promoter. The activity and/or function of said proteins
(SSN6-like related and SSN6-like) results in that the genes that
are under control of said promoter (LLP-promoter) are
overexpressed.
[0221] The different activity and/or function of the SSN6-like
related protein or SSN6-like protein can be any different activity
and/or function that results in an overexpression of the respective
gene that is under control of the affected regulatory sequence. For
example an SSN6-like related protein or SSN6-like protein might be
altered in its activity and/or function by "gain of function" or
"loss of function" modifications. If several copies of different
modified versions or wild-type SSN6-like related protein or
SSN6-like protein are present within a cell at the same time,
certain SSN6-like related proteins or SSN6-like proteins might be
dominant regarding their activity and/or function.
"Loss-of-function" mutants comprise a mutation that results in
reduced or abolished protein function. "Gain-of-function" mutants
comprise a mutation that results in an abnormal activity on a
protein. Preferably, it is a reduced activity and/or function of
the SSN6-like related protein or of the SSN6-like protein. It can
also be that there is no SSN6-like related protein or SSN6-like
protein at all present in the modified eukaryotic cell, or that
there is no SSN6-like related protein or SSN6-like protein within
the detection limit of commonly know methods such as for example
RT-PCR, qPCR or other PCR-related techniques, western blotting,
ELISA, or other immunological detection assays, reporter gene
assays, mass spectrometric detection methods, chip assays detecting
proteins or nucleic acids, etc.
[0222] In order to arrive at a modified eukaryotic cell that
exhibits different, preferably reduced, more preferably essentially
no, SSN6-like related protein or SSN6-like protein function and/or
activity, the ssn6-like related gene or ssn6-like gene can be
modified. This modification can for instance have taken place in a
coding sequence encoding the respective protein, and/or in a
respective regulatory sequence. A regulatory sequence that can be
modified is for instance a promoter, enhancer, terminator,
silencer, IRES-sequence, ribosome-binding site, and sequence
stabilizing or destabilizing the mRNA by secondary structures or
enhancer.
[0223] According to the present invention, the above modifications
on the regulatory and/or coding regions of the genes encoding
SSN6-like related protein and/or SSN6-like protein have an effect
on the expression level of the SSN6-like related protein or
SSN6-like protein in that the modified eukaryotic cell is not able
to express said proteins, or that the amount of said proteins is
different, preferably reduced, when compared with its wildtype
form. The above modifications can also have an effect on the
function and/or activity of SSN6-like related protein or SSN6-like
protein in that said proteins do not exert their wildtype function
and/or activity. It is also possible to arrive at a modified
eukaryotic cell exhibiting impaired SSN6-like related/SSN6-like
protein as defined above by impairing the transcription or
translation of the gene encoding SSN6-like related/SSN6-like
protein. Transcription and translation can be impaired by any
suitable method that is known to a person skilled in the art, for
instance translation can be impaired by hybridizing a complementary
sequence or a partial sequence thereof to the mRNA transcript. The
partial sequence of the complementary sequence is for example
selected from siRNA, anti-sense RNA, ribozyme, and triplex RNA or
DNA, etc.
[0224] Irrespective of whether the regulatory and/or coding regions
of the ssn6-like related/ssn6-like gene is modified, or whether the
transcription and/or translation of the gene encoding SSN6-like
related/SSN6-like protein is impaired, as a result, the modified
eukaryotic cell is not able to provide an SSN6-like related protein
or an SSN6-like protein that exerts its wildtype function and/or
wildtype activity, the amount of SSN6-like related protein or of
SSN6-like protein being present in the modified eukaryotic cell
differs from the amount of SSN6-like related protein or of
SSN6-like protein being present in its wildtype form, and/or no
SSN6-like related protein or SSN6-like protein is present in the
modified cell, as it is disclosed elsewhere herein.
[0225] The ssn6-like related/ssn6-like gene and/or the expression
level of the SSN6-like related/SSN6-like protein can be modified by
any suitable method that is known to a person skilled in the art.
For instance, the ssn6-like related/ssn6-like gene and/or the
expression level of the SSN6-like related/SSN6-like protein is
modified by [0226] introduction of one or more point mutations,
e.g. substitution, insertion or deletion of a single or more
nucleotides in the polynucleotide sequence of the ssn6-like related
gene or of the ssn6-like gene, [0227] partial or complete deletion
of the polynucleotide sequence of the ssn6-like related gene or of
the ssn6-like gene, and/or [0228] partial or complete replacement
of the polynucleotide sequence of the ssn6-like related gene or of
the ssn6-like gene by a different, e.g. heterologous, nucleotide
sequence.
[0229] In a preferred embodiment, the total activity, function,
preferably gene regulatory function, and/or amount of SSN6-like
related protein or SSN6-like protein of the modified eukaryotic
cell is at most 20%, preferably at most 15%, more preferably at
most 10%, even more preferably at most 5% of the activity, function
and/or amount of SSN6-like related protein or SSN6-like protein of
its wildtype form, and most preferably there is no activity and/or
no function of the SSN6-like related protein or SSN6-like protein
of the modified eukaryotic cell at all.
[0230] Within the meaning of the present invention, the term "total
activity, function, and/or amount of SSN6-like related protein or
SSN6-like protein of the modified eukaryotic cell" defines the
overall activity, function, and/or amount of SSN6-like related
protein or SSN6-like protein of a sample of modified eukaryotic
cells. For instance, in order to assess whether the activity,
function, and/or amount of SSN6-like related protein or SSN6-like
protein of the modified eukaryotic cells differs from the
corresponding feature of the wildtype cells, the total amount of
SSN6-like protein being present in a sample of a certain number of
wildtype cells is compared with the total amount of SSN6-like
protein being present in a sample of essentially the same number of
modified eukaryotic cells.
[0231] Within the meaning of the present invention, the modified
eukaryotic cell can be any suitable cell, i.e. any suitable
eukaryotic cell that comprises, in its wildtype form, a
polynucleotide sequence encoding SSN6-like related/SSN6-like
protein. In a preferred embodiment, said suitable cell additionally
comprises a nucleotide sequence representing the LLP promoter. In
order to determine whether a eukaryotic cell comprises the above
defined sequences, any suitable method that is known to a person
skilled in the art can be used, for instance techniques such as
northern blotting, real time PCR, reverse transcriptase
quantitative PCR (RT-qPCR), or microarray hybridization
experiments. Microarray hybridization experiments can be used to
quantify the expression of hundreds to thousands of genes at the
same time. Suitable protocols can for instance be found in Sambrook
et al.
[0232] In a preferred embodiment of the present invention, the
modified eukaryotic cell is a fungal cell, preferably a yeast cell,
more preferably selected from the group consisting of Saccharomyces
species (e.g., Saccharomyces cerevisiae), Kluyveromyces species
(e.g., Kluyveromyces lactis), Torulaspora species, Yarrowia species
(e.g., Yarrowia lipolitica), Schizosaccharomyces species (e.g.,
Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris
or Pichia methanolica), Hansenula species (e.g., Hansenula
polymorpha), Torulopsis species, Komagataella species, Candida
species (e.g., Candida boidinii), and Karwinskia species, even more
preferably the eukaryotic cell is a Pichia cell, most preferably a
Pichia pastoris cell according to current classification.
[0233] In a further preferred embodiment, the modified eukaryotic
cell is a P. Pastoris cell, preferably a cell of a strain selected
from the group consisting of NRRL Y-11430, CBS704, GS115, and
KM71.
[0234] As disclosed herein, the modified eukaryotic cell according
to the present invention can comprise a modified ssn6-like related
gene or a modified ssn6-like gene. Thus, in a further embodiment,
the modified eukaryotic cell comprises a polynucleotide sequence
which represents a modification of SEQ ID NO: 1.
[0235] As explained elsewhere herein, it has been found that a
modification of ssn6-like related/ssn6-like gene according to the
present invention, i.e. a modification that results in a modified
expression of SSN6-like related/SSN6-like protein, results in an
overexpression of genes that are under control of a promoter that
is regulated by SSN6-like related/SSN6-like protein, such as the
LLP promoter. Thus, the present invention also refers to a
polynucleotide sequence comprising [0236] a modified ssn6-like
related gene or a modified ssn6-like gene comprising a coding
sequence and a regulatory sequence,
[0237] wherein, if said polynucleotide sequence is expressed in a
suitable expression system, [0238] essentially no SSN6-like related
protein or SSN6-like protein is expressed, or [0239] SSN6-like
related protein or SSN6-like protein is expressed that does not
exert its wildtype function and/or wildtype activity, preferably
with respect to its activity and/or function in regulating gene
expression, more preferably in regulating the expression of genes
that are under control of the LLP promoter, and/or [0240] an amount
of SSN6-like related protein or SSN6-like protein is expressed that
differs from the amount of SSN6-like related protein or of
SSN6-like protein that is expressed when the corresponding wildtype
polynucleotide sequence is expressed under comparable or the same
conditions.
[0241] In a preferred embodiment, the amount of SSN6-like related
protein or SSN6-like protein that is expressed differs from the
amount of SSN6-like related protein or of SSN6-like protein that is
expressed when the corresponding wildtype polynucleotide sequence
is expressed under comparable or the same conditions in that it is
reduced.
[0242] In a further embodiment, the above polynucleotide consists
of said modified ssn6-like/ssn6 gene.
[0243] In a preferred embodiment, the amount of SSN6-like
related/SSN6-like protein is reduced, or there is no SSN6-like
related protein or SSN6-like protein present at all, compared to
the amount of SSN6-like related/SSN6-like protein that is expressed
when the corresponding wildtype polynucleotide sequence is
expressed under comparable or the same conditions.
[0244] The modifications can be carried out in the regulatory
sequence (regulatory region) and/or in the coding sequence (coding
region) of the ssn6-like related/ssn6-like gene. The regulatory
sequence can be any regulatory sequence, preferably the regulatory
sequence is selected from the group comprising or consisting of
promoters, enhancers, terminators, silencers, IRES-sequences,
ribosome-binding sites, and sequences stabilizing or destabilizing
the mRNA by secondary structures, more preferably said regulatory
sequence is selected from the group consisting of promoters,
enhancers, and terminators.
[0245] The ssn6-like related/ssn6-like gene has been modified as
disclosed elsewhere herein. In a preferred embodiment, the
polynucleotide comprises, preferably consists of, modifications of
SEQ ID NO: 1. An example of a modified ssn6-like gene is depicted
in FIG. 1 G (SEQ ID NO: 7). Thus, in a further preferred
embodiment, the polynucleotide according to the present invention
comprises, preferably consists of, SEQ ID NO: 7.
[0246] As it is known to a person skilled in the art, it is
possible to propagate and/or express a certain nucleotide sequence
by inserting this sequence into a vector, which is then, in turn,
introduced in a host cell such as a bacterium, with this bacterium
preferably being Escherichia coli (E. coli) or a eukaryotic cell
for instance as disclosed elsewhere herein. Thus, the present
invention also refers to a vector comprising the polynucleotide
sequence comprising the modified ssn6-like related/ssn6-like gene,
as well as to a host cell comprising said vector or parts of said
vector.
[0247] Depending on the respective intended use, said vector
contains specific structural elements. If it is for instance
intended to propagate the nucleotide sequence, then the
corresponding vector has to contain an ori.
[0248] The present invention also refers to a polypeptide encoded
by the polynucleotide sequence according to the present invention.
If the polynucleotide sequence according to the present invention,
comprising a modified ssn6-like related/ssn6-like gene as disclosed
above, is expressed in a suitable expression system, the expression
of a gene product, e.g. of a polypeptide or protein, depends on the
modifications that have been carried out. For instance, if the
modification results in a shift of the complete reading frame, in
all likelihood no gene product can be expressed. It can however
also be that the modification only results in a truncated
("shortened") nucleotide sequence, which in turn results in the
expression of a truncated gene product which may also contain a
short heterologous amino acid sequence at its C-terminus which is
coded by the inserted heterologous nucleotide sequence (see FIG. 2
B, SEQ ID NO: 9, with 7 heterologous C-terminal amino acids
(EWYLQLR, underlined in FIG. 2 B; SEQ ID NO: 139), as compared to
the non-mutated sequence in FIG. 2 A, SEQ ID NO: 8).
[0249] In the present invention it has been surprisingly found that
by partially or completely inhibiting the wildtype function and/or
activity of SSN6-like related/SSN6-like protein, or by effecting
that SSN6-like related/SSN6-like protein is essentially not
present, respectively in a cell that in its wildtype form comprises
a gene encoding said protein, a gene being under the control of a
promoter that is repressed in the presence of SSN6-like
related/SSN6-like protein can be overexpressed in a suitable
expression system.
[0250] Thus, the present invention further refers to an expression
vector comprising a promoter, wherein said promoter is
characterized in that it is repressed in the presence of SSN6-like
protein or SSN6-like related protein if said expression vector is
introduced into a suitable expression system, e.g. into a
eukaryotic cell as defined elsewhere herein. By using this
expression vector, a gene under control of a promoter that is
repressed in the presence of SSN6-like related/SSN6-like protein
can be overexpressed in a suitable expression system. In order to
determine whether a promoter is repressible by SSN6-like
related/SSN6-like protein, any suitable method that is known to a
person skilled in the art can be used. For instance, expression
vectors can be designed that comprise the promoter to be tested,
and an indicator gene (also called reporter-gene) such as
luciferase that is under control of the promoter to be tested. If,
in the presence of SSN6-like related/SSN6-like protein, the
expression of the gene product is reduced, then the candidate
promoter is repressed in the presence of SSN6-like
related/SSN6-like. Determination of the reduced candidate promoter
activity for example can be measured by quantifying the indicator
gene product or its activity, for example luciferase, by methods
known in the art. There are a number of assays know in the art,
which can be used to determine promoter-activity or measure
interaction of proteins with promoter-sequences and which thereby
are suitable to test whether the activity of a certain promoter is
influenced by an SSN6-like or SSN6-like related protein. Examples
of such assays are reporter gene assays, electrophoretic mobility
shift assays (EMSA, gel shift assays), double-stranded DNA
pull-down assay, chromatin immune-precipitation assays, and DNase
foot printing assays, etc.
[0251] In a preferred embodiment of the present invention, the
expression vector comprises one or more gene(s) of interest that
are under control of said promoter, i.e. the promoter that is
repressed in the presence of SSN6-like related/SSN6-like protein.
By applying suitable protocols that are known to a skilled person,
such as a protocol as disclosed above, a person skilled in the art
is able to assess whether a promoter is repressed in the presence
of SSN6-like related/SSN6-like protein, or not. In a preferred
embodiment of the present invention, the promoter being comprised
in the expression vector and controlling the gene(s) of interest is
an LLP promoter or a modified version thereof. Preferably, the LLP
promoter comprises, preferably consists of, the polynucleotide
sequence depicted in SEQ ID NO: 2 or 12. The term "modified
version" of the LLP promoter defines that said modified LLP
promoter still exhibits the function of an LLP promoter in terms of
regulating the expression of the gene that is under its control. It
is further possible that the promoter is an LLP promoter, and that
LLP is encoded or is not encoded by the polynucleotide sequence of
the expression vector. In either case, and also in case the
promoter is not LLP promoter but a different promoter that is
repressed in the presence of SSN6-like related/SSN6-like protein,
the gene of interest preferably is selected from the group
consisting of genes encoding enzymes, antibodies or fragments
thereof, hormones, structural proteins, and protein-antigens being
present in vaccines.
[0252] Preferably, the expression vector further comprises features
selected from any one of the following: [0253] (i) a selection
marker; [0254] (ii) a purification marker; [0255] (iii) a signal
sequence, preferably an alpha-factor secreting signal sequence,
more preferably the MFalpha pre-pro signal sequence, even more
preferably a signal sequence comprising or consisting of,
preferably consisting of, SEQ ID NO: 14 or SEQ ID NO: 21; [0256]
(iv) an origin of replication; and/or [0257] (v) a nucleotide
sequence for targeted and/or random integration into the genome of
a host cell.
[0258] The expression vector can be comprised in a host cell as
described herein, preferably in a bacterium such as E. coli, for
instance for propagation purposes.
[0259] The expression vector can also be introduced into the
modified eukaryotic cell according to the present invention,
resulting in the introduction of the expression vector's expression
cassette into the modified eukaryotic cell's genome. The
introduction of the expression vector into the host cell can be
carried out according to any suitable method that is known to a
skilled person, for instance by electroporation. In addition,
transient expression, i.e. the expression vector is not integrated
into the genome, can also be considered.
[0260] The present invention also refers to a modified eukaryotic
cell comprising the expression vector as defined herein.
[0261] The introduction of the expression cassette of the
expression vector into the modified eukaryotic cell is also denoted
as "recombination" and can be a targeted, i.e. homologous,
recombination, or a random recombination. If a random recombination
takes place, the expression cassette integrates in such a way into
the genome of the modified eukaryotic cell that the nucleotide
sequence encoding LLP protein present in the modified eukaryotic
cell is not affected. In other words, the modified eukaryotic cell
expresses not only the gene of interest, but also the LLP protein.
In this case, the amount of LLP protein being present in the
modified cell, the supernatant of the modified cell or in
subfractions thereof can be used as an indicator of the purity of
the expression product of the gene of interest, i.e. the protein of
interest, for example during purification of the protein of
interest. This situation, where the LLP protein is expressed in
addition to the gene(s) of interest, is referred to as "Option 2"
(FIG. 3 D).
[0262] Thus, the present invention also refers to a eukaryotic cell
comprising [0263] a modified ssn6-like related gene or a modified
ssn6-like gene, wherein said gene has inserted a foreign nucleotide
sequence, to the effect that [0264] (i) the eukaryotic cell is not
able to provide an SSN6-like related protein or an SSN6-like
protein that exerts its wildtype function and/or wildtype activity,
preferably with respect to its activity and/or function in
regulating the LLP promoter, [0265] (ii) the amount of SSN6-like
related protein or of SSN6-like protein being present in the
eukaryotic cell differs from the amount of SSN6-like related
protein or of SSN6-like protein being present in its wildtype form,
preferably the amount of the SSN6-like related protein or of the
SSN6-like protein is reduced, and/or [0266] (iii) no SSN6-like
related protein or SSN6-like protein is present in the eukaryotic
cell, [0267] a gene of interest under control of the LLP promoter,
and [0268] an llp gene,
[0269] preferably wherein said eukaryotic cell exhibits reduced
SSN6-like related protein or SSN6-like protein activity and/or
function, or no SSN6-like related protein or SSN6-like protein
activity and/or function at all.
[0270] If a targeted, i.e. homologous, recombination takes place,
and if additionally the promoter of the expression cassette is an
LLP promoter or a modification thereof, then the expression
cassette is inserted in such a way that it replaces (partially or
completely) the nucleotide sequence of the gene encoding the LLP
protein. In this case, there is no expression of LLP. This
situation, where only the gene(s) of interest, but no LLP protein,
is expressed, is referred to as "Option 1".
[0271] Thus, the present invention also refers to a eukaryotic cell
comprising [0272] a modified ssn6-like related gene or a modified
ssn6-like gene, wherein said gene has inserted a foreign nucleotide
sequence, to the effect that [0273] (i) the eukaryotic cell is not
able to provide an SSN6-like related protein or an SSN6-like
protein that exerts its wildtype function and/or wildtype activity,
preferably with respect to its activity and/or function in
regulating the LLP promoter, [0274] (ii) the amount of SSN6-like
related protein or of SSN6-like protein being present in the
eukaryotic cell differs from the amount of SSN6-like related
protein or of SSN6-like protein being present in its wildtype form,
preferably the amount of the SSN6-like related protein or of the
SSN6-like protein is reduced, and/or [0275] (iii) no SSN6-like
related protein or SSN6-like protein is present in the eukaryotic
cell,
[0276] and [0277] a gene of interest that replaces a part or all of
the coding region of the gene encoding the LLP protein, to the
effect that essentially no LLP protein is present in the eukaryotic
cell, and that is under control of the LLP promoter,
[0278] preferably wherein said eukaryotic cell exhibits reduced
SSN6-like related protein or SSN6-like protein activity and/or
function, or no SSN6-like related protein or SSN6-like-protein
activity and/or function at all.
[0279] The present invention further refers to an expression
system, comprising [0280] a) the modified eukaryotic cell as
defined herein; [0281] b) the expression vector as defined herein,
wherein said expression vector can also be present in linearized
form and/or at least parts of the vector being integrated into the
genome of the modified eukaryotic cell.
[0282] In a further preferred embodiment of the present invention,
the modified eukaryotic cell additionally comprises a promoter,
wherein said promoter is repressed in the presence of SSN6 protein
or SSN6-like protein. Preferably, said promoter is an LLP promoter,
preferably an LLP promoter comprising, preferably consisting of,
SEQ ID NO: 2 or 12, or modified versions thereof, said modified
versions being characterized in that they still exhibit essentially
the same promoter function as a wildtype LLP promoter.
[0283] In a further embodiment, the modified eukaryotic cell
additionally comprises (a) gene(s) of interest, preferably selected
from the group consisting of genes encoding enzymes, antibodies or
fragments thereof, hormones, structural proteins, and
protein-antigens being present in vaccines, wherein said gene(s) of
interest is (are) under control of a promoter being repressed in
the presence of SSN6-like protein or SSN6-like related protein.
Preferably, this promoter is an LLP promoter, preferably an LLP
promoter comprising, preferably consisting of, SEQ ID NO: 2 or 12,
or modified versions thereof, said modified versions being
characterized in that they still exhibit essentially the same
promoter function as a wildtype promoter.
[0284] As already explained above, it is possible that in said
expression system the LLP gene is not expressed in addition to the
gene(s) of interest (option 1), and it is also possible that the
LLP gene is expressed in addition to the gene(s) of interest
(option 2). In the first case (option 1), the LLP promoter only
controls the expression of (a) gene(s) of interest, and in the
latter case (option 2), the LLP promoter controls the expression of
(a) gene(s) of interest (which are different from genes encoding
LLP), and LLP.
[0285] The present invention further refers to the use of the
modified eukaryotic cell, the modified polynucleotide sequence, the
expression vector, the host cell, or an expression system as
defined herein in a method of expressing, preferably
overexpressing, gene(s) of interest.
[0286] As disclosed above, the amount of LLP protein being present
in a composition comprising the protein of interest is an indicator
of the purity of said composition regarding said protein of
interest. Thus, the present invention also refers to a method for
determining the purity of a composition comprising the expression
product of a gene of interest, comprising the following steps:
[0287] (a) expressing gene(s) of interest by using the modified
eukaryotic cell and the expression vector, respectively according
to the present invention, wherein [0288] (a1) the modified
eukaryotic cell comprises a gene encoding LLP protein under control
of an LLP promoter, and wherein [0289] (a2) the expression vector
comprises one or more gene(s) of interest under control of an LLP
promoter or a modified LLP promoter, wherein said gene of interest
does not encode LLP, [0290] thereby obtaining a composition
comprising the expression product of the gene(s) of interest, i.e.
the protein(s) of interest, and the LLP protein; [0291] (b)
determining the amount of the expression product of the gene(s) of
interest, i.e. the amount of the protein(s) of interest, and the
amount of LLP protein being present in the composition obtained in
step (a), wherein the amount of LLP protein compared to the amount
of expression product of the gene(s) of interest, i.e. of the
protein(s) of interest, is indicative for the purity of the
composition obtained in step (a); and, optionally, [0292] (c)
subjecting the composition of step (a) to one or more downstream
purification step(s), followed by step b), i.e. determining the
amount of the expression product of the gene(s) of interest, i.e.
the protein(s) of interest, and the amount of LLP protein being
present in the composition obtained after having carried out said
downstream purification step.
[0293] In order to obtain a composition that comprises LLP, in
addition to the gene(s) of interest, the integration of the
expression cassette of the expression vector represents a random
integration into the genome of the modified eukaryotic cell.
[0294] The composition comprising the protein(s) of interest and
the LLP protein can be any composition that arises in the course of
the process of producing the protein(s) of interest. Accordingly,
such a composition can for instance be a supernatant of the cell
culture used in expressing the protein(s) of interest
[0295] The present invention further refers to a method of
expressing one or more gene(s) of interest in a eukaryotic cell
comprising an ssn6-like related gene or an ssn6-like gene, wherein
the translation of the mRNA transcript of the ssn6-like related
gene or the ssn6-like gene is prevented by hybridizing a
complementary sequence or a partial sequence thereof to the mRNA
transcript. In a preferred embodiment, the partial sequence of the
complementary sequence is a siRNA, anti-sense RNA, a ribozyme, or
triplex RNA or DNA.
[0296] In a further embodiment, the present invention refers to a
method of expressing one or more gene(s) of interest in a
eukaryotic cell comprising an SSN6-like expression cassette or
SSN6-like related expression cassette, wherein the SSN6-like
protein or the SSN6-like related protein in said eukaryotic cell is
modified or inhibited in its function and/or activity. Preferably,
the SSN6-like protein or SSN6-like related protein of said
eukaryotic cell is modified or inhibited in its function and/or
activity in regulating the LLP promoter, more preferably the
SSN6-like protein or SSN6-like related protein exhibits reduced
SSN6-like related protein or SSN6-like protein activity and/or
function, or no SSN6-like related protein or SSN6-like protein
activity and/or function.
DESCRIPTION OF THE FIGURES
[0297] FIG. 1: Polynucleotide sequences
[0298] FIG. 1 shows various polynucleotide sequences.
[0299] FIG. 1 A shows the nucleotide sequence of the coding region
of the ssn6-like gene of P. Pastoris (SEQ ID. NO: 1).
[0300] FIG. 1 B shows the nucleotide sequence of the LLP promoter
of P. pastoris, 605 bp 3' from the ATG start codon of LLP (SEQ ID
NO: 2).
[0301] FIG. 1 C shows the nucleotide sequence of the signal
sequence of the LLP protein of P. Pastoris (SEQ ID NO: 3).
[0302] FIG. 1 D shows the nucleotide sequence of the terminator
sequence of llp gene of P. Pastoris (SEQ ID NO: 4).
[0303] FIG. 1 E shows the nucleotide sequence of the SSN6-like
promoter of P. Pastoris, 1000 bp 3' from the ATG start codon of
SSN6-like (SEQ ID NO: 5).
[0304] FIG. 1 F shows the nucleotide sequence of the terminator
sequence of ssn6-like gene of P. Pastoris (SEQ ID NO: 6).
[0305] FIG. 1 G shows the nucleotide sequence of the SSN6-like
modified DNA of P. Pastoris (from the ATG start to the TAA stop
codon) as present in P. Pastoris strain SSS1. This results in a
ssn6-like coding region with an internal insertation of a
heterologous nucleotide sequence, which disrupts the coding
sequence of ssn6-like coding sequence (SEQ ID NO: 7).
[0306] FIG. 1 H shows the nucleotide sequence of the Kozak start
sequence (SEQ ID NO: 11).
[0307] FIG. 1 I shows the nucleotide sequence of the LLP promoter,
1000 bp 3' from the ATG start codon of LLP (SEQ ID NO: 12).
[0308] FIG. 1 J shows the amino acid sequence of the SSN6-like
protein (SEQ ID NO: 13)
[0309] FIG. 1 K shows MFalpha pre-pro signal sequence: FIG. 1 K (A)
shows said signal sequence without EAEA repeat (SEQ ID NO: 14), and
FIG. 1 K (B) shows said signal sequence with EAEA repeat (SEQ ID
NO: 21).
[0310] FIG. 1 L shows the codon optimized DNA sequence of encoding
a single chain antibody fragment DLX521 (SEQ ID NO: 15).
[0311] FIG. 1 M shows the coding region of the DNA sequence of the
llp-gene including the signal sequence (SEQ ID NO: 16).
[0312] FIG. 1 N shows the codon optimized nucleotide sequence of
the human Growth Hormone (hGH) as used in the examples of this
application (SEQ ID NO: 17)
[0313] FIG. 1 O shows the nucleotide sequence of the human serum
albumin (HSA) as used in the examples of this application (SEQ ID
NO: 18).
[0314] FIG. 1 P shows the nucleotide sequence of the penicillin V
amidase (PVA) as used in the examples of this application (SEQ ID
NO: 19).
[0315] FIG. 1 Q shows the nucleotide sequence of the vector pGAPk
used for generating the SSS1 yeast cell line (plasmid without a GOI
and with a Geneticin resistance marker) (SEQ ID NO: 20)
[0316] FIG. 1 R shows the sequence of a pLLP vector containing a
Geneticin resistance marker (pLLPk) corresponding to SEQ ID NO:
22.
[0317] FIG. 1 S shows SEQ ID NO: 23, which is part of the
nucleotide sequence of chromosome 1 of the genomic sequence of
yeast strain YJK_PVA_021 after random integration of a PVA- and a
Zeocin-expression cassette into the coding region of ssn6-like
(reverse-complement sequence of ssn6-like) at position 807,480 of
chromosome 1 of the reference strain Pichia pastoris CBS 7435. The
ssn6-like sequence is underlined, the start codon is shown in bold
and double-underlined (ATG.fwdarw.reverse-complement.fwdarw.CAT).
Shown is the interrupted ssn6-like coding sequence including 10
nucleotides flanking 5' and 3' to the ssn6-like coding sequence.
The sequence shown is part of the Illumina-sequenced genome of
strain YJK_PVA_021 (SEQ ID NOs: 67-115) and the sequence of SEQ ID
NO: 23 is part of the genomic sequence SEQ ID NO: 103 obtained by
Illumina Inc. Sequences of oligo2395 (reverse-complement sequence
of oligo2395) and oligo2398 are labeled in grey.
[0318] FIG. 1 T shows the LLP signal sequence fused to the 3' end
of HSA coding sequence replacing the native HSA-signal sequence
resulting in the sequence according to SEQ ID NO: 121.
[0319] FIG. 1 U shows SEQ ID NO: 129, which is the shortened
LLP-promoter sequence delta29 corresponding to a 576 bases length
of the shortened LLP-promoter.
[0320] FIG. 1 V shows SEQ ID NO: 130, which is the shortened
LLP-promoter sequence delta93 corresponding to a 512 bases length
of the shortened LLP-promoter.
[0321] FIG. 1 W shows SEQ ID NO: 131, which is the shortened
LLP-promoter sequence delta133 corresponding to a 472 bases length
of the shortened LLP-promoter.
[0322] FIG. 1 X shows SEQ ID NO: 132, which is the shortened
LLP-promoter sequence delta201 corresponding to a 404 bases length
of the shortened LLP-promoter.
[0323] FIG. 1 Y shows SEQ ID NO: 133, which is the shortened
LLP-promoter sequence delta233 corresponding to a 372 bases length
of the shortened LLP-promoter.
[0324] FIG. 1 Z shows SEQ ID NO: 134, which is the shortened
LLP-promoter sequence delta300 corresponding to a 305 bases length
of the shortened LLP-promoter.
[0325] FIG. 2: Amino acid sequences
[0326] FIG. 2 shows various amino acid sequences.
[0327] FIG. 2 A shows the amino acid sequence of the SSN6-like
protein of P. Pastoris (SEQ ID NO: 8).
[0328] FIG. 2 B shows the amino acid sequence of the SSN6-like
modified protein of P. Pastoris YJK_PVA_021 (SEQ ID NO: 9). The
underlined 7 amino acids represent heterologous, non-SSN6-like
amino acids originating from the vector used to inactivate the
SSN6-like protein.
[0329] FIG. 2 C shows the amino acid sequence of the LLP protein
including LLP signal sequence (SEQ ID NO: 10).
[0330] FIG. 2 D shows the amino acid sequence of the LLP signal
sequence of p. pastoris (SEQ ID NO: 140).
[0331] FIG. 3: Mechanism of super-secretor Pichia pastoris
(SSS1)
[0332] wt=wild-type, ssn6-like=ssn6-like gene, LLP=LLP coding
sequence, PI=LLP-promoter, LLP-prot.=LLP-protein, Pg=GAP-promoter,
AR=antibiotic resistance, GOI=coding sequence of gene of interest,
GOI-prot.=gene of interest protein
[0333] FIG. 3 A illustrates a polynucleotide sequence which is a
wt-strain (wild-type strain, NRRL Y-11430) which contains an intact
ssn6-like gene which according to our interpretation suppresses the
promoter (PI) of the lectine like protein (LLP). The ssn6-like gene
is on the reverse strand of chromosome 1 (coding sequence is from
806379-808244) and the LLP gene is on the forward strand of
chromosome 1 (coding sequence is from 2492530 to 2493945).
[0334] FIG. 3 B illustrates an expression construct with a randomly
integrated Pg/AR sequence into the coding sequence of ssn6-like
gene of strain NRRL Y-11430 was obtained. The resulting strain
contains a disrupted ssn6-like gene (inact. ssn6-like). According
to our interpretation, disruption of the ssn6-like resulting in
inact. ssn6-like removes the suppressing effect of ssn6-like on the
PI-promoter, thereby activating of the PI-promoter finally
resulting in high expression of LLP-protein or of other GOIs (Gene
Of Interest) under control of a LLP-promoter.
[0335] FIG. 3 C shows a gene of interest (GOI) which can be
introduced into the yeast strain of Fig. B by either homologous
recombination thereby replacing the coding region of the LLP-gene
by the coding sequence of the GOI (FIG. 3C, =Option 1), and
resulting in expression of the GOI (GOI-protein), whereas no
LLP-protein is produced.
[0336] FIG. 3 D alternatively shows a gene of interest (GOI) fused
to the LLP-promoter (PI, GOI) which can be introduced into the
yeast strain of Fig. B by random recombination leaving the LLP
intact and inserting the GOI under control of the LLP-promoter (PI)
into a random position of the yeast genome. This results in
concomit expression of the GOI (GOI-prot.) and of LLP
(LLP-prot.).
[0337] FIG. 4: SDS-PAGE analysis
[0338] A codon optimized (done by DNA2.0 Inc., Menelo Park, USA)
DNA sequence encoding the model protein DLX521 (an scFv=single
chain variable Fragment of an antibody) was inserted into a plasmid
with the GAP-promoter (pGAP=pJ905 from DNA2.0 Inc.) between
restriction sites EcoRI and NotI, and a plasmid with the
LLP-promoter (pLLP) between restriction sites EcoRV and NotI. The
pGAP plasmid was linearized with SwaI and transformed in NRRL
Y-11430 and the pLLP plasmid was linearized with AvrII and
transformed in SSS1. For the pGAP plasmid, transformants were PCR
screened and 8 clones with a positive PCR signal were randomly
picked and used for glycerol stock preparation. For the pLLP
plasmid, transformants were PCR screened and 9 clones with a
positive PCR signal were randomly picked and used for glycerol
stock preparation. The glycerol stocks of the 8 pGAP and 9 pLLP
strains were subjected to expression studies at microtiter plate
scale (25.degree. C., 350 rpm, 70 h). The OD (optical density) at
600 nm was comparable at harvest (OD was determined at harvest to
check for biomass variability). Subsequently, 15 .mu.L of the
samples (supernatant mixed with NuPAGE.RTM. LDS sample buffer incl.
2-mercaptoethanol according to manufacturers (Invitrogen/Life
Technologies) instructions) were loaded onto the gel.
[0339] The SDS-polyacrylamide gel (SDS-PAGE) system used was the
Novex NuPAGE.RTM. Bis-Tris 4-12% gradient gel (Invitrogen/Life
Technologies) using the MES-buffer system (Invitrogen/Life
Technologieis). The protein molecular weight marker (M) used was
from AppliChem GmbH, Darmstadt, Germany. The used Protein Marker
VI, prestained (AppliChem), shows the indicated approximate
molecular weights in kilo Dalton (kDa) of the marker proteins if
separated in a 10% SDS polyarcrylamide gel (SDS-PAGE) using
Bis-Tris 10% MES buffer according the the manufacturer AppliChem.
Loading volume was 15 .mu.l yeast culture supernatant/lane. All
pGAP- and pLLP-strains were pre-tested by PCR for scFv. Only
scFv-positive strains were subsequently tested for expression of
scFv protein. The table below the SDS PAGE gel lists the protein
bands at the molecular weight positions of LLP and scFV, the
intensity of the protein band (++ strong band, + clear band, o
faint band, - no band) as well as the Option according to FIG. 1 to
which this yeast clone belongs (Option 1: only GOI is expressed, no
LLP; Option 2: GOI and LLP are expressed) [0340] M: Protein Marker
VI prestained (AppliChem) [0341] Lane 1 scFv control strain that
did not grow very well in deep well plates [0342] Lanes 2-9: strain
YJK_PVA_021, expressing low amounts of PVA under control of the
GAP-promoter (faint band of scFV protein can be seen in lanes 2-9)
[0343] Lanes 10-18: strain PP_ESBA521_010 expressing scFv protein
under control of the LLP-promoter. [0344] Lanes 10 to 14: strong
protein band at the molecular weight of scFv (see .rarw.FscFv)
(".rarw." indicates the protein band corresponding to the scFv
protein) no protein band at the molecular weight of LLP (see
.rarw.FLLP), example of Option 1 (only expression of GOI) [0345]
Lanes 15 and 16: strong protein band at molecular weight of LLP
(see .rarw.FLLP) (".rarw." indicates the protein band corresponding
to the LLP protein) and clear band of scFv at molecular weight of
scFv (see .rarw.FscFv), example of Option 2 (parallel expression of
GOI and of LLP) [0346] Lane 17: clear protein band at molecular
weight of LLP (see .rarw.FLLP) and strong protein band of scFv at
molecular weight of scFv (see .rarw.FscFv), example of Option 2
[0347] Lane 18: no protein band at molecular weight of LLP (see
.rarw.FLLP) and strong protein band of scFv at molecular weight of
scFv (see .rarw.FscFv), example of Option 1
[0348] The high molecular weight protein band in the gel at the
position of about the molecular weight marker 125 kDa represents a
dimer of the LLP-protein. The identity of the LLP-protein was
proven by the following experiments and evidences. First the
LLP-band was cut out of the gel and the C-terminal amino acid
sequence was determined by peptide sequencing using standard
methods know in the art. Furthermore the LLP-gel band was analyzed
by mass spectrometry using standard methods known in the art. Both
methods proved that this protein band in the gel indeed is
LLP-protein. Furthermore LLP was treated with an enzyme removing
glycostructures from proteins, namely PNGase F. Treatment of LLP
with PNGase F lowered the molecular weight of the LLP-dimer from
about 125 kDa to about 110 kDa. Furthermore it is known from the
literature (IUBMB Live, 2009, 61:252-60), that lectins can form
very stable multimers such as dimers. Therefore we conclude the
high molecular LLP-protein band in our SDS-PAGE gels is a stable
LLP-protein dimer.
[0349] FIG. 5 A: SDS PAGE Analysis
[0350] A codon optimized DNA sequence (SEQ ID NO: 17) encoding the
model protein hGH was inserted into a plasmid with the GAP-promoter
(pGAP) and one plasmid with the LLP-promoter (pLLP). The pGAP
plasmid was transformed in NRRL Y-11430 and the pLLP plasmid in
SSS1. For pGAP, 16 transformants were randomly picked and subjected
to expression studies at microtiter plate scale (25.degree. C., 350
rpm, 70 h). For pLLP, 28 transformants were randomly picked and
subjected to expression studies at microtiter plate scale
(25.degree. C., 350 rpm, 70 h). The OD at 600 nm was comparable at
harvest (OD was determined at harvest to check for biomass
variability). Subsequently, 15 .mu.L of the samples (supernatant
mixed with NuPAGE.RTM. LDS sample buffer incl. 2-mercaptoethanol)
were loaded onto the gel. [0351] M: Protein Marker VI 10-245 from
AppliChem. [0352] pGAP: a protein band corresponding to hGH was
detected by visual inspection of SDS-PAGE gels in 0/16 randomly
picked clones (without prior PCR screening) [0353] pLLP: a protein
band corresponding to hGH was detected by visual inspection of
SDS-PAGE gels in 8/28 randomly picked clones (without prior PCR
screening)
[0354] This result indicates that the pLLP system is superior to
the pGAP system.
[0355] FIG. 5 B: hGH Western Blot
[0356] 15, 20 or 30 .mu.l supernatant samples of deep well plate
cultures of Pichia pastoris cells transformed as described under
FIG. 5 A were directly loaded on SDS-PAGE gels (Novex NuPage 4-12%
Bis-Tris Gels from Invitrogen with MES-running buffer). The
proteins were then transferred to a PVDF membrane. After transfer
and blocking, the membrane was incubated with a solution containing
anti-hGH antibody (Zymed Cat.Nr. 18-0090, dilution: 1:1000) for 2
h. After washing the membrane, a secondary antibody coupled with
alkaline phosphatase (anti-rabbit IgG alkaline-phosphatase
conjugate, Sigma Cat. No. A3687, dilution: 1:16.000) was added for
1 h. After washing the membrane, NBT/BCIP (nitro-blue tetrazolium
chloride/5-bromo-4-chloro-3-indolylphosphate toluidine salt) was
added to detect hGH. [0357] M: Protein Marker VI; [0358] NRRL
Y-11430 Control: wild type strain without hGH; [0359] SSS1 Control:
supersecretor strain without hGH; [0360] pGAP: expression of hGH
under control of GAP-promotor; [0361] pLLP: expression of hGH under
control of the LLP-promoter
[0362] FIG. 6: SDS PAGE Analysis
[0363] Lane 6 contains a sample of SSS1-cells not transfected with
a pLLP construct (negative control).
[0364] SDS-PAGE analysis. A codon optimized DNA sequence encoding
the model protein HSA was inserted into on plasmid with the
GAP-promoter (pGAP) and one plasmid with the LLP-promoter (pLLP).
The pGAP plasmid was transformed in NRRL Y-11430 and the pLLP
plasmid in SSS1. Colony PCR was applied to screen for transformants
which were then subjected to expression studies at microtiter plate
scale (25.degree. C., 350 rpm, 70 h). The OD at 600 nm was
comparable at harvest (OD was determined at harvest to check for
biomass variability). Subsequently, 15 .mu.L of the samples
(supernatant mixed with NuPAGE.RTM. LDS sample buffer incl.
2-mercaptoethanol) were loaded onto the gel. [0365] M: Protein
Marker VI 10-245 from AppliChem. [0366] pGAP: a faint protein band
corresponding to HSA was detected by visual inspection of SDS-PAGE
gels in 2/5 selected clones (lanes 3 and 5) (with prior PCR
screening) [0367] pLLP: a protein band corresponding to HSA was
detected by visual inspection of SDS-PAGE gels in 4/4 selected
clones (with prior PCR screening)
[0368] This result indicates that the pLLP system is superior to
the pGAP system.
[0369] FIG. 7 Comparison of pGAP expression system with pLLP
expression system
[0370] This result indicates that the pLLP system is superior to
the pGAP system.
[0371] 7A:
[0372] Pichia pastoris strains transformed with HSA either under
control of the GAP promoter (pGAP=P. pastoris
glyceraldehyde-3-phosphate dehydrogenase promotor) or under control
of the LLP promoter (pLLP=P. pastoris Lectin Like Protein promotor)
were cultured in a 5 liter fermenter for 144 h at 25.degree. C., pH
6.0 and 30% oxygen, using glycerol in batch growth phase and using
constant glucose feed in main growth stage. Culture supernatant was
analyzed for HSA employing a Human Serum Albumin ELISA (Enzyme
Linked Immuno Sorbent Assay) kit from Cygnus Technologies (Cat. Nr.
F055) according to the manufacturer's instructions. Mixtures of
transformed yeast strains (pGAP) and mixtures of transformed yeast
strains (pLLP) were tested in order to account for expression
differences in individual strains, e.g. to determine the average
expression rate of pGAP- and of pLLP-strains.
[0373] 7B:
[0374] PVA enzyme assay. A codon optimized DNA sequence encoding
PVA (Pleurotus ostreatus--Penicillin V Amidase) was inserted into a
plasmid with the GAP-promoter (pGAP) and into a plasmid with the
LLP-promoter (pLLP). The pGAP plasmid was transformed in NRRL
Y-11430 and the pLLP plasmid in SSS1 yeast cells. For each plasmid,
16 transformants were randomly picked and subjected to expression
studies at microtiter plate scale (25.degree. C., 350 rpm, 70 h).
The PVA titer (g/kg phenoxy acetic acid) and OD at 600 nm were
determined at harvest and used for normalization (g/kg phenoxy
normalized). The diagram shows the mean value.+-.SEM (standard
error of the mean) of PVA activity as measured by
PVA-mediated-conversion of phenoxymethylpenicillin into phenoxy
acetic acid g/kg. The highest individual value in each group is
labeled with an asterix (*). For expression purpose one would
usually choose those strains which do express the highest amount of
PVA-activity (labeled with * in the diagram). In this case the best
strain expressing PVA under control of the pLLP expressed about
2.9-fold more PVA-activity as compared to the best pGAP strain
(pGAP: n=13, max. value was 0.17 g/kg, pLLP: n=15, max. value was
0.49 g/kg). The pGAP vector from DNA2.0 Inc. and the pGAP vector
from Sandoz are almost identical. All Vector elements such as
promoters, terminators, resistance-marker, pUC ori, etc. are the
same in both vectors. Only minor differences regarding the used
restriction enzyme sites and short nucleotide sequences which
connect some of these elements within the vectors and are slightly
different between both vectors.
[0375] FIG. 8: Expression of gene of interest (GOI) in fermentor
yeast cultures
[0376] 8A
[0377] A codon optimized DNA sequence encoding the enzyme PVA
(Penicillin V Amidase) was inserted into a plasmid under the
control of the GAP-promoter (pGAP). The pGAP plasmid was
transformed in NRRL Y-11430 yeast cells (Pichia pastoris), and
cells were grown in a fermentor under standard conditions using
standard media and culturing conditions as know in the art. The
pGAP vector integrated randomly into the SSN6-gene, thereby
activating the LLP-promoter. The supernatant of the fermentation
culture was subsequently treated with the enzyme PNGase F (Peptide
N-Glycosidase F; cleaves asparagine-linked high mannose as well as
hybrid complex oligosaccharides from glycoproteins; New England
Biolabs, Catalogue Number P0704S). Samples of the untreated cell
culture supernatant (lane 1) and of PNGase F-treated cell culture
supernatant (lane 2) were separated in SDS-PAGE. Lane 1 shows the
glycosylated LLP-dimer as a very strong band at about 125 kDa,
whereas the PVA-band is somewhat diffuse at around 72 kDa, probably
because individual molecules of PVA are glycosylated to a different
extend. Lane 3 shows at about 36 kDa the protein band of the added
PNGase F, at about 110 kDa the very strong band of the
deglycosylated LLP-dimer (.rarw.LLP de-glyc.) and at about 52 kDa a
now very clear, distinct band representing deglycosylated PVA
(.rarw.PVA de-glyc.).
[0378] 8B
[0379] A codon optimized DNA sequence encoding a single chain
antibody fragment (scFv), in this case the scFv named DLX521, was
inserted into a plasmid under control of the LLP-promoter (pLLP).
The pLLP plasmid containing a Geneticin resistance was randomly
transformed into YJK_PVA_021 (the already PVA-transformed yeast
cells of FIG. 8 A), and cells were grown in a fermentor under
standard conditions using standard media and culturing conditions
as know in the art. Supernatant of the fermentation was diluted
20-fold and 15 .mu.l were loaded onto SDS-PAGE in the presence of
2-mercaptoethanol. A very strong protein band at the molecular
weight position of the scFv is visible in the gel in lane 1. In
addition a minor band at the position of the LLP-dimer is seen at
about 125 kDa and a strong protein band of PVA is seen at about 72
kDa. No PNGase was added so all proteins are glycosylated. 20-fold
dilution of the cell culture supernatant was done in order to
depict the very high expression rate of the scFv relative to the
LLP. This yeast strain is an example of the "Option 2" as depicted
in FIG. 3 D, except that 2 GOI are expressed (PVA and DLX521). PVA
is under control of the GAP promoter and randomly integrated by
chance into the ssn6-like gene.
[0380] This is an example showing, that besides the LLP promoter
also other promotors, such as the GAP promotor, can be used in
conjunction with the expression system of the invention.
[0381] 8C
[0382] This scheme shows the expression construct used in FIG. 8 A.
A codon optimized DNA sequence encoding the enzyme PVA under
control of the GAP-promoter was inserted into a plasmid (pGAP).
This pGAP plasmid was transformed in NRRL Y-11430 yeast cells
(Pichia pastoris), and cells were grown in a fermentor under
standard conditions using standard media and culturing. OD600
measurements were used to adjust cell densities. The pGAP vector
integrated randomly into the ssn6 gene, thereby activating the
LLP-promoter and resulting in an expression of LLP protein.
[0383] This expression construct is a proof-of-principle, again
showing that interrupting the ssn6 gene results in an improved
expression of the gene being under control of the LLP promoter, in
this case in an improved expression of the LLP protein, in addition
to the expression of the PVA protein.
[0384] FIG. 9:
[0385] A:
[0386] Partial alignment of 39 different protein sequences showing
sequence homology to P. pastoris SSN6-like protein (CCA36593.1)
(SEQ ID NO: 8)
[0387] B:
[0388] P. pastoris SSN6-like consensus sequence 1 (SEQ ID NO:
63)
[0389] C:
[0390] P. pastoris SSN6-like consensus sequence 2 (SEQ ID NO:
64)
[0391] FIG. 10: Expression vector pLLP
[0392] This figure shows the expression vector pLLP without
inserted GOI (FIG. 10 A), and its full length sequence (FIG. 10 B)
(SEQ ID NO: 65).
[0393] Expression vector (without inserted GOI): The gene of
interest (GOI) can be inserted into this vector using the
restriction endonuclease cleavage sites NotI and/or EcoRV,
resulting in the GOI being under control of the LLP promoter and
the ADH terminator. In order to insert the expression vector into
the yeast genome, the vector can be linearized by cleaving with the
restriction endonuclease AvrII, which restriction site is located
between the LLP promoter and the LLP terminator sequence. The
resulting linearized vector contains at its 5'-end the LLP promoter
and at its 3'-end the LLP terminator. If inserted into the yeast
genome by homologous recombination via the LLP promoter- and the
LLP terminator-sequences, this homologous recombination removes the
native LLP-coding sequence from the yeast genome. At the same time
the GOI under control of the LLP promoter and the ADH terminator as
well as a Zeocin expression cassette and all other parts of the
pLLP vector are inserted into the yeast genome.
[0394] FIG. 11: Expression vector pGAP with DLX521 as GOI This
figure shows the expression vector pGAP with DLX521 as GOI (FIG. 11
A), and its full length sequence (FIG. 11 B) (SEQ ID NO: 66).
[0395] Linearization of the vector has been carried out with
SwaI.
[0396] FIG. 12: Expression of HSA using LLP-signal sequence
[0397] This figure shows the expression of human serum albumin
(HSA) utilizing the secretion signal sequence of LLP. The
LLP-signal sequence was fused N-terminally to the coding sequence
of HSA (llps-HSA), transformed into yeast cells. 10 randomly picked
clones were grown in deep well plates and the supernatant checked
for HSA expression (FIG. 12 A). One clone was expressed also in a 1
liter fermenter for 48, 72 and 96 hours (FIG. 12 B). Expression was
shown by SDS-PAGE followed by Coomassie blue staining.
[0398] FIG. 13 A-D: Analysis of the functionality of LLP-promoter
fragments (Truncation analysis)
[0399] The LLP-promoter was successively shortened by cloning of
PCR-generated LLP-promoter fragments into YJK_PVA_021 yeast strain.
11 randomly picked clones for each LLP-promoter length were picked
and grown in deep well plates and the supernatant analyzed by
SDS-PAGE followed by Coomassie blue staining. The lengths of the
tested LLP-promoter fragments were as follows: Fig. A: 576 bp, Fig.
B: 512 bp, Fig. C: 472 bp, and Fig. D: 372 bp. Each figure shows
two representative lines, with "Option 1" denoting that only scFv
is expressed, and with "Option 2" denoting that scFv and LLP is
expressed.
[0400] FIG. 14: Sequences S. cerevisae ssn6 and TUP1 (complete
coding region)
[0401] This figure shows the respective complete coding region of
the following:
[0402] A. Ssn6 nucleic acid, of S. cerevisiae (SEQ ID NO: 135);
[0403] B: SSN6 Protein, of S. cerevisiae (SEQ ID NO: 137);
[0404] C: Tup-1 nucleic acid of S. cerevisiae (SEQ ID NO: 136);
[0405] D: Tup-1 protein of S. cerevisiae (SEQ ID NO: 138).
METHODS
[0406] 1. Assessing the Amount of a Candidate Ssn6-Like Related
Gene
[0407] The level of expression of a candidate ssn6-like related
gene can be measured for example by measuring the level of mRNA of
said ssn6-like related gene by northern blotting or by quantitative
Polymerase Chain Reaction (qPCR) or reverse transcriptase qPCR, or
measuring the activity of the promoter of said ssn6-like related
gene for example by using luciferase reporter gene assays or by
using ssn6-like related promoter-green fluorescent protein (GFP)
constructs, etc. All these methods are well known to a person
skilled in the art and represent routine work. A textbook
comprising protocols for routine methods is for instance Sambrook
et al., "Molecular Cloning: A Laboratory Manual", 4.sup.th Edition,
Cold Spring Harbor Laboratory Press, (2012), referred to herein as
Sambrook et al.
[0408] 2. Assessing Whether a Candidate Ssn6-Like Related Gene
Resembles the Ssn6-Like Gene as Defined Herein with Respect to
Function, Activity and Sequence
[0409] 2.1 Measuring the Amount of LLP Protein Expressed in Cell
Culture:
[0410] The amount of a protein (or its expression level) can be
determined according to any suitable method that is known to a
person skilled in the art, for instance by measuring the amount of
LLP protein in the supernatant of a cell culture by ELISA, by
western blotting, or SDS PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) analysis of LLP protein.
[0411] In the present invention, the amount of LLP protein in the
supernatant has been measured by carrying out a qualitative SDS
PAGE analysis (Novex NuPage 4-12% Bis-Tris Gels with MES running
buffer, from Invitrogen).
[0412] Alternatively the level of LLP protein can be determined
indirectly by measuring LLP-mRNA using the same methods as
described above in paragraph 1 "Assessing the amount of a candidate
ssn6-like related gene".
[0413] 2.2 Measuring the Amount of a Gene of Interest (GOI)
Expressed Under Control of the LLP-Promoter in Cell Culture
[0414] The same methods for measuring the amount of LLP-protein, as
described above under 2.1, can also be used for measuring
GOI-protein expressed under control of a LLP-promoter according to
the invention.
[0415] 2.3 Measuring Activity and Function of a Protein
[0416] In order to measure the function and activity of SSN6-like
protein or SSN6-like related protein, any suitable protocol that is
known to a skilled person can be used. Especially any method
suitable to measure the functioning of the LLP-promotor can be use
to measure the function and activity of SSN6-like protein or
SSN6-like related protein, such as the methods mentioned under 2.1
and 2.2 above.
[0417] Once an SSN6-like protein or a SSN6-like related protein has
been identified, its LLP-promoter-suppressing activity can be
inhibited by inactivating the corresponding ssn6-like gene or
ssn6-like related gene by methods described elsewhere in this
application. Once this blockage has been performed the functioning
(meaning the reduced amount of, or the complete lack of) the
corresponding SSN6-like protein or mRNA or SSN6-like related
protein or mRNA can be measured. Suitable methods are described
elsewhere in this application.
[0418] PVA enzyme assay: an aliquot of the supernatant was mixed
with the substrate Phenoxymethylpenicillin-Kalium and incubated at
22.degree. C. The presence of active PVA enzyme will result in
cleavage of the substrate in a titer-dependent manner. The reaction
was stopped after 60 min by addition of ice-cold methanol. The
amount of the cleavage product phenoxy acetic acid was determined
by quantitative HPLC.
[0419] 3. Comparison of Different Expression Systems
[0420] In order to compare the protein expression of different
expression systems, any suitable method that is known to a person
skilled in the art can be carried out. In the present invention,
the following protocol has been applied for this purpose:
[0421] The supernatants obtained from the pLLP expression system
and from the pGAP system (same volume) were directly loaded on
SDS-PAGE gels for relative comparisons of the pLLP vs. pGAP
system.
[0422] 4. Identification of Regulatory DNA Sequences
[0423] There are various tools available to predict regulatory DNA
sequences, reviewed for example by Wassermann et al., Nature
Reviews Genetics 5, 276-287. There are also tools for the
prediction the total length of a promotor sequence, as well as
tools predicting distinct positions within such a promoter, which
supposedly bind to certain transcription factors, etc. One of these
online tools is available from the University of Copenhagen,
Bioinformatics Center, under http://jaspar.binf.ku.dk/. We used the
JASPAR development server, Version 5.0_ALPHA. Settings of this
online tool were: JASPAR CORE fungi, all JASPAR matrix models were
chosen (we searched for all transcription recognition sites listed
for fungi in the JASPER CORE fungi database), profile score
threshold was set to 95%. Our input sequence was the LLP-promoter
sequence SEQ ID NO: 12. This analysis resulted in 63 predicted
binding sites for transcription factors within the tested sequence
as shown in the table below.
[0424] More details on transcription factor binding site prediction
can be found in Nat Rev Genet. 2004:4, 276-87.
TABLE-US-00002 predicted Model site name Score Start End Strand
sequence SKO1 14.890 274 281 1 ACGTAATG ABF1 13.893 204 219 -1
CCGTAAAAA GCGATAC (SEQ ID NO: 116) CST6 13.595 271 279 1 ATGACGTAA
SUM1 11.962 346 354 1 ATAATTTTT SPT23 11.708 750 757 -1 AAAATCAA
SPT23 11.708 516 523 1 AAAATCAA YOX1 11.696 638 645 -1 TTAATTAT
RFX1 11.668 39 46 -1 CGTTGCTA STE12 11.624 917 923 -1 TGAAACG YHP1
11.563 499 504 1 TAATTG AFT2 11.464 69 76 -1 CACACCCT TEA1 11.362
265 272 1 GCGGACAT YML08 11.251 72 80 -1 ACCCCACAC 1W ARR1 11.154
684 691 -1 ATTTGAAT TOS8 11.143 380 387 1 GTGTCAAA MBP1:: 10.880
717 723 1 TCGCGTT SWI6 PHD1 10.721 104 113 -1 ACCTGCATCA (SEQ ID
NO: 117) YPR02 10.671 73 79 -1 CCCCACA 2C YGR06 10.573 67 80 -1
ACCCCACAC 7C CCTAC (SEQ ID NO: 118) ACE2 10.293 333 339 1 CCCAGCA
ADR1 10.260 74 80 -1 ACCCCAC MIG3 10.179 73 79 -1 CCCCACA STB5
10.040 835 842 -1 CGGTATTA MIG2 10.037 73 79 -1 CCCCACA MIG1 9.822
74 80 -1 ACCCCAC YAP5 9.428 704 709 -1 AAGCAT YAP5 9.428 698 703 1
AAGCAT YAP5 9.428 456 461 1 AAGCAT SIG1 9.387 222 226 -1 ATATA ARR1
9.314 106 113 -1 ACCTGCAT MOT3 9.052 296 301 1 AAGGTA YAP5 8.787
259 264 1 AAACAT HAP2 8.684 424 428 1 TTGGT HAP2 8.684 159 163 1
TTGGT SKN7 8.580 401 406 -1 GGCCAT HAP2 8.495 665 669 -1 TTGGC HAP2
8.495 622 626 1 TTGGC HAP2 8.495 404 408 -1 TTGGC PHO2 8.463 436
441 -1 ATAATA GLN3 8.378 570 574 1 GATAA GLN3 8.378 6 10 1 GATAA
MBP1 8.257 716 722 -1 ACGCGAT PHO2 8.131 119 124 1 ATATTA PHO2
8.131 90 95 -1 ATATTA SKN7 8.026 403 408 1 GGCCAA ARG80 7.974 956
961 -1 TGACAC ARG80 7.974 380 385 -1 TGACAC YAP5 7.933 185 190 1
AGACAT FZF1 7.932 551 556 -1 CTATCA PHO2 7.806 488 493 1 TTATTA
PHO2 7.806 435 440 1 TTATTA PHO2 7.733 638 643 1 ATAATT PHO2 7.733
346 351 1 ATAATT PHO2 7.733 51 56 1 ATAATT PHO2 7.408 640 645 -1
TTAATT PHO2 7.402 587 592 -1 ATATTT GLN3 7.272 552 556 1 GATAG GLN3
7.272 28 32 -1 GATAG GLN3 7.272 2 6 1 GATAG HAP2 7.252 592 596 -1
TTGGA HAP2 7.252 529 533 1 TTGGA HAP2 7.252 453 457 -1 TTGGA HAP2
7.252 196 200 -1 TTGGA
[0425] 5. Transformation and Cultivation of Strains
[0426] Expression constructs were linearized by digestion with
suitable restriction enzyme and transformed in Pichia strains by
electroporation. The transformants were subsequently plated on agar
plates containing Zeocin (final concentration: 100 mg/L) and/or
Geneticin (final concentration: 300 mg/L).
[0427] Single colonies or glycerol stocks were subjected to
expression studies in 48-well plates using a starch/amylase based
cell culture medium. OD (Optical Density) at 600 nm at harvest was
around 10.
[0428] 6. Testing of Promoter Activity
[0429] Promoter activity can be measured by any suitable method
that is known to a person skilled in the art. An example of such a
method is the use of qPCR or reporter gene assays (e.g. Luciferase,
Green Fluorescent Protein (GFP) etc.), both of which are standard
methods that are known to a person skilled in the art. For example
the most suitable part of the LLP-promoter for high level
expression of a GOI can be determined by successively shortened
versions of the LLP-promoter sequence according to SEQ ID NO. 12,
by inserting such shortened LLP-promoter sequences together with a
Kozak sequence and a model protein sequence such as DLX521 (scFv),
hGH, HSA, PVA, etc. and together with a signal sequence such as the
MF-alpha pre-pro signal sequence with or without EAEA repeat, the
natural signal sequence of said model protein, etc. into a pLLP
vector carrying a resistance-marker such as Geneticin, Zeocin, etc.
and transfecting such pLLP vector into a suitable yeast cell such
as for example YJK_PVA_021-cells, SSS1-cells, NRRL Y-11430-cells,
etc. Individual clones or pooled clones of such transformed yeast
cells then can be grown under standard growth conditions in deep
well plated, shaker flasks, fermentors, etc. and the amount of
expression of said model protein being measured using methods such
as SDS-PAGE, ELISA, or protein-activity assays such as the
PVA-assay described elsewhere in this application, etc. Shortened
versions of the promoter could represent parts of the promoter
sequence disclosed in SEQ ID NO: 12, for example a LLP-promoter
having a length of 1000, 775, 675, 605, 576, 512, 472, 415, 404,
372, 305, 285, 235, 165, 100 nucleotides, etc. counted in each case
from the 3'-end of SEQ ID NO: 12.
[0430] 7. Assessing Degree of Identity of Nucleotide Sequences or
Amino Acid Sequences
[0431] "Sequence identity" or "% identity" refers to the percentage
of residue matches between at least two polypeptide or
polynucleotide sequences aligned using a standardized algorithm.
Such an algorithm may insert, in a standardized and reproducible
way, gaps in the sequences being compared in order to optimize
alignment between two or more sequences, and therefore achieve a
more meaningful comparison of the sequences. For purposes of the
present invention, the sequence identity between two amino acid or
nucleotide sequences is determined using the NCBI BLAST program
version 2.2.29 (Jan. 6, 2014) (Altschul et al., Nucleic Acids Res.
(1997) 25:3389-3402). Sequence identity of two amino acid sequences
can be determined with blastp set at the following parameters:
Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost:
Existence=11, Extension=1; Filter=low complexity activated; Filter
String: L; Compositional adjustments: Conditional compositional
score matrix adjustment. For purposes of the present invention, the
sequence identity between two nucleotide sequences is determined
using the NCBI BLAST program version 2.2.29 (Jan. 6, 2014) with
blastn set at the following exemplary parameters: Word Size: 11;
Expect value: 10; Gap costs: Existence=5, Extension=2; Filter=low
complexity activated; Match/Mismatch Scores: 2,-3; Filter String:
L; m.
[0432] 8. Generation of the Super-Secretor Strain (SSS1)
[0433] An expression cassette encoding the enzyme PVA (Penicillin V
Amidase) under the control of the GAP promoter and another
expression cassette coding for Zeocin was transformed into the P.
pastoris strain NRRL Y-11430 (see table below). Clones secreting
high levels of active PVA were screened by the following enzyme
assay:
[0434] The cleavage of the PVA substrate penicillin V to the
products 6-APA (6-Aminopenicillanic acid) and phenoxyacetic acid
was determined by quantifying the phenoxyacetic acid amount by
HPLC.
[0435] One clone showing high PVA titers was identified and named
YJK_PVA_021. Surprisingly, this clone not only secreted high
amounts of PVA but also the LLP protein at even higher levels.
Thus, YJK_PVA_021 was further characterized by whole genome
sequencing done by the company Illumina Inc., San Diego, Calif.,
USA. After de-novo assembly of the genome done by Illumina Inc.
(see SEQ ID NOs: 67-115, showing the results of genomic
sequencing), the localization of the sequence of the PVA expression
construct was identified to be in SEQ ID NO: 103 and the adjacent
genomic sequences were compared to the reference strain (P.
Pastoris CBS 7435, gi|32835130|emb|FR839629.1|) for identification
of the insertion site. A single copy of the expression cassette
encoding PVA was found to be integrated randomly at position
807,480 of chromosome 1 of the reference strain Pichia pastoris CBS
7435. This position lies within the ssn6-like gene sequence
resulting in disruption of the ssn6-like gene leading to a
C-terminal truncated protein (see FIG. 2 B, see SEQ ID NO: 9) and
to a 3'-truncated coding region of the ssn6-like gene, respectively
(for details see FIG. 1 S, see SEQ ID NO: 23. No further obvious
deviations from the reference sequence were found. Thus, this
single random integration event resulted in high level secretion of
LLP. The PVA expression cassette was removed from YJK_PVA_021
resulting in clone SSS1 (see also FIG. 3).
[0436] The vector pGAPk was PCR-amplified (linearized by PCR) using
oligo2395 (TCCTCGTCCAATCAGGTAG; SEQ ID NO: 119) and oligo2398
(AGTGGTACCTGCAGCTAAG; SEQ ID NO: 120) and the PCR-product was
transformed into yeast strain YJK_PVA_021. Homologous recombination
replaced the pGAP-PVA expression vector containing the
Zeocin-resistance marker with the empty vector sequence of pGAPk
(empty expression cassette and Geneticin-resistance marker). Clones
with Geneticin-resistance and without PVA activity were screened.
PVA was measured as indicated above. One strain which does not
express PVA but expresses high amounts of LLP was denoted SSS1 and
used for subsequent expression studies of different GOIs.
[0437] 9. Characterization of the LLP Signal Sequence
[0438] The secreted LLP protein was N-terminally sequenced by Edman
degradation to identify the LLP signal sequence cleavage site (Seq.
ID NO. 3, FIG. 1 C). In order to proof the general functionality of
the LLP signal sequence, resulting in the secretion of heterologous
proteins fused to the LLP signal sequence, the following
experiments were performed: The LLP signal sequence was fused to
the 3' end of HSA coding sequence replacing the native HSA-signal
sequence resulting in the sequence according to SEQ. ID NO: 121,
(FIG. 1 T) and cloned into the pLLP plasmid via EcoRV and NotI
restriction enzyme sites. The HSA coding sequence was codon
optimized (done by DNA2.0 Inc., Menelo Park, USA) and a silent
point mutation was inserted into the LLP-signal sequence at
position 45 exchanging G for a C, which does not change the amino
acid sequence of the LLP-signal peptide, but which deletes the
restriction enzyme site PstI from the LLP-signal sequence. The
resulting plasmid was transformed into SSS1 yeast strain. 10 clones
were randomly picked and subjected to cultivation in deep well
plates. 15-20 .mu.l/lane supernatant of deep well plate cultures
were directly loaded on SDS-PAGE gels (Novex NuPage 4-12% Bis-Tris
Gels from Invitrogen with MES-running buffer). FIG. 12 A shows the
results of this experiment: Lanes 3 and 7 represent clones bearing
no HSA insert, lanes 2, 4, 8 and 10 represent clones according to
option 1 (only HSA expressed), and lanes 1, 5, 6 and 9 represent
clones according to option 2 (HSA expressed in parallel to LLP).
FIG. 12 B shows the result of a 1 liter fermenter expression using
the same clone used in lane 2 of the gel in FIG. 12 A. A sample
from the fermenter supernatant was collected after 48, 72 and 96
hours and 15 .mu.l of each sample subjected to SDS-PAGE, followed
by Coomassie blue staining using the "SimplyBlue SafeStain" system
from Life Technologies according to the manufacturer's
instruction.
[0439] 10. Characterization of the Ssn6-Like/LLP-Promoter
Expression System
[0440] The table below shows the basic characteristics of the
expression constructs/vectors and the yeast cells used in order to
evaluate the functioning of the expression system for various
classes of proteins/genes of interest (GOIs), namely hormones,
antibodies, enzymes and structural proteins. The expression results
of GOI depicted in the figures were generated with the expression
constructs/vectors/host strain combinations listed in the table
below. Comparison of the expression of a GOI using a standard
GAP-promotor or the LLP-promoter was done using SDS-PAGE, ELISA
and/or enzymatic activity assays, as described in the figures.
TABLE-US-00003 GOI Codon inserted in Plasmid Resist- Protein
Protein usage Signal vector (linearized ance Host name class for
GOI sequence between with) marker strain Comments hGH Hormone codon
S. EcoRV and pLLP Zeocin SSS1 pLLP from optimized cerevisiae Notl
(Avrll) Sandoz for Pichia MF-alpha EcoRI and pGAP Zeocin NRRL pGAP
from pastoris (without Notl (Swal) Y- DNA2.0 EAEA 11430 (pJ905)
repeat) DLX521 Antibody codon S. EcoRV and pLLP Zeocin SSS1 pLLP
from (fragment optimized cerevisiae Notl (Avrll) Sandoz scFv) for
Pichia MF-alpha EcoRI and pGAP Zeocin NRRL pGAP from pastoris
(without Notl (Swal) Y- DNA2.0 EAEA 11430 (pJ905) repeat) EcoRV and
pLLP Geneticin YJK_P pLLP from Notl (Avrll) VA_021 Sandoz PVA
Enzyme codon S. EcoRV and pLLP Zeocin SSS1 pLLP from optimized
cerevisiae Notl (Avrll) Sandoz for Pichia MF-alpha EcoRI and pGAP
Zeocin NRRLY- pGAP from pastoris (with Notl (Bglll* 11430 Sandoz
EAEA repeat) HSA Structural codon Human EcoRV and pLLP Zeocin SSS1
pLLP from protein optimized HSA pre- Notl (Avrll) Sandoz for Pichia
pro signal EcoRI and pGAP Zeocin NRRLY- pGAP from pastoris sequence
Notl I (Swal) 11430 DNA2.0 (pJ905) Pichia EcoRV and pLLP Zeocin
SSS1 pLLP from pastoris Notl (Avrll) Sandoz LLP- signal sequence**,
*** *This plasmid was also linearized by PCR as described elsewhere
in this application (see paragraph 8. ''Generation of the super
secretor strain (SSS1)''). **HSA coding sequence was independently
codon optimized resulting in identical amino acid sequences but in
slightly different nucleotide sequences as compared to the
HSA-sequence one row above ***P. pastoris signal sequence contains
a silent point mutation at position 45 changing G to C in order to
delete a restriction enzyme site
[0441] 11. Identification of Ssn6-Like Consensus Sequences
[0442] The ssn6-like sequence of Pichia pastoris strain NRRL
Y-11430 was used for a BLAST-search of similar sequences. The BLAST
was performed on http://blast.ncbi.nlm.nih.gov/. BLAST parameters
were "automatically adjust parameters for short input sequences",
Expect threshold: 10, Word size: 3, Max matches in a querry range:
0, Matrix: BLOSUM62, Gap Costs: Existence: 11 Extension:1,
Compositional adjustments: "Conditional compositional score matrix
adjustment. The top 39 sequences originate from the following
organisms (also see FIG. 9A): Komagataella pastoris CBS 7435
(Synonym/other names: Pichia pastoris, Pichia pastoris CBS 7435),
Komagataella pastoris GS115 (Synonym/other names: Pichia pastoris,
Pichia pastoris GS115), Scheffersomyces stipitis CBS 6054
(Synonym/other names: Pichia stipitis, Pichia stipitis CBS 6054),
Millerozyma farinosa CBS 7064 (other name: Pichia farinosa CBS
7064), Candida parapsilosis, Candida orthopsilosis Co 90-125,
Debaryomyces hansenii CBS767, Spathaspora passalidarum NRRL
Y-27907, Candida albicans, Candida albicans SC5314, Candida maltosa
Xu316, Candida tropicalis MYA-3404 (other name: Candida tropicalis
T1), Lodderomyces elongisporus NRRL YB-4239 (other name:
Saccharomyces elongisporus), Clavispora lusitaniae ATCC 4272
(genebank anamorph: Candida lusitaniae ATCC 42720), Meyerozyma
guilliermondii ATCC 6260 (genebank anamorph: Pichia guilliermondii
ATCC 6260), Wickerhamomyces ciferrii, Ogataea parapolymorpha DL-1
(synonym and other names: Hansenula polymorpha, Hansenula
polymorpha DL-1, Ogataea angusta DL-1, Ogataea parapolymorpha ATCC
26012, Ogataea parapolymorpha DL-1, Pichia angusta DL-1),
Cyberlindnera fabianii (synonyms and other names: Hansenula
fabianii, Pichia fabianii, . . . ), Kuraishia capsulata CBS 1993,
Dictyostelium discoideum AX4 (belongs to social amoebae),
Tetrapisispora phaffii CBS 4417 (synonym: Fabospora phaffii,
Dictyostelium purpureum (belongs to social amoebae), Pseudozyma
flocculosa PF-1, Malassezia globosa CBS 7966, Botryobasidium
botryosum FD-172 SS1 (basidiomycete), Naumovozyma dairenensis CBS
421 (synonyme: Saccharomyces dairenensis), Tetrapisispora blattae
CBS 6284, Mucor circinelloides f. circinelloides 1006PhL (Early
diverging fungal lineage), Malassezia sympodialis ATCC 42132,
Kazachstania naganishii CBS 8797 (Saccharomyces naganishii),
Saccharomyces cerevisiae YJM789, Saccharomyces cerevisiae FostersB,
Saccharomyces cerevisiae, Saccharomyces cerevisiae S288c, Ustilago
hordei (Corn smut fungus, basidiomycete), Meyerozyma guilliermondii
ATCC 6260 (synonym/other names: Candida guilliermondii, Pichia
guilliermondii ATCC 6260), Ustilago maydis 521, (Corn smut fungus,
basidiomycete).
[0443] The top 39 identified amino acid sequences were aligned to
the ssn6-like-amino acid sequence (=identical to CCA36593.1)
revealing significant similarities (see FIGS. 9 B and 9 C). The
alignment was done using the online tool Clustal Omega provided by
the EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalo/ which is
described by Sievers et al., Molecular Systems Biology 7, article
number: 539, Valentin et al., Nucleic acids research, 2010, 38,
Suppl. W695-9, and by McWilliam et al., Nucleic acids research,
2013, July; 41 (Web Server issue): W597-600. Clustal Omega uses the
HHaliqn algorithm and its default settings as its core alignment
engine. The algorithm is described in Soding, J. (2005) `Protein
homology detection by HMM-HMM comparison`. Bioinformatics 21,
951-960. The default transition matrix is Gonnet, gap opening
penalty is 6 bits, gap extension is 1 bit. The symbols used for the
consensus sequence at the bottom of the alignment are as follows.
An "*" (asterisk) indicates amino acid positions which have a
single, fully conserved residue across all 40 aligned sequences. A
":" (colon) indicates conservation between groups of amino acids of
strongly similar properties--scoring>0.5 in the Gonnet PAM 250
matrix. A "." (period) indicates conservation between groups of
amino acids of weakly similar properties--scoring .gtoreq.0.5 in
the Gonnet PAM 250 matrix. Sequence CCA36593.1 corresponds to the
Pichia pastoris SSN6 sequence identified in this application. FIG.
9 A shows only that part of the alignment with the highest
similarity between all 40 sequences. FIG. 9 B shows the consensus
sequence corresponding to Pichia pastoris SSN6-like (CCA36593.1)
amino acids 352 to 372. FIG. 9 C shows the consensus sequence
corresponding to Pichia pastoris SSN6-like (CCA36593.1) amino acids
394 to 417.
[0444] Amino acids which are identical in all 40 sequences are
written in white with black background-labelling. The consensus
sequence shows either individual amino acids which are identical in
all 40 sequences or shows a group of amino acids in brackets "( )"
and each of the amino acids in such a set of brackets can be chosen
alternatively for that position in the consensus sequence. A "-"
(dash) indicates that the amino acid at this position may also be
omitted from the consensus sequence, which is for example one
possibility for positions 406 and 407 of the consensus sequence
depicted in FIG. 9 C. For example the starting amino acid at
position 352 of the consensus sequence in FIG. 9 B is "W", meaning
that the consensus sequence at this position contains a Tryptophan,
position 353 of the consensus sequence in FIG. 9 B is written as
"(CGL)" meaning that at this position of the consensus sequence can
either be located Cysteine, Glycine or Leucine, position 354 is
labelled "(SLTA)" meaning that at this position there can either be
located Serine, Leucine, Threonine or Alanine, etc. The same
nomenclature is used for the second consensus sequence depicted in
FIG. 9 C. Consensus sequences can be shorter or longer as the two
exemplary consensus sequences shown in FIGS. 9 B and 9 C. Consensus
sequences can be deduced from the sequence alignment of FIG. 9A or
can be deduced from other parts of the sequence alignment prepared
from the 40 above mentioned sequences using the sequence alignment
method as described above. Preferably the consensus sequence
contains at least 24 amino acids, preferably 23, 22, 21, 20, 19,
18, 17 amino acids, more preferably at least 16, 15, 14, 13, 12,
11, or 10 amino acids, most preferably at least 9, 8, 7, 6, 5 or 4
amino acids. Preferably a SSN6-like protein contains at least one
or two consensus sequences, more preferably both consensus
sequences shown in FIG. 9 B and FIG. 9 C, more preferably at least
one consensus sequence, most preferably a consensus sequence
selected from the sequences shown in FIGS. 9 B and 9 C.
[0445] 12. Characterization of the Functionality of the
LLP-Promoter
[0446] The plasmid pLLPk containing DLX521 with MFalpha signal
sequence was used as PCR-template in combination with the following
PCR-primers to generate shortened/truncated versions of the
LLP-promoter (A-fragment).
[0447] Used Reverse Primer:
TABLE-US-00004 Sequence SEQ Primer (5' to ID name 3' end)
.DELTA.-fragment NO.: 2892 TGTCGAAC used for all 122 CACCACTA see
FIG. 13 A C to FIG. 13 D fragments
[0448] Used Forward Primer:
TABLE-US-00005 Sequence Length of SEQ Primer (5' to 3' Promoter ID
name end) .DELTA.-fragment fragment NO.: Yo_218 TATACCTAGG
.DELTA.29 576 123 TGGTGGAACT TTATTATTCT TTC Yo_219 TATACCTAGG
.DELTA.93 512 124 TATTAGCTGG TAATTGAGCG Yo_220 TATACCTAGG
.DELTA.133 472 125 TTGGAGGGT ATGGTCAGAG Yo_221 TATACCTAGG
.DELTA.201 404 126 TTTCATTCCA TCTTGCCATC Yo_222 TATACCTAGG
.DELTA.233 372 127 CTTACATCAA TAATTAAAAC Yo_223 TATACCTAGG
.DELTA.300 305 128 GCAAGCATAT GCTTAAAAGG
[0449] The resulting PCR products were ligated in via SpeI and
AvrII restriction enzyme sites into the vector pLLPk_containing
DLX521. The correct sequences of the resulting plasmids were
confirmed by DNA sequencing. The plasmids were linearized with
AvrII and transformed in strain YJK_PVA_021. 11 clones per plasmid
were randomly picked and subjected to cultivations in deep well
plates using a synthetic medium. At harvest, 15-30 .mu.l
supernatant samples were directly loaded on SOS-PAGE gels (Novex
NuPage 4-12% Bis-Tris Gels from Invitrogen with MES-running buffer)
to analyze expression of scFv by staining the gels (Simply Blue
Safe Stain). The 00600, meaning the yeast cell numbers per ml
culture medium at harvest, was comparable for all clones
analyzed.
[0450] M=Protein Marker VI.
[0451] Results are shown in FIG. 13 A to FIG. 13 D.
[0452] 13. Examples of Ssn6-Like Mutants
[0453] The yeast strain contains the modified ssn6-like gene coding
for amino acids 1 to 367 and in addition seven amino acids
(EWYLQLR; SEQ ID NO: 139) originating from the vector inserted into
the ssn6-like gene in order to disrupt the ssn6-like gene.
Alternatively, modified versions of ssn6-like might contain a
modified ssn6-like gene coding for the following regions of amino
acids of SSN6-like protein, to the effect that the modified
SSN6-like protein is not able to exert its wildtype function and/or
wildtype activity:
[0454] Modified versions of SSN6-like protein comprising amino
acids 1 to 44, 1 to 77, 1 to 100, 1 to 122, 1 to 155, 1 to 189, 1
to 235, 1 to 275, 1 to 315, 1 to 348, 1 to 400, 1 to 450, 1 to 500,
1 to 550, 1 to 600, 1 to 650, 1 to 367, 1 to 400, 1 to 450, 1 to
500, 1 to 550, 1 to 600, 1 to 650, and/or 1 to 700 of SSN6-like
protein according to SEQ ID NO. 9.
[0455] In another alternative version the modified versions of
ssn6-like might contain the region of the ssn6-like gene coding for
blocks of SSN6-like amino acids according to SEQ ID NO. 9, namely
amino acids 1 to 44, 45 to 77, 78 to 100, 101 to 122, 123 to 155,
156 to 189, 190 to 235, 236 to 275, 276 to 315, 316 to 348, 348 to
367, 368 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600,
601 to 650, 651 to 700, and/or 701 to 736. Each of these blocks of
amino acids might be combined with one or more other blocks of
amino acids, preferably in the same order as they occur in SEQ ID
NO. 9, wherein none, one or more amino acid block(s) is/are lacking
in between two amino acid blocks.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220220161A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220220161A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References