IE83463B1 - Process for preparing a protein by a fungus transformed by multicopy integration of an expression vector - Google Patents
Process for preparing a protein by a fungus transformed by multicopy integration of an expression vectorInfo
- Publication number
- IE83463B1 IE83463B1 IE1998/0989A IE980989A IE83463B1 IE 83463 B1 IE83463 B1 IE 83463B1 IE 1998/0989 A IE1998/0989 A IE 1998/0989A IE 980989 A IE980989 A IE 980989A IE 83463 B1 IE83463 B1 IE 83463B1
- Authority
- IE
- Ireland
- Prior art keywords
- gene
- lipase
- multicopy
- dna
- protein
- Prior art date
Links
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Abstract
ABSTRACT A process is disclosed for preparing a protein by a eukaryote transformed by multicopy integration of an expression vector into the genome of a yeast, such as Saccharomyces, Hansenula and Kluyveromyces, or of a mould such as Aspergillus, Rhizopus and Trichoderma, said expression vector containing both an "expressible gene" encoding said protein and a so-called "deficient selection marker needed for the growth of the yeast or mould in a specific medium", such as the LEU2d, TRP1d or URA3d gene, in combination with a ribosomal DNA sequence, resulting in stable high copy integration of 100-300 copies per cell. This multicopy integration results in at increased production of the desired protein, which can be guar 0L-galactosidase, an oxidase or a hydrolytic enzyme such as a lipase.
Description
PATENTS ACT, 1992
980989
PROCESS FOR PREPARING A PROTEIN BY A FUNGUS TRANSFORMED BY
MULTICOPY INTEGRATION OF AN EXPRESSION VECTOR
UNILEVER PLC
Process for preparing a protein by a fungus transformed
by multicopy integration of an expression vector.
In a major aspect, the invention relates to a process
for preparing a, homologous or heterologous, protein by
a yeast, transformed by multicopy integration of an
expression vector into the genome of the yeast, said
expression vector containing both an "expressible gene"
encoding said protein and a so—cal1ed "deficient
selection marker needed for the growth of the yeast in a
specific medium".
Although most experiments have been carried out with
yeasts, it is envisaged that the invention is also
applicable to moulds. Therefore in this specification in
addition of either yeast or mould the term "fungus", or
its plural form "fungi", will be used which covers both
yeasts and moulds.
In this specification the expression "expressible gene"
means a structural gene encoding a protein, either
homologous or heterologous to the host organism, in
combination with DNA sequences for proper transcription
and translation of the structural gene, and optionally
with secretion signal DNA sequences, which DNA sequences
should be functional in the host eukaryote.
In this specification the expression "deficient
selection marker needed for the growth of the yeast or
mould in a specific medium" is used for a marker gene
containing a promoter and a structural gene encoding a
polypeptide or protein, said polypeptide or protein
- either being needed for the production of an ingre-
dient, such as amino acids, vitamins and nucleo-
tides, which ingredient is essential for the
growth of the yeast or mould; in this specification
such ingredient is also called "essential
nutrient",
- or being needed for the protection of the cell
against toxic compounds, such as antibiotics or
Cu2+ ions, present in the medium,
provided that the deficient selection marker results
- either in sub—optimal de novo synthesis of said
polypeptide or protein, which in turn results in a
sub—optimal production of the essential ingredient
or in sub—optimal protection against said the toxic
compound, respectively,
— or in de novo synthesis of a modification of said
polypeptide or protein having a sub-optimal
efficiency in the production of said essential
ingredient or in sub-optimal protection against
said toxic compound, respectively.
Thus the word "deficient" is used to indicate both the
sub—optimal synthesis of the polypeptide or protein, and
the production of a polypeptide or protein having sub-
optimal efficiency in the actions for the cell as-
mentioned above;‘
Examples of such marker genes include auxotrophic
markers such as the LEU2, the TRP1 and the URA3 genes,
antibiotic resistance genes such as the G418 resistance
gene and the chloramphenicol resistance gene, and the
gene encoding the enzyme catalase which can protect the
cell against H202.
BACKGROUND OF THE MULTICOPY INTEGRATION ASPECT OF THE
INVENTION
An example of a so—called "deficient selection marker
needed for the growth of the yeast" is the LEU2d gene
described by Kingsman c.s. (reference 1), who described
the development of a multicopy integrative vector which
was dispersed throughout the genome using the transposa-
ble Ty element Tyl-15 (reference 2). The element was
engineered to contain two selectable markers, TRP1
(reference 3) and LEU2 from pMA3a, and the PGK expres-
sion signals from pMA91 (reference 4) with an IFN-a2
coding sequence (reference 5). A single copy of the
engineered Ty was integrated into the genome using a
linear fragment to stimulate recombination across the
ends of the element and thereby replacing an endogenous
element. Transformants were selected for the TRP1
marker. Few transformants were obtained by selecting for
LEU2 as insufficient enzyme was produced by a single
copy of this gene. The transformant was then grown in
decreasing concentrations of leucine to select for an
increase in the copy number of the LEU2 gene, presumably
by spread of the Ty element throughout the genome by
gene conversion and transposition (reference 6). A
strain was constructed which produced 8 x 105 molecules
of IFN per cell; this being intermediate between yields
from single copy ARS/GEN vectors (105 molecules/cell)
and from multicopy vectors such as pMA9l (6 x 10
molecules/cell).
For a practical stable production system with a
transformed yeast the use of Ty elements has certain
disadvantages.
- For example, Ty elements are homologous to
retroviral sequences, which are more or less suspect
materials for production of a protein suitable for
products for human consumption or in the preparation
thereof. Thus it is preferable to find solutions whereby
these more or less suspect materials are not used.
- Another disadvantage is their property of being
transposable elements. This has the consequence that an
appreciable risk exists that the resulting strain is not
genetically stable, because the transposable TY elements
integrated in the chromosome of the yeast can transpose
and integrate at other sites of the genome which has
negative implications for the production process and can
give problems in obtaining clearance from responsible
companies and the authorities..
- In view of their retroviral.properties-Ty.elements
may result in virus-like particles.-This is highly
undesirable for practical production processes, because
instability of genetically modified organisms should be
avoided.
- Ty elements only occur in the yeast saccharomyces
cerevisiae. Therefore it is doubtful, whether they can
be used for other yeasts or even moulds. It is unknown
whether transposable elements occurring in other
organisms can be used in a similar way. But even if they
could, they have the same disadvantages as indicated
above.
- The copy number obtained with Ty integration is
about 20-30 with a single maximum of about 40 copies per
cell. A higher number of 100-300 copies per cell would
be highly advantageous for commercial production
systems, as higher copy numbers, in general, will result
in higher expression levels.
Therefore a need exists for other systems by which
multicopy integration of heterologous genes in fungi
such as yeast and moulds can be achieved.
SUMMARY OF THE MULTICOPY INTEGRATION ASPECT OF THE
INVENTION A
It has now been found that stable multicopy integration
in S. cerevisiae can be obtained by use of an expression
vector containing both an expressible heterologous gene
and a "deficient selection marker needed for the growth
of the yeast" as above defined and additionally a
ribosomal DNA sequence, of which the ribosomal DNA
sequence enables stable multicopy integration of said
expression vector in the ribosomal DNA locus of the
yeast genome. Surprisingly it appeared to be possible
with such a system to obtain multicopy integration of
over 200 copies per cell, which were stable over more
than 70 generations in both batch and continuous
cultures.
It has surprisingly been found that not only the known
LEU2d system but also other "deficient markers" can be
used, in particular a TRP1d or URA3d gene.
It has further been found that this technique can also
be applied to other yeasts, in particular of the genera
Hansenula and Kluyveromyces.
Thus the principle of using an expression vector
containing a "deficient marker" combined with a
ribosomal DNA sequence for obtaining multicopy integra-
tion in a yeast as disclosed above appears to have a
more general application, for example for other yeasts
like Picbia or moulds e.g. belonging to the genera
Aspergillus, Rhizopus or Tricboderma, in particular if
the multicopy integration vectors contain ribosomal DNA
originating from the host organism. Thus this principle
is applicable for fungi in general. ‘
The multicopy integration aspect of.the.presentg
invention provides a process for preparing a heteroloee
gous protein, e.g. a lipase, by a eukaryote transformed
by multicopy integration of an expression vector into
the genome of the eukaryote, said expression vector
containing both an expressible gene encoding said
heterologous protein and a so-called "deficient
selection marker needed for the growth of the
eukaryote", in which process said expression vector
contains ribosomal DNA sequences enabling multicopy
integration of said expression vector in the ribosomal
DNA locus of the eukaryote genome.
It has further been found that an expression vector as
herein before described can be stably maintained at a
high copy number, when a fungus transformed according to
the invention is grown in a so-called "complete" or non-
selective medium, which contains all the ingredients
necessary for growth of the fungus. Normally one would
expect that de novo synthesis is not required due to the
presence of the essential ingredient in the medium,
which would result in decreasing the proportion of
multicopy-integrated yeast cells in the total yeast
population and thus would lead to a decreased production
of the desired polypeptide or protein. Surprisingly,
despite a situation in which de novo synthesis is not
required, the multicopy integration is stably maintained
and the polypeptide or protein was produced in relative-
ly large quantities.
Although the invention is not limited by any explana-
tion, it is believed that the effects observed are
based on the following theory. For unknown reasons it
seems that in such a system the uptake of the essential
ingredient is limited. Therefore, de novo synthesis is
still needed when the fungus is grown at a growth—rate
above a certain minimum value. This will result in a
selection advantage for those cells which have a high
copy number of the "deficient marker". Possibly the
active uptake of the essential ingredient, e.g. leucine,
is negatively influenced by the presence of other
components in the medium, such as peptides and valine.
Thus, in general, the process can be described as a
process in which said transformed fungus is grown in a
medium containing said essential ingredient at a
concentration below a certain limit whereby the uptake
of said ingredient is rate-limiting, so that de novo
synthesis of said ingredient is required for a growth-
rate above a certain minimum value.
A plasmid suitable for use in a process for preparing a
desired protein by a fungus transformed by multicopy
integration of an expression vector in the ribosomal DNA
of the fungus was described before the present invention.
Lopes et al. have published in 1988 and 1989 on their
findings with a yeast vector suitable for high-level
expression of heterologous proteins, which vector they
called pMIRY2.
In a poster published in 1988 the vector was described
for the first time. The penultimate sentence of the
poster abstract was
" pM]RY2 derivatives containing the PGK or thaumatin
gene appeared to be significantly less stable than the
original plasmid.
The results indicated in the 1988 poster were published
in a full publication in Gene, 19 (No. 2; 15 July 1989)
199-206. However, the sentence onthe instability of the»
pMIRY2 plasmid containing an inserted gene mentioned_in
the 1988 poster was neither repeated nor contradicted in
the 1989 Gene publication.
Other relevant passages in this Gene article are
" The best candidate seemed to be the xibosomal DNA
which encompasses about '140 copies of a 9.1 kb-unit
repeated in tandem on chromosome XII (Petes, 1979).
Experiments using this system to introduce either homolo-
gous or heterologous genes into yeast cells demonstrate
expression levels comparable to those previously achieved
by the use of YBp vectors.
(see page 200, right hand column)
" ...,we conclude that multiple copies of the plasmid have
integrated into the rDNA locus in a tandem arrangement
.at a very limiter number of sites.
(see page 202, right hand column)
This pMIRY2 vector has been the basis for plasmids used
in the development of the present invention. The stabil-
ity problem mentioned in the 1988 poster was solved by
the present inventors in developing a process as
described in claim 1 and the claims dependent thereon.
An example of a complete medium is an industrially
applied growth medium such as molasses, whey, yeast
extract and combinations thereof.
Another embodiment of this -invention is the fermentative
production of one of the various forms ofienzymes
described above or related .ho'sts',._ Such a fermentation
can either be a normal batch fermentation, a fed—batch
fermentation or a continuous fermentation. The selection
of which process has to be used depends on the host
strain and the preferred down stream process.
According to this embodiment it is preferred that the
enzyme is secreted by the microorganism into the
fermentation broth, whereafter the enzyme can be
recovered from the broth by first removal of the cells
either by filtration or by centrifugation.
In a further aspect, the invention relates to enzymes
to recombinant DNA techniques applicable for example for
their modification and production.
In particular embodiments this aspect of the invention
relates to the production of modified enzymes or
modified enzymes, especially modified lipases. Thus this
aspect as described below provides inter alia techniques
for production of lipase, e.g. lipases of the genus
Pseudomonas, e.g. lipase from P. glumae (alias P.
gladioli) and further provides genetically modified
forms of such lipases.
SPECIFIC EMBODIMENTS OF THE MULTICOPY ASPECT OF THE
INVENTION
More specifically the invention provides a process for
preparing a, homologous or heterologous, protein by a
eukaryote transformed by multicopy integration of an
expression vector into the genome of a host eukaryote,
said expression vector containing both an "expressible
gene" as herein before defined encoding said homologous
or heterologous protein and a so-called "deficient
selection marker needed for the growth of the yeast or
mould in a specific medium" as herein before defined,
wherein said expression vector contains ribosomal DNA
sequences enabling multicopy integration of said
expression vector in the ribosomal DNA locus of the
eukaryote genome. Preferably said deficient selection
marker is a LEU2d gene, a TRP1d gene, or a URA3d gene.
The eukaryote can be a fungus such as a yeast,
preferably one of the genera Saccbaromyces, Kluyvero-
myces or Hansenula, or a mould, preferably one of the
genera Aspergillus, Rhizopus and Tricboderma.
In a preferred process said transformed eukaryote is
grown in a medium containing an ingredient, which is
essential for the growth of the eukaryote, at a concen-
tration whereby the uptake of said ingredient is rate-
limiting, so that de novo synthesis of said ingredient
is required for a growth-rate above a certain minimum
value which value depends on the host organism and the
process conditions. Preferably such medium is a so-
called "complete" or non-selective medium, which
contains all the ingredients necessary for growth of the
eukaryote, for example an.industrially applied growth
medium, such as molasses, whey, yeast extract and
mixtures thereof.
In order to obtain sufficient production of a selected
protein in the process according to the invention it is
preferred that the transformed eukaryote contains the
gene or genes required for expression of said protein in
a multimeric form in one of its chromosomes in, or
directly linked to, a locus coding for a ribosomal RNA
while at the same locus also multimeric copies of a
deficient gene encoding a protein required in the
biochemical pathway for the synthesis of said "essential
nutrient" are present. Examples of such expressible gene
are those encoding an enzyme, preferably a hydrolytic
enzyme, in particular a lipase, or a genetically
modified form of such enzyme. Particularly preferred
lipases that can be produced with a process according
to the present invention are lipases that cross-react
with antisera raised against a lipase from Chromobacter
viscosum var lipolyticum NRRL B-3673, or with antisera
raised against lipase from Alcaligenes PL-679, ATCC
31371 or FERM-P 3783, or with antisera raised against a
lipase from Pseudomonas fluorescens IAM 1057, and
modified forms of such cross-reacting lipase.
A specially preferred lipase is encoded by a gene having
the nucleotide sequence given in Figure 2 or any
nucleotide sequence encoding the same amino acid
sequence as specified by that nucleotide sequence or
encoding modified forms of this amino acid sequence
resulting in a lipase with a better overall performance
in detergents systems than the original lipase.
The transformed eukaryote used in a process according to
the invention is preferably a eukaryote being deficient
for the synthesis of an "essential nutrient" as herein
before defined and whereby the deficient selection
marker can contribute to complementation of the
synthesis of the "essential nutrient". The deficiency of
the parent strain can be achieved by replacement of a
gene coding for an enzyme effective in the biosynthetic
pathway of producing said essential nutrient. It is
particularly advantageous if the enzyme, for which the
parent strain is deficient, catalyses a reaction in a
part of the biosynthetic pathway that is not branched
until the essential nutrient is formed. Examples of
essential nutrients are amino acids, nucleotide or
vitamins, in particular one of the amino acids leucine,
tryptophan or uracil.
Another embodiment of the invention is a process as
described above, in which the expression vector contains
(i) a ds ribosomal DNA or part thereof e.g. a ds DNA
sequence that codes for a ribosomal RNA, and
(ii) a DNA sequence containing in the 5’—-> 3’ direction
in the following order:
(ii)(a) a powerful promoter operable in the host
organism,
(ii)(b) optionally a signal sequence facilitating the
secretion of said protein from the host
eukaryote,
(ii)(c) a structural gene encoding the protein,
(ii)(d) an efficient terminator operable in the host
eukaryote,
in addition to the sequences normally present in a
vector.
The ribosomal DNA can be ribosomal DNA’s occurring in
moulds, in particular moulds of the genera Aspergillus,
Rhizopus and Trichoderma, or those occurring in yeasts,
in particular yeasts of the genera Saccharomyces,
Kluyveromyces, Bansenula and Pichia.
Experiments have shown that the best results are
obtained when the vector has approximately the same
length as one ribosomal DNA unit of the host organism.
For example, if the ribosomal unit in the chromosomal
DNA is about 9 kb, vectors of about 14 kb or 5 kb were
not stably maintained, but vectors of about 8-10 kb were
stably maintained.
The promoter controlling the expressible gene is
preferably
(i) the Ga17 promoter, the GAPDH promoter, or the PGK
promoter, if the host belongs to the genus
Saccharomyces,
the inulinase promoter, the PGK promoter or the
LAC4 promoter, if the host belongs to the genus
Kluyveromyces,
(iii) the DHAS promoter or MOX promoter, if the host
belongs to the genus Hansenula,
the glucoamylase promoter, glucose-oxidase promoter
or the GAPDH promoter, if the host belongs to a
mould of the genus Aspergillus, or
the cellulase promoter or the GAPDH promoter, if
the host belongs to moulds of the genera Rhizopus
and Trichoderma.
If the structural gene encodes an oxidase, the host cell
preferably belongs to the genera Hansenula or Pichia or
Aspergillus.
Another preferred embodiment relates to a process in
which the expressible structural gene encodes the light
or heavy chain of an immuno-globulin or preferably both
genes, or part of the light or heavy chain of an
immunoglobulin, preferably that part coding for what
normally is called FAB fragment, or that part thereof
that codes for the variable regions. Related to this
embodiment is the use of a gene or genes modified by
genetic engineering resulting in modified immuno-
globulins or immunoglobulins with catalytic activity
(Abzymes).
(ii)
(iv)
or uracil, a nucleotide or a vitamin.
A process according to the invention can be carried out
as a normal batch fermentation, a fed—batch fermenta-
tion, or a continuous fermentation. It is preferred that
the medium contains the essential nutrient in such a
concentration that at least 20, but preferably at least
50, copies of the deficient gene are maintained in the
chromosome, said deficient gene encoding an enzyme
involved in the biosynthesis of that essential nutrient.
Good yields of the protein to be produced by the
transformed eukaryote can be obtained when the growth
rate of the host is between 20 and 100 %, preferably
between 80 and 100 %, of the maximum growth rate of a
similar host not deficient for said essential nutrient
under the same fermentation conditions.
BACKGROUND OF THE LIPASE ASPECT OF THE INVENTION
Lipases and proteases are both known as ingredients of
detergent and cleaning compositions. Proteases are
widely used.
Examples of known lipase—containing detergent composi-
tions are provided by EPA 0 205 208 and EPA 0 206 390
(Unilever) which relates to a class of lipases defined
on basis of their immunological relationship and their
superior cleaning effects in textile washing. The
preferred class of lipases contains lipases from a.o. P.
fluorescens, P. gladioli and Chromobacter species.
EPA o 214 751 (NOVO) and EPA o 258 063 (NOVO), each give
detailed description of lipases from certain microor-
ganisms, and also certain uses of detergent additives
and detergent compositions for the enzymes described.
EPA O 214 761 gives detailed description of lipases
derived from organisms of the specimen P. cepacia, and
certain uses therefor. EPA 0 258 068 gives detailed
description of lipases derived from organisms of the
genus Thermomyces (previous name Humicola) and certain
uses therefor.
A difficulty with the simultaneous incorporation of both
lipases and proteases into detergent compositions is
that the protease tends to attack the lipase.
Measures have been proposed to mitigate this disad-
vantage.
One such attempt is represented by EPA O 271 154
(Unilever) wherein certain selected proteases with
isoelectric points less than 10 are shown to combine
advantageously with lipases.
Another attempt is described in we 89/04361 (NOVO),
which concerns detergent compositions containing a
lipase from Pseudomonas species and a protease from
Fusarium or proteases of subtilisin type which has been
mutated in its amino acid sequence at positions 166,
169, or 222 in certain ways. It was reported that there
was some reduction in the degree of attack upon the
lipase by the particular proteases described.
THE LIPASE ASPECT OF THE INVENTION
The invention in one of its aspects provides lipases
produced by recombinant DNA techniques, which carry at
least one mutation of their amino acid sequences,
conferring improved stability against attack by
protease.
For example, the invention provides lipases showing
immunological cross-reactivity with antisera raised
against lipase from Chromobacter viscosum var.
lipolyticum NRRL B-3673 or against lipase from Pseudo-
monas fluorescens IAM 1057 and produced by an artifi-
cially modified microorganism containing a gene made by
recombinant DNA techniques which carries at least one
mutation affecting the amino acid sequence of the lipase
thereby to confer upon the lipase improved stability
against attack by protease.
The artificially modified microorganisms include
Escherichia coli, Pseudomonas aeruginosa, P. putida and
P. glumae in which the original gene for the lipase has
been deleted, Bacillus subtilis and various varieties of
the genus Aspergillus, Rhizopus and Tricboderma,
Saccharomyces cerevisiae and related species, Hansenula
polymorpha, Pichia and related species, Kluyveromyces
marxianus and related species. As these host cells
reflect a broad range of different micro—organisms other
microorganisms not described in detail in the examples
can be used as well as host cells.
The modified lipase can bring advantage in both activity
and stability when used as part of a detergent or
cleaning composition.
In such lipase, the mutation can for example be selected
from
introduction (e.g. by insertion or substitution) of
one or more proline residues at a location
otherwise vulnerable to proteolytic attack:
an increase of the net positive charge of the
lipase molecule (e.g. by insertion of positively-
charged amino acid residues or by substitution of
neutral or negatively—charged amino acid fesiduesx;
introduction (e.g. by insertion or substitution) of
a combination of amino acid residues of the lipase
capable of becoming glycosylated in the selected
host cell, thereby improving the stability of the
glycosylated lipase against proteolytic attack.
Also provided by the invention is a method for the
production of a modified microorganism capable of
producing an enzyme by recombinant DNA techniques,
characterized in that the gene coding for the enzyme
that is introduced into the microorganism is fused at
its 5’-end to a (modified) pre—sequence.
In particular embodiments of the invention, the gene of
bacterial origin is introduced with an artificial pre-
sequence into eukaryotic organisms.
Accordingly, in certain aspects the invention provides
artificially modified microorganisms containing a gene
coding for an enzyme and able to produce that enzyme
derived originally from one of the organisms mentioned
above or a modified form of such enzyme by use of
recombinant DNA techniques and fermentative processes
for enzyme production based on such artificially
modified microorganisms.
The fermentation processes in themselves apart from the
special nature of the microorganisms can be based on
known fermentation techniques and commonly used
fermentation and down stream processing equipment.
According to a further aspect of the present invention
it is found that modified (mutant) lipases from
Pseudomonas or another of the preferred class of
lipases, with amino acid sequence modification(s) chosen
to increase the stability of the enzyme to protease
digestion are of value in detergent and cleaning
compositions, especially for example in combination with
proteases, e.g. proteases of the subtilisin type.
A suitable and presently preferred example of such a
mutation is embodied in a mutant lipase from Pseudomonas
glumae with a His 154 Pro mutation, which is believed to
replace a site vulnerable to protease digestion in one
of the loops of the tertiary structure of the lipase
with a less vulnerable site.
According to a further aspect of the present invention
it is found that modified (mutant) lipases from
Pseudomonas or another of the preferred class of lipases
with amino acid sequence modification(s) chosen to
increase the net positive charge of the lipase and its
pI, are of value in detergent and cleaning compositions,
especially for example in combination with proteases,
e.g. proteases of the subtilisin type. '
Suitable mutations include for example the deletion of
negatively charged residues (e.g. aspartate or
glutamate) or their substitution by neutral residues
(e.g. serine, glycine and proline) or by the
substitution of neutral or negative residues by
positively-charged amino acid residues (e.g. arginine or
lysine) or the insertion of positively-charged residues.
Suitable examples of such mutations increasing the net
positive charge and pI include D157R, D55A and I110K.
Suitable examples of the introduction (e.g. by insertion
or substitution) of a combination of amino acid residues
capable of becoming glycosylated in the selected host
and thereby improving its stability against proteolytic
attack are given by mutations D157T and insertion of G
between N155 and T156.
To avoid over-glycosylation or to remove glycosylation
on less desirable positions the potential glycosylation
sites of the original lipase can be removed.
Within the preferred class of lipases the lipase
produced by Pseudomonas glumae (formerly and more
usually called Pseudomonas gladioli) is a preferred
basis for the processes and products of this invention.
Neither the amino acid sequence nor the nucleotide
sequence of the gene coding for the preferred lipase was
previously known. The present inventors have isolated
the gene coding for the preferred lipase of this
bacterium as will be illustrated below.
The invention also provides genetic derived material
from the introduction of this gene into cloning vectors,
and the use of these to transform new host cells and to
express the lipase gene in these new host cells.
Usable heterologous new host cells include for example
Escherichia coli, Pseudomonas aeruginosa, P. putida.
Also P. glumae in which the original lipase gene has
been deleted is a suitable host. The preferred host
systems for large scale production are Bacillus
subcilis, Saccharomyces cerevisiae and related species,
Kluyveromyces marxianus and related species, Hansenula
polymorpha, Picbia and related species and members of
the genera Aspergillus, Rhizopus and Trichoderma. Also
suitable hosts for large scale production are Gram (—)
bacteria specially selected and/or modified for
efficient secretion of (mutant) lipases.
As these host cells reflect a broad range of different
microorganisms other microorganisms not described in
detail in the examples can be used as well as host
cells. —
Another embodiment of the invention relates to vectors
able to direct the expression of the nucleotide sequence
encoding a gene coding for—an¥en;yme as described4above
in one of the preferred»hosts*preferably-comprise:
ds DNA coding for mature enzyme or pre—enzyme
directly down stream of a (for the selected host
preferred) secretion signal; in cases where the
part of the gene that should be translated does not
start with the codon ATG, an ATG should be placed
in front. The translated part of the gene should
always end with an appropriate stop codon;
an expression regulon (suitable for the selected
host organism) situated upstream of the plus
strand of the ds DNA of (a);
a terminator sequence (suitable for
host organism) situated down stream
strand of the ds DNA of (b);
nucleotide sequences which facilitates integration,
preferably multicopy integration, of the ds DNA of
(a—c) into the genome of the selected host which
host is deficient for an essential nutrient. The
nucleotide sequence that facilitates multicopy
integration is ds ribosomal DNA or at least part of
this sequence. Moreover a ds DNA sequence
containing the deficient gene coding for—the
enzyme that is absent in the host cell has to
be present on the integration vector, and
optionally a ds DNA sequence encoding proteins
involved in temporary inactivation or unfolding
and/or in the maturation and/or secretion of one of
the precursor forms of the enzyme in the host
selected.
the selected
(C)
of the plus
The invention will be illustrated by the following
examples.
Example 1. Isolation and characterization of the
gene encoding (pre)-lipase of P. glumae.
Example 2. Construction of the lipase negative P.
glumae strains PG2 and PG3.
Example 3. Construction of a synthetic gene encoding
P. glumae (pre)—lipase.
Example 4. Introduction of the (wild type) synthetic
lipase gene in the lipase negative P. glumae PG3.
Example 5. Production of mutant lipase genes and
their introduction in PG3.
Example 6. Expression of the synthetic lipase genes
in Saccharomyces cerevisiae using autonomously replicat-
ing smids.
Example 7. Expression of synthetic lipase genes in
saccharomyces cerevisiae using multicopy integration.
Example 8. Production of guar.a-galactosidase in
Saccharomyces cerev1siae.uSing multicopy integration.’
Example 9. Multicopy integration in Saccharomyces
cerevisiae using other deficient selection markers.
Example 10. Stability of the multicopy integrant in
continuous cultures .
Example 11. Parameters affecting the stability of
multicopy integrant SUSOB
Example 12. Expression of the synthetic lipase genes
in Hansenula polymorpha.
Example 13. Production of guar a-galactosidase in
Hansenula polymorpba using multicopy integration.
Example 14. Multicopy integration in Kluyveromyces.
Examples 1-6 and 12 relate to the isolation, cloning and
expression of lipase genes in the yeasts Saccharomyces
cerevisiae and Hansenula polymorpha using plasmid
vectors.
Example 7 relates to the expression of a lipase gene in
the yeast Saccharomyces cerevisiae after multicopy
integration of an expression vector according to the
invention.
Examples 9-11 relate to other aspects of the multicopy
integration.
Examples 8 and 13 relate to the expression of guar a-
galactosidase in the yeasts Saccharomyces cerevisiae and
Hansenula polymorpha.
Example 14 shows that multicopy integration can be
achieved in a Kluyveromyces yeast.
EXAMPLE 1. ISOLATION AND CHARACTERIZATION OF THE
GENE ENCODING (PRE)°LIPASE OF P. GLUHAE.
Isolation of P. glumae chromosomal DNA.
Cells of a 15 ml overnight culture in LB medium were
collected by centrifugation (sorvall HB4 rotor, 10,000
rpm for 10 min). The cell pellet was stored at — 20 °C
overnight.
After thawing the cells were re—suspended in 10 ml SSC
(0.15 M NaCl, 0.015 M Na-citrate) containing 2 mg/ml
lysozyme. After incubation for 30 min at 37 °C, 0.5 ml
of 10% SDS was added, followed by an incubation at 70 °C
for 10 min. After cooling to 45 °C, 1 ml proteinase K (2
mg/ml Tris—HCl pH 7.0, pre-incubated for 30 min at 45
°C) was added and the mixture was incubated at 45 °C for
another 30 min. Next, 3.2 ml 5 M NaClO4 was added,
followed by two extractions with 15 ml CHC13/iso-C5H11OH
(24:1), each of which was followed by a centrifugation
step (Sorvall H54, 5,000 rpm/10 min). The DNA was
precipitated from the supernatant by adding 10 mlt
ethanol. After a wash in:75% ethanol the, DNA pellet was.
re—suspended in 2 ml H20.
Preparation of a gene bank.
A DNA preparation of P. glumae was partially digested
with the restriction enzyme Sau3A, as described by
Maniatis (7). The cosmid vector c2RB (8) was digested
to completion with SmaI and BamHI, both enzymes having
one recognition site in the cosmid. Excess vector
fragments were ligated, using T4 DNA ligase (in 50 mM
Tris—HCl pH 7.5, 10 mM dithiotreitol (DTT), 10 mM MgCl2
and 0.5 mM rATP), with DNA fragments from P. glumae. The
recombinant DNA thus obtained was packaged in phage par-
ticles as described by Hohn (9). The complete phage
particles obtained this way were used to transform E.
coli 1046 met, gal, lac, hsdR, pbx, supE, hsdM, recA)
by transfection.
Sml fresh LB medium containing 0.4 % maltose, was
inoculated with 0.5 ml of a overnight culture of E. coli
1046 and incubated for 6 h. at 37 °C under continuous
shaking. Before infection with phage particles, MgCl2
and CaCl were added to a final concentration of 10 mM.
In a typical experiment 50 pl phage particles were mixed
with 50 pl of cells and the mixture was incubated at 37
°C for 15 min: 100 pl LB medium was added and incubation
at 37 °C continued for 30 min. The cells were plated
directly an LB—agar plates containing 75 pg/ml ampicil—
lin (Brocacef). After overnight growth at 37 °C ca. 300
colonies were obtained.
oligonucleotide svnthesis.
As probes for the lipase encoding DNA fragment, we used
oligonucleotides based on the sequences of the 24 N-
terminal amino acids (see below), determined by Edman
degradation, using an Applied Biosystems Gas Phase
Protein Sequencer.
Based on the established amino acid sequence, all the
possible nucleotide sequences encoding the amino acid
sequence were derived. Deoxy-oligonucleotides containing
all or part of the possible nucleotide sequences (so
called mixed-probes) were synthesized on a DNA syn-
thesizer (Applied Biosystems 380 A) using the Phospho-
amidit technique (10). oligonucleotides were purified on
% or 20% polyacrylamide gels (7).
Radio-labeled oligonucleotide probes.
Typically, 0.1-0.3 pg of the purified oligonucleotide
was labelled by incubation for 30 minutes at 37 °C in 50
mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 10 mM DTT,
70 pCi gamma-32P-ATP (3000 Ci/mmol, Amersham) and 10
units T4 polynucleotide kinase (Amersham) in a final
volume of 15 pl. The reaction was terminated with 10 pl
0.5 M EDTA pH 8.0 and.passed through a Sephadex G25
column of 2.5 ml (disposable syringe) equilibrated_with
TE buffer (10 mM Tris-Hcl pH 8.0 and 1 mM EDTA).
Fractions of 250 pl were collected, from which the
first two radioactive fractions, usually fractions 4 and
, were pooled and used for hybridization.
Screening of the gene bank.
From several packaging and transfection experiments,
performed as described above, a total of ca 1000
separate colonies were obtained. These colonies were
transferred to ELISA plates (Greiner, F—form) containing
150 pl LB-medium (100 pg ampicillin/ml)/well. After
overnight growth at 37 °C duplicates were made using a
home-made template, consisting of 68 pins, arranged to
fit in the microtiter wells. To the wells of the
masterplates 50 pl 50 % glycerol was added, and after
careful mixing with the aid of the template, these
plates were stored at -80 °C.
"The duplicates were used to transfer the gene bank to
nitro—cellulose filters (Millipore, type HATF, 0.45 pm,
¢ 14 cm). To this end the cellulose filters were pre-
wetted by laying them on LB-agar plates with 100 pg/ml
ampicillin. After transfer of the bacteria with the aid
of the template, colonies were grown overnight at 37 °C.
The colonies on the filters were lysed by placing them
on a stack of Whattman 3 MM paper, saturated with 0.5 M
NaOH, 1.5 M NaCl for 15 min. After removal of excess
liquid by placing the filters on dry paper, they were
neutralised by placing them on a stack of 3 MM paper,
saturated with 1 M Tris—HCl pH 7.0, 1.5 mM NaCl for 2-3
min. Finally the filters were dunked into 10 x SSC (1.5
M Nacl, 0.15 M Na—citrate) for 30 sec, air dried and
baked at 80 °C under vacuum for 2 hours. Prior to
(pre)hybridization the filters are washed extensively in
3 x ssc, 0.1% sos at 65 °c for 16-24 h with several
changes of buffer. The washing was stopped when the
colonies were no longer visible.
Pre-hybridization of the filters was performed in 5 x
SSC, 5 x Denhardts (10 x Denhardts = 0.2 % ficoll, 0.2 %
polyvinyl-pyrrolidone, 0.2 % bovine serum albumin), 0.1
% SDS, 50 mM sodium phosphate pH 7.5, 1 % glycine, 100
pg/ml calf-thymus DNA (sheared and heat denatured), 500
pg/$1 tRNA and 50 % de-ionized formamide for 2 hours at
37 C.
Hybridization with a radio—active labelled (see above)
mixed probe (visO2, 32 nucleotides) was performed in 5 x
SSC, 1 x Denhardts, 0.1 % SDS, 20 mM sodium phosphate pH
7.5, 100 pg/ml calf—thymus DNA, 500 pg/ml tRNA and 50 %
deionized formamide, for 16 h. at 39 °C. After the
hybridization, the filters are washed: 3 x 15 min with 6
x SSC at room temperature, 1 x 15 min 2 x ssc, 0.1% SDS
and subsequently at a room temperature dependent on the
properties of the oligonucleotide probe. For vis02
washing was extended.for_l5 min at 37 90 in preheated
.1 SSC 0.1% SDS.
Upon screening the gene bank as described above, several
cosmid clones were isolated. Clone 5G3 (hereinafter
called pUR6000) was chosen for further investigations.
Sequencing of the lipase qene.
DNA fragments resulting from digestion of pUR6000 with
BamHI were ligated in plasmid pEMBL9 (11) which was
also cleaved with BamHI and the obtained recombinant
DNA was used to transform E. coli JMl0l (12), with the
CaCl2 procedure and plated on LB-agar plates
supplemented with X-gal and IPTG (7).
white colonies were transferred to microtiter plates
and subjected to the same screening procedure as
described for the cosmid bank. Several positive clones
could be isolated. A representative plasmid isolated of
one of these colonies is depicted in Fig. 1 and is
referred to as pUR6002. Upon digesting this plasmid with
EcoRI, two fragments were found on gel, ~4.1 kb and
~2.1 kb in length, respectively. Another plasmid,
pUR6001, contained the BamHI fragment in the opposite
orientation. After digestion with EcoRI, this plasmid
resulted in fragments with a length of ~6.l kb and ~70
bp, respectively.
In essentially the same way pUR6006 was constructed. In
this case pUR6000 was digested with EcoRI after which
the fragments were ligated in the EcoRI site of plasmid
pLAFRI (13). After screening the transformants, a
positive clone was selected, containing a EcoRI
fragment of ~6 kb, designated pUR6006 (Fig. 1).
The purified DNA of pUR6001 and pUR6002 was used for the
establishment of the nucleotide sequence by the Sanger
dideoxy chain termination procedure (14) with the
modifications as described by Biggin et al. (15), using
alpha—35S—dATP (2000Ci/mmol) and Klenow enzyme (Amer-
sham), ddNTP’s (Pharmacia—PL Biochemicals) and dNTP’s
(Boehringer). We also used the Sequenase kit (United
states Biochemical Corporation), with substitution of
the dGTP for 7~deaza-dGTP. The sequencing reaction
products were separated on a denaturing polyacrylamide
gel with a buffer gradient as described by Biggin et al.
(15).
The complete nucleotide sequence (1074bp) of the P.
glumae lipase (hereafter also called: glumae lipase)
gene is given in Fig. 2.
The nucleotide sequence contains an open reading frame
encoding 358 amino acid residues followed by a stop
codon.
The deduced amino acid sequence is shown in the IUPAC
one-letter notation below the nucleotide sequence in
Fig. 2.
The NH2-terminal‘amino acid sequence of the lipase ‘
enzyme as purified from the P- glumae culture broth has
been identified as AlaAspThrTyrAlaA1aThrArgTyrProVal—
I1eLeuValHisGlyLeuAlaGlyThrAspLys (= ADTYAATRYPV—
ILVHGLAGTDK). This amino acid sequence is encoded by
nucleotides 118-183 (Fig. 2). Firstly, from these
findings it can be concluded that the mature lipase
enzyme is composed of 319 amino acid residues, and has a
calculated molecular weight of 33,092 dalton.
secondly, the enzyme is synthesized as a precursor, with
a 39 amino acid residue N—terminal extension (numbered -
39 to -1 in Fig. 2).
From the scientific literature it is well known that
most excreted proteins are produced intracellular as
precursor enzymes (16). Most commonly these enzymes have
a N-terminal elongation, the so-called leader peptide or
signal sequence. This peptide is involved in the initial
interaction with the bacterial membrane.
General features of the signal sequence as it is found
in gram negative bacteria are:
. an amino-terminal region containing (on average) 2
positively charged amino acid residues;
. a hydrophobic sequence of 12 to 15 residues;
. a cleavage site region, ending with serine, alanine
or glycine
. the total length is approximately 23 amino acids.
Surprisingly, the lipase signal sequence comprises 39
amino acids, which is rather long. Furthermore, it
contains four positively charged amino acids at the N-
terminus.
For gram negative bacteria, this seems to be an
exceptional type of signal sequence.
Isolation of genes from other organisms, encoding
related lipases.
As mentioned earlier, the P. glumae lipase belongs to a
group of immunologically related lipases. From this it
can be expected that these enzymes, although produced by
different organisms, contain stretches of highly
conserved amino acids sequences.
As a consequence there has to be certain degree of
homology in the DNA-sequence.
Having the P. glumae lipase gene at our disposal, it is
easy to isolate related lipase genes from other
organisms.
This can be done in essentially the same way as
described above.
From the organism of interest a gene bank (for example
in a cosmid or phage Lambda) is made. This genome bank
can be screened using (parts of) the -2.2 kb BamHI
fragment (described above) as a probe. Colonies giving a
positive signal, can be isolated and characterized in
more detail.
EXAMPLE 2. CONSTRUCTION OF THE LIPASE NEGATIVE P.
GLUHAE STRAINS PG2 AND PG3.
The construction of PG2, from which the lipase gene has
been deleted; and PG3, in which the lipase gene has been
replaced with a tetracycline resistance (Tc—res) gene,
comprises three main steps.
A ~ construction of pUR6l06 and pUR6l07 (in E. coli),
starting from pUR6001 (see example 1):
pUR6001 contains a BamHI fragment from the P. glumae
chromosome of ~2.2kb. The lipase gene (1074 base pairs)
situated on this fragment, has a 5’— and a 3’— flanking
sequence of ~480 and ~660 base pairs, respectively.
Subsequent construction steps were:
a. partial digestion of pUR6001 (isolated from E. coli
KA816 dam-3, dcm-6, thr, leu, chi, LacY, galK2,
galT22, are-14, tonA31, tsx-78, supE44) (also named
GM418 [17]) with C1aI, to obtain linearized
plasmids
b. phenol extraction and ethanol precipitation (7) of
the DNA, followed by digested with PstI
c. isolation of a 4.5 kb plasmid DNA fragment (having
C131 and a PstI sticky ends), and a PstI fragment
of ~670 bp from agarose gel after gel electropho-
resis followed by electro-elution in dialysis bags
(7) ’
d. the obtained plasmid DNA fragment with a C131 and
a PstI sticky end was ligated with a synthetic
linker fragment (shown below), with a C1aI and a
PstI sticky end.
C1aI CGAIGAGAflCETCEflCE£EGCA PstI
T%CKH$G¥KEMHG
This synthetic fragment contains a recognition site
for the restriction enzymes Bell and Bg1II.
After transformation of the ligation mixture to E.
coli SAl01 (is JM10l with recA, hdsR), selection
on LB-Ap (100 pg ampicillin/ml) agar plates, and
screening of the plasmids from the obtained
transformants by restriction enzyme analysis, a
correct plasmid was selected for the next construc-
tion step. Upon digesting this correct plasmid with
BamHI and HindIII a vector fragment of ~4kb and an
insert fragment of ~500 bp were found.
e. the plasmid construct obtained as described in d.
was digested with PstI, and ligated together with
the ~670 bp Pstl fragment isolated as described in
50 c.
f. transformation of the ligation mixture to E. coli
SA101, selection on LB-Ap (100 pg ampicillin/ml)
agar plates, and screening of the plasmids from the
obtained‘transformants. Since the PstI fragment
can have two different orientations this had to be
analysed by means of restriction enzyme analysis"
In the construct we were looking for, the orienta-
tion should be thus that digestion with BamHI
results in a vector fragment of ~4 kb and an
insert-fragment of ~1.2 kb.
A representative of the correct plasmids is
depicted in Fig. 3 and was called pUR6102.
pUR6102 was digested to completion with Bg1II
pBR322 (18) was digested to completion with AvaI
and EcoRI, after which the DNA fragments were
separated by agarose gel electrophoresis. A
fragment of ~l435 base-pairs, containing the
tetracycline resistance gene was isolated from the
gel by electro-elution
upon filling in the sticky ends (in a buffer
containing 7 mM tris-Hcl pH7.5, 0.1 mM EDTA, 5 mM
fi—mercapto-ethanol, 7 mM MgCl2, 0.05 mm dNTPs and
0.1 ¢/pl Klenow polymerase) of the DNA fragment
containing the Tc—res gene and the linearized
pUR6102 they were ligated.
transformation of E. coli SA101 with the ligation
mixture, selection on LB-Tc (25pg tetracycline/ml)
agar plates, and screening of the plasmids from the
obtained transformants by restriction enzyme
analysis.
The construction route of pUR6102 and pUR6103 is
depicted in Fig. 3.
pUR6102 was digested with BamHI and pUR6103 was
partially digested with BamHI; the obtained
fragments were separated by agarose gel electropho-
resis and the desired fragments (~1145 bp and ~255O
bp resp.) were isolated out of the gel by electro-
elution.
pRZl02 (19) was digested to completion with BamHI
and ligated to the BamHI fragments obtained in
step k.
transformation of the ligation mixtures to E. coli
Sl7—1 (20), selection on LB-km,Tc (25 p/ml each)
and screening of the plasmids from the obtained
transformants, by restriction enzyme analysis. The
resulting plasmids were called pUR6106 and pUR6107
(Fig. 4), respectively.
Deletion of the lipase gene of the P. glumae
chromosome.
Introduction of pUR6106 in P. glumae via biparental
conjugation with E. coli S17—1(pUR6l06) (which is
the notation for E. coli S17-1 containing plasmid
pUR6106) .
A P. glumae colony was transferred from a MME plate
(0.2 g/l MgSO4—7H2O, 2 g/l Citrate—H2O, 10 g/l
KZHPO4, 335 g/l NaNH4HPO4.4H20, 0.5%‘glucose and
1.5% agar) to 20 ml Luria Broth (LB) culture medium
and grown overnight at 30 9C. E. col; S175
1(pUR6106) was grown overnight in 3 ml is medium,
pg/ml Km, at 37 °C.
The next day the P. glumae culture was diluted 1:1
and grown for 4 to 5 hours at 30 °C until OD660 is
2.0 -0 2.5. E. coli s17-1 (pUR6106) was diluted 1:50
and grown for 4 to 5 hours at 37 °C until OD660 is
1.5 - 2.0
For the conjugation 50 OD units (1 unit = 1 ml with
OD = 1) (20 to 25 ml) P. glumae cells and 2.5 OD
units (1.2 - 1.6 ml) E. coli s17-1 (pUR6106) were
mixed and spun down for 10 min at 5,000 rpm (H54-
rotor). The cell pellet was divided over 3 LB
plates and incubated overnight at 30 °C.
Subsequently the cell material was removed from the
plate and re-suspended in 3 ml 0.9% Nacl solution
and pelleted by centrifugation (10 min, RT, HB4-
rotor, 4krpm).
The cell pellet was re-suspended in 1.8 ml 0.9%
Nacl solution and divided over 3 plates MME, 0.5%
glucose, 1.5% agar, 50 pg/ml kanamycin (Km) and
grown at 30 °C.
Since pUR6106 does not replicate in P. glumae, Km
resistant trans-conjugants can only be obtained by
integration. In these strains the plasmid pUR6106
is integrated into the bacterial chromosome by a
single recombination event at the 5’— or 3’-
flanking region. Due to the fact that these strains
still contain a functional lipase gene, their
phenotype is lipase positive.
Two such strains (PG-RZ21 and PG—RZ25) were
selected for further experiments.
To delete the plasmid and the functional lipase
gene out of the chromosomal DNA, a second recom-
bination event should take place. This can be
achieved by growing said strains for several days
on LB-medium without Km (without selective
pressure), plate the cells on BYPO-plates (10 g/l
trypticase peptone, 3 g/l yeast extract, 5 g/l beef
extract, 5 g/l Nacl, 7 g/l KHZPO4, 50 ml/1 olive
oil emulsion and 1.5% agar) in a density which
assures separate colonies, and screen for lipase
negative colonies. Upon plating these lipase
negative colonies on selective plates (MME—KM 50
pg/ml), they should not grow. A strain obtained in
this way could be called PG-2.
Replacement of the lipase gene of the P. glumae
chromosome bv the Tc-res gene.
Introduction of pUR6107 in P. glumae via
conjugation with E. coli S17-1 (pUR6107) as
described in B. Selection of trans-conjugants
was performed at 30 °C on MME-medium contain-
ing 50 pg/ml Tc.
Trans—conjugants obtained in this way were dupli-
cated to BYPO—plates containing 50 pg/ml Tc and to
MME—plates containing 100 pg/ml Km. Several trans-
conjugants exhibited a Km sensitivity (no growth on
MME Km—100 plates) and lipase negative (no clearing
zone on BYPO—p1ates) phenotype. Due to a double
cross over (at the 5’- and at the 3’—flanking
region) the lipase gene was replaced by the Tc
resistance gene.
One representative strain was selected for further
investigation and was called PG—3.
EXAMPLE 3. CONSTRUCTION OF A SYNTHETIC GENE ENCODING
P. CLUHAE (PRE)'LIPASE.
Based on the nucleotide sequence of the P. glumae (pre)-
lipase gene a new gene was designed, containing several
silent mutations. Due to these mutations the amino acid
sequence of the enzyme was not changed. It was however
possible to lower the GC-content, which facilitates
enzyme engineering and enabled us to use the synthetic
gene in a variety of heterologous host systems.
An other point, facilitating enzyme engineering, was the
possibility to introduce restriction enzyme recognition
sites at convenient positions in the gene.
The sequence of the new gene is given in Fig. 5(A).
The new gene was divided in restriction fragments of
approximately 200 nucleotides, so—called cassettes. An
example of such a cassette is depicted in Fig. 6.
Each cassette was elongated at the 5’ and 3' end to
create an EcoRI and HindIII site respectively.
The coding strands of these cassettes were divided in
oligo-nucleotides (oligos) with an average length of 33
bases. The same was done for the non coding strands, in
such a way that the oligos overlapped for ~ 50% with
these of the coding strand.
The oligos were synthesized as described in example 1.
Before assembling the fragments, the 5’ ends of the
synthetic oligos had to be phosphorylated in order to
facilitate ligation. Phosphorylation was performed as
follows:
Equimolar amounts (50 pmol) of the oligos were pooled
and kinated in 40 pl reaction buffer with 8 Units
polynucleotide kinase for 30-45 minutes at 37 °C. The
reaction was stopped by heating for 5 minutes at 70 °C
and ethanol precipitation.
subsequently the mixture was placed in a water bath at
65 °C for 5 minutes, followed by cooling to 30 °C over a
period of 1 hour. Mgclz was added to a final concentra-
tion of 10 mmol/1. T4 DNA—Ligase (2.5 Units) was added
and the mixture was placed at 37 °C for 30 minutes or
o/n at 16 °C. After this the reaction mixture was heated
for 10 minutes at 70 °C.
After ethanol precipitation the pellet was dissolved in
digestion buffer and cut with EcoRI and HindIII.
The mixture was separated on a 2% agarose gel and the
fragment with a length corresponding to the correctly
assembled cassette was isolated by electro-elution.
The fragments were ligated in pEMBL9 (digested with
EcoRI/HindIII) as described in example 1, and they were
checked for correctness by sequence analysis. In
subsequent cloning steps the various cassettes were put
together in the proper order, which resulted in pUR6038.
This is a pEMBL9 derivative containing the complete
synthetic lipase gene.
To be able to make the constructions as described in
example 4, a second version of the synthetic gene was
made, by replacing fragment 5. In this way construct
pUR6600 was made, having the 3' PstI site at position
1069 instead of position 1091 (See Fig. 5B).
EXAMPLE 4. INTRODUCTION OF THE (WILD TYPE) SYNTHETIC
LIPASE GENE IN THE LIPASE NEGATIVE P. GLUHAE PG3.
In order to test whether the synthetic lipase gene is
functional in P. glumae, the gene was introduced in
strain PG3.
To simplify fermentation procedures, it was decided to
stably integrate this gene in the PG3 chromosome, rather
than introducing on a plasmid.
For this reason the synthetic lipase gene had to be
equipped with the 5’ and 3’ border sequences of the
original P. glumae lipase gene.
This was achieved in the following way (see Fig. 7):
a. From pUR6002 (ex E. coli KA816) a vector with ClaI
and PstI sticky ends was prepared in the same way
as described in example 2.
b. pUR6600 (ex E. coli KA816) was digested to
completion with C1aI and partial with PstI. After
separating the fragments by agarose gel electropho-
resis a fragment of ~1050 bp was isolated.
c. The fragment thus obtained, was ligated in the
pUR6002 derived vector and used to transform E.
coli SAIOI. In this way construct pUR6603 was
obtained.
d. pUR6603 was digested to completion with BamHI,
After separating the fragments by agarose gel _
electrophoresis a fragment of r2.2kb was isolated.
lipase gene
This fragment contains the synthetic
of the wild
with the 5' and 3' flanking regions,
type P. gladioli lipase gene.
e. pRZ102 was also digested to completion with BamHI.
f. The 2.2 kb fragment obtained in d. was ligated in
pRZ102 as described in example 2. ‘
g. The resulting construct, pUR6131 was transferred to
E. coli S17-l.
Integration of this construct in the chromosome of PG3
was accomplished in the same way as described for
pUR6106 in example 2 section B-a.
From the obtained Km-resistant trans—conjugants, several
were transferred to BYPO plates. They all appeared to
have the lipase positive phenotype, since clearing zones
occurred around the colonies. A typical representative
was called PGL26.
Obviously the same route can be followed to integrate
construct (pUR6131) in a lipase negative P. glumae PG2
(see example 2B—b) strain.
From the examples 2 and 4 it might be clear that the P.
glumae strain PG1 (and derivatives thereof, e.g. PG2
and PG3; or derivatives of PG1 obtained via classical
mutagenesis having an improved lipase production) can be
manipulated easily by deleting or introducing (homolo-
gous or heterologous) DNA fragments in the bacterial
chromosome.
By using these techniques it is possible to construct a
strain optimized for the production of lipase. In this
respect one could think of:
— replacing the original lipase promotor, by a
stronger (inducible) promotor,
- introduction of more than one copy of the lipase
gene (eventually, encoding different lipase
mutants),
- replacing the original promoter, or introduction of
more copies of genes encoding functions involved in
the production and excretion of the lipase enzyme
(eg. chaperon proteins, "helper proteins" involved
in the export of the lipase enzyme),
— deletion of the gene encoding extracellular
protease (A Tn5 mutant of PG1 (PGT89) which does
not produce a clearing zone or skimmilk plates has
been deposited),
— manipulating the rhamnolipid production.
EXAMPLE 5. PRODUCTION OF MUTANT LIPASE GENES AND
THEIR INTRODUCTION IN PG3.
To improve the lipase, it is necessary to have the
possibility to introduce well-defined changes in the
amino acid sequence of_the_protein.
A preferred method to achieve this is via the replace-
ment of a gene fragment of the synthetic gene encoding‘
wild type lipase or of the wild type P. glumae lipase
gene, with a corresponding chemically synthesized
fragment containing the desired mutation.
In the case of the synthetic wild type lipase gene, a
cassette (or fragment thereof) can be replaced with a
corresponding cassette (or fragment thereof) containing
the desired mutation.
The cassette, comprisir~ the codon(s) for the amino
acid(s) of interest, wa: assembled once more (as
described in Example 3). This time however, the oligos
of the coding and the non-coding DNA strands, comprising
the codon(s) of interest, were replaced by oligomers
with the desired mutation. The new oligos were syn-
thesised as described in Example 1.
The thus obtained mutant cassette, or a fragment thereof
was introduced at the corresponding position in the
synthetic wild type lipase gene of constructs like
pUR6038 or pUR6603.
To introduce a synthetic mutant lipase gene in PG2 or
PG3, the route as described in Example 4 has to be
followed, starting at step d.
A typical example of the production of a mutant gene is
described below. In this case the His at position 154 of
the wild type lipase gene has been replaced by a Pro.
To accomplish this, two new oligomers were synthesized.
The codon encoding amino acid 154 of the mature lipase
is changed to CCT.
These oligomers were used to assemble fragment 3(H154P),
as described in example 3. After cloning the fragment in
pEMBL9, the DNA sequences was determined as described in
example 1. The thus obtained construct was called
UR607l.
Plasmid pUR6071 was digested to completion with FspI
and Sa1I. Upon separation of the obtained DNA fragments
via gel electrophoresis (as described in example 2), a
fragment of ~90 bp was isolated out of agarose gel.
pUR6002 was partially digested with FspI and partially
with Sa1I. After gel electrophoresis a vector of ~6000
nucleotides was isolated out of the agarose gel in
example 2.
The isolated ~90 bp fragment was ligated in the pUR6002
vector to obtain pUR6077A.
The BamHI fragment (~2200 bp) of pUR6077A was ligated
in pRZl02 as described in examples 3 and 4. In this way
pUR6127 was obtained.
Introduction of this construct into the chromosome of
PG3 was accomplished as described in example 4. A
resulting lipase producing P. glumae trans-conjugant,
was called PGL24.
The modified lipase produced by this strain proved to be
significantly more stable than the parent lipase in an
actual detergents system (Fig. 8).
In essentially the same-way several'other,mutant lipase
genes have been made. In some cases this resulted.in a
altered net charge of the encoded protein (eg. Dl57R
(+2), DSSA (+1), I1lOK (+1), R6lP (-1), Tl09D (-1), R8D
(-2)). In other cases amino acids have been introduced
or deleted (eg. PGL40 in which 152$-154H has been
replaced by A1aLeuSerGlyHisPro = ALSGHP).
Furthermore potential glycosylation sites have been
removed (eg. N485 and/or N238S) and/or introduced (eg.
Dl57T and insertion of G between N155 and T156).
EXAMPLE 6. EXPRESSION OF THE SYNTHETIC LIPASE GENES IN
SACCHAROMYCES CEREVISIAE USING AUTONOMOUSLY REPLICATING
PLASMIDS.
To illustrate the production of P. glumae lipase by
eukaryotic micro—organisms, vectors suited for expres-
sion of P. glumae lipase in the yeast S. cerevisiae
using the GAL7 promoter (21) were constructed. The P.
glumae lipase is produced by the yeast S. cerevisiae
using two different expression systems. An expression
system based on autonomously replicating plasmids with
the lipase expression cassette and an expression system
based on multicopy integration of the lipase expression
cassette in the host genome.
The plasmid pUR2730 (21) was used as the basis for the
lipase expression plasmids. The plasmid pUR2730 consists
of the GAL7 promoter, S. cerevisiae invertase signal
sequence, a—galactosidase gene (the a-galactosidase
expression cassette), 2pm sequences for replication in
S. cerevisiae, the LEU2d gene for selection in S. cerev-
isiae and pBR322 sequences for replication and selection
in E. coli-
The plasmid pUR6038 was used as the source for the
lipase gene.
The following S. cerevisiae expression plasmids were
constructed, encoding:
. mature lipase preceded by the invertase signal
sequence (pUR6801), _
2. mature lipase preceded by a KEX2 cleavage site, a
glycosylation site and the invertase signal
sequence (pUR6802).
In order to obtain the above mentioned constructs, the
routes followed were (Fig. 9; the used restriction
recognition sites are marked with an asterisk):
ad 1 and 2.
a. The plasmid pUR2730 was digested with SacI and
HindIII and the vector fragment was isolated.
b. The plasmid pUR6038 was digested with EcoRV and
HindIII and the fragment with theglipase gene was
isolated.
c. Synthetic SacI-EcoRV DNA fragments were synthesized
and constructed as described in example 3,
consisting of the following sequences:
In the case of pUR680l:
I.
' CATCACACAAACAAACAAAACAAAATGATGCTTTTGCAAGCCTTCCTTTTCCTT-
' TCGAGTAGTGTGTTTGTTTGTTTTGTTTTACTACGAAAACGTTCGGAAGGAAAAGGAA-
-TTGGCTGGTTTTGCAGCCAAAATATCTGCCGCGGACACATATGCAGCTACGAGAT 3'
-AACCGACCAAAACGTCGGTTTTATAGACGGCGCCTGTGTATACGTCGATGCTCTA 5’
This fragment gives a correct junction of the GAL7
promoter and the lipase gene with in between the
sequence encoding the invertase signal sequence.
In the case of pUR6802:
II.
' CATCACACAAACAAACAAAACAAAATGATGCTTTTGCAAGCCTTCCTTTTCCT-
’ TCGAGTAGTGTGTTTGTTTGTTTTGTTTTACTACGAAAACGTTCGGAAGGAAAAGGA-
—TTTGGCTGGTTTTGCAGCCAAAATATCTGCCTCCGGTACTAACGAAACTTCTGAIAA-
-AAACCGACCAAAACGTCGGTTTTATAGACGGAGGCCATGATTGCTTTGAAGACTATT-
-GAGATGAAGCGAAGCTGCTGACACATATGCAGCTACGAGAT 3’
-CTCTCTTCGACTTCGACGACTGTGTATACGTCGAIGCTCTA 5’
This fragment gives a correct junction of the GAL7
promoter and the lipase gene with in between the
sequences encoding a KEX2 cleavage site, a glycosylation
site and the invertase signal sequence.
d. The sacI-HindIII vector fragment, one of the sacI-
EcoRV synthetic fragments (I) and the EcoRV~
HindIII DNA fragment with the lipase gene were
ligated. For the construction of pUR6801 this is
shown in Fig. 9. (pUR6802 is constructed in the
same way, using synthetic fragment II)
e. The ligation mixture was transformed to E. coli.
From single colonies, after cultivation, the
plasmid DNA was isolated and the correct plasmids,
as judged by restriction enzyme analysis, were
selected and isolated in large amounts.
f. The plasmids pUR6801 and pUR6802 were transformed
to S. cerevisiae strain SUIO (21) using the
spheroplast procedure (22) using selection on the
presence of the LEU2d gene product.
g. The transformants were grown overnight in defined
medium ( 0,68% Yeast Nitrogen Base w/o amino acids,
2% glucose, histidine and uracil), diluted 1 : 10
in induction medium (1% yeast extract,_2% bacto-
peptone, 5% galactose) and grown for 40 “
hours. ;,_“;,
h. The cells were isolated by centrifugation and cell
extracts were prepared (23).
i. The cell extracts were analysed by SDS-gel-
electrophoresis (7) and blotted on nitrocellulose.
j. The nitrocellulose blots were incubated with lipase
antibodies and subsequently with I125 labelled
protein A followed by fluorography (Fig. 10).
As shown in Fig. 10, SU10 cells containing the plasmid
pUR6801 produce lipase enzyme with the correct molecular
weight as compared to lipase from P. glumae. In addition
to the correct protein also not processed and glycosy-
lated lipase protein can also be seen. The P. glumae
lipase produced by s. cerevisiae is enzymatically
active.
EXAMPLE 7. PRODUCTION OF P.lHDHAE LIPASE BY S.
CEREVISIAE USING MULTICOPY INTEGRATION.
The multi-copy integration vector was derived from the
plasmid pARES6 (24) by replacing the 335 bp yeast RNA
polymerase I promoter element with the 4.5 Bg1II B
fragment of S. cerevisiae rDNA (25). Also the 2pm origin
of replication was removed and the Bg1II-HindIII DNA
fragment comprising chloroplast DNA from S. oligorhiza
was replaced by a polylinker DNA sequence. This resulted
in plasmid pUR2790 from which a detailed picture is
shown in Fig. 11.
The essential sequences for multicopy integration in the
yeast genome of pUR2790 are: 1. rDNA sequences for
multicopy integration in the yeast genome, 2. the S.
cerevisiae LEU2d gene (26): this is the LEU2 gene with a
deficient promoter.
the following multicopy integration
Amongst others,
were constructed, encoding:
expression plasmids
. mature lipase preceded by the invertase signal
sequence_(pUR6803),
2. mature lipase preceded by a KEX2 cleavage site, a
glycosylation site and the invertase signal
sequence (pUR6804).
In order to obtain the above mentioned constructs, the
routes followed were (Fig. 12; the used restriction
recognition sites are marked with an asterisk):
ad 1 and 2.
a. The plasmid pUR2790 was partially digested with
HindIII. The linear plasmid was isolated d
digested to completion with Bg1II and the HindIII-
BglII vector fragment was isolated by agarose gel-
electrophoresis and_electrojelution.
b. The plasmid pUR6801 was digested'partially.with
Bg1II and to completion_with Hipdlll and the .
Bg1II—HindIII DNA fragment with the lipase gene was
isolated (pUR6804 is constructed in the same way
using plasmid pUR6802 instead of pUR6801).
c. The Bg1II-HindIII vector fragment of pUR2790 and
the Bg1II—HindIII fragment with the lipase gene
were ligated (Fig. 12), resulting in plasmid
pUR6803.
d. The ligation mixture was transformed to E. coli.
From single colonies, after cultivation, the
plasmid DNA was isolated and the correct plasmids,
pUR6803 and pUR6804, as judged by restriction
enzyme analysis were selected and isolated in large
ounts.
e. The plasmids pUR6803 and pUR6804 were transformed
to S. cerevisiae strain YT61 L (26) = SU50 with
the spheroplast procedure (22) using selecting for
the presence of the LEU2d gene product. The host
strain SU50 is deficient for the essential nutrient
leucine (IEUZ), which means that strain SU50 is not
capable of producing leucine. Thus it can only grow
when the growth medium contains sufficient amounts
of leucine. .
The deficient promoter of the LEU2 gene present in
vectors pUR6803 and pUR6804 is essential for
multicopy integration of the plasmid vectors in the
yeast genome. The multicopy integration occurs at
the rDNA locus of the yeast genome due to homolo-
gous recombination of the rDNA sequences of the
plasmids and the rDNA sequences of the yeast
genome.
f. The integrants were grown overnight in defined
medium ( 0,68% Yeast Nitrogen Base w/o amino acids,
2% glucose, histidine and uracil), diluted 1 : 10
in induction medium (1% yeast extract, 2% bacto—
peptone, 5% galactose) and grown for 40 hours.
g. The cells were isolated by centrifugation and cell
extracts were prepared (23).
h. The cell extracts were analysed by SDS-gel-
electrophoresis (7) and blotted to nitrocellulose
filters.
i. The nitrocellulose blots were incubated with lipase
antibodies and subsequently with I125 labelled
protein A followed by fluorography (Fig. 13).
As shown in Fig. 13, integrants of SU50 with the plasmid
pUR6803 produce lipase enzyme with the correct molecular
weight as compared to lipase from P. glumae. In addition
to the correct protein, not processed and glycosylated
lipase protein can also be seen. The P. glumae lipase
produced by S. cerevisiae is enzymatically active.
integration system is stable even under non-selective
conditions.
EXAMPLE 8; PRODUCTION OF GUAR a-GALACTOSIDASE
IN SACCHAROHYCES CEREVISIAE USING MULTICOPY INTEGRATION.
In this example the expression of a heterologous
protein, a-galactosidase from guar (Cyamopsis tetrago-
noloba) in Saccbaromyces cerevisiae, using multicopy
integration , is described. The gene encoding guar a-
galactosidase was fused to homologous expression
signals as is described by Overbeeke (21) resulting in
the expression vector pUR2730. The a—ga1actosidase
expression cassette of pUR2730 consists of the s.
cerevisiae GAL7 promoter, the S. cerevisiae invertase
signal sequence and the a—galactosidase gene encoding
mature a—ga1actosidase. The multicopy integration vector
used is pUR2770, which is identical to pMIRY2.1 (27).
The a-galactosidase expression cassette was isolated and
inserted in the multicopy integration vector pUR2770
resulting in pUR2774. This multicopy integration vector
contains the a—ga1actosidase expression vector, S.
cerevisiae ribosomal DNA sequences and the S.
cerevisiae deficient LEU2 gene (LEU2d) as a selection
marker. The multicopy integration vector was
transformed to S. cerevisiae and multicopy integrants
were obtained. The multicopy integrants were mitotically
stable and the multicopy integrants expressed and
secreted the plant protein a—galactosidase. This example
clearly demonstrates that it is possible to obtain
multicopy integration in the genome of S. cerevisiae and
that the multicopy integrants can be used for the
production of proteins. All DNA manipulations were
carried out as described in Maniatis (7).
. Construction of multicopy integration vector
pUR2774.
The multicopy integration vector pUR2770 was partially
digested with HindIII and the linearized vector fragment
was isolated. The linear vector fragment was digested to
completion with BamHI and the resulting 8 kb vector
fragment was isolated. The a—galactosidase expression
cassette was isolated from pUR2730 by digestion with
HindIII and Bg1II and isolation of the 1.9 kb DNA
fragment. The a-galactosidase expression cassette was
ligated in the isolated vector fragment of pUR2770
resulting in the multicopy integration vector pUR2774
(see also Fig. 14). The ligation mixture was transformed
to E. coli. From single colonies, after cultivation, the
plasmid DNA was isolated and the correct plasmids, as
judged by restriction enzyme analysis, were selected and
isolated_in large amounts. The multicopy integration -“
vector pUR2774, lineafiied with SmaI was transformed to
the s. cerevisiae strain YT61 L (25) using the 1
spheroplast method (22) by selecting for the presence of
the LEU2d gene product.
. Analysis of the integration pattern of the
multicopy integrants.
The ribosomal DNA of S. cerevisiae is present in i 150
identical copies of the rDNA unit, comprising the genes
that specify the 17S, 5.85 and 26S rRNA components of
the ribosomes of s. cerevisiae These rDNA units are
tandemly repeated in a large gene cluster on chromosome
XII of S. cerevisiae. The complete sequence of the rDNA
unit is known, the rDNA unit is 9.0 kB large and
contains two BglII sites (28, 29, 30). When chromosomal
DNA isolated from S. cerevisiae is digested with BglII,
the rDNA gene cluster gives rise to two fragments, with
a length of 4.5 kb. This gene organization is schemati-
cally represented in Fig. 15A. The 4.5 kb band cor-
responding to the ribosomal DNA fragments is detectable
in the restriction pattern on an ethidium-bromide
stained agarose gel, because of the large number of
ribosomal DNA units present in a haploid genome. The
plasmid pUR2774 has a length of 9.8 kb and contains one
single Bg1II restriction enzyme recognition site. If
plasmid pUR2774 is tandemly integrated in a high copy-
number, digestion of the chromosomal DNA with Bg1II will
give rise to a 9.8 kb DNA fragment. With an ethidium-
bromide stained agarose gel a comparison can be made
between the intensity of the 4.5 kb DNA band, cor-
responding to t 150 copies of the ribosomal DNA unit,
and the 9.8 kb DNA band, derived from the integrated
plasmid. This gene cluster organization is shown in Fig.
15B. This comparison will give a reasonable estimation
of the number of integrated pUR2774 plasmids. pUR2774
was linearized and transformed to the yeast strain YT6-
2-1 L (SU50) which is LEU2'. Transformants were streaked
on MM(defined)-medium without leucine for an extra check
for the LEU2+ phenotype. To examine whether integration
of the multi—copy vector pUR2774 actually occurred,
chromosomal DNA was isolated from independent integrants
SUSOA, SUSOB, SUSOC and SU50D. The total DNA was
digested with Bg1II and analyzed by gel-electrophoresis.
An example of such a ethidium-bromide stained gel is
shown in Fig. 16. As expected, in the restriction
patterns of integrants SUSOB and SUSOC, two main bands
can be distinguished at 4.5 and 9.8 kb. The parent
strain gives to a single band only of 4.5 kb; the rDNA
unit. So, we can conclude that in addition to the
multiple ribosomal DNA units, these integrants surpris-
ingly also contain multiple integrated copies of the 9.8
kb plasmid pUR2774. Different multicopy integrants were
found to contain different copy-numbers of the plasmid
pUR2774 varying'from 10 to 100. To confirm the presence
of the a-galactosidase gene, hybridization with radio-
labelled probe-was performed._The probe for the a- _
galactosidase genevwas.isolated'fr9m,pUR2731, a pUR2730
derivative, by digestion with PvuII and HindIII and
isolation of the 1.4 kb fragment containing the a-
galactosidase gene. To identify the rDNA sequences a
probe was prepared by digestion of pUR2770 with SmaI
and HindIII, followed by isolation and labelling of the
2.0 kb fragment. Upon hybridisation with the a-
galactosidase probe (see Fig. 17) it was found that the
9.8 kb band as detected in the ethidium bromide stained
gel indeed corresponds to the a-galactosidase gene since
this single band was present in the autoradiographs,
while in the lane containing digested total DNA from the
YT61 L parent strain no hybridisation signals were
detected. Since we could detect the 9.8 kb band no
extensive re-arrangements and/or deletions can have
occurred in the integration process. Hybridisation with
the rDNA probe resulted in signals corresponding to a
4.5 kb band and a 9.8 kb band, from which it follows
that indeed the 4.5 kb band contains the expected
ribosomal DNA sequences. As proven with the a-
galactosidase probe the 9.8 kb band results from the
multicopy integration of pUR2774. This 9.8 kb DNA band
also gives a positive signal with the rDNA probe,
because pUR2774 also contains ribosomal DNA sequences.
From the results shown in Fig. 18, assuming that the 4.5
kb ribosomal DNA band represents 150 copies of the rDNA,
the 9.8 kB band containing pUR2774 can be estimated to
contain 50-100 copies. Thus, by transformation of the
mu1ti—copy integration plasmid pUR2774, it is indeed
possible to direct 50-100 copies per cell of the a-
galactosidase expression cassette to the genome of S.
cerevisiae.
. Production of a-galactosidase by multicopy
integrants.
Multicopy integrants SUSOA, SUSOB, SU50C and SUSOD
chosen for having high copy numbers of the integrated
plasmid were examined for a—galactosidase activity by
growing them on 0.67% Yeast Nitrogen Base w/o amino
acids, 2% glucose overnight, followed by induction of
the GAL7 promoter by a 1:10 dilution in 1% Yeast
Extract, 2% Bacto-peptone, 2% galactose (YPGal). The a-
galactosidase activity in the supernatants of the
cultures was determined by means of an enzyme activity
test, as described by Overbeeke et al. (21), at 24 and
48 hours after start of the induction. The results are
shown in the following table:
hours induction 48 hours induction
Integrant 0D660 a-gal OD66O a»ga1
mg/1 mg/1
SUSOA 2%ga1 9 S8 8 82
SUSOB 2%gal 15 101 13 235
SUSOC 2%gal 6 45 4 101
SUSOD 2%gal 14 41 10 54
The results shown in the table clearly demonstrate that
it is possible to obtain high levels of expression of a
foreign gene using a multicopy integrant. Moreover,
SUSOB (235 mg/l) gives rise to a higher level of a-
galactosidase production as compared to a expression
system with extrachromosomal plasmids (see pUR2730 in
reference 21). In spite of the fact that all four multi-
copy integrants had been elected for having a high copy-
number of integrated a-galactosidase expression
cassettes, their expression levels vary from 54 to 235
mg/l.
4. Genetic stability of the SUSOB multicopy integrant.
To test whether integration of multiple copies a-
galactosidase expression plasmids in the s. cerevisiae
genome is genetically stable the complete test procedure
was repeated under non-selective conditions. Integrant
SUSOB was streaked on an YPD-agar plate and a pre-
culture was inoculated and grown overnight in YPD at 30
°C. Subsequently, the pre-culture was diluted 1:10, in
YPGal. Samples were taken, optical density measured at
660 nm and the a-galactosidase content of the culture-
broth was determined by the enzyme activity assay.
Surprisingly, the expression level of a-galactosidase
was stable during the whole experiment. This experiment
shows that indeed the multiple integrated expression
plasmids are maintained very stable under non-selective
conditions for many generations. Another important
finding was that the multi-copy integrants were stable
for months on non-selective YPD-agar-plates kept at 4
°C. When the pre-culture of the SUSOB integrant is
diluted 1:1000 in YP with 2% galactose, grown at 30 °C
to an identical OD 660 nm, the a-galactosidase expres-
sion is 250 mg/l. In this experiment the pre-culture of
the multicopy integrant SUSOB is diluted to a larger
extend in YPGal; and the cells in the induced culture
have to make more divisions before the same biomass and
related to this the a-galactosidase production_is
achieved as with a 1:10 dilution. Thus, we can conclude
that the stability of-the=argalactosidase;production;and;
therefor the genetic stability of the multicopy
integrants is very good compared to the stability of
extrachromosomal plasmids under non—selective condi-
tions.
We have also found that for multicopy integration and
genetic stability of the multicopy integrants the length
of the multicopy integration vector is an important
parameter. The use of multicopy integration vectors with
a length of about 12 kb have a tendency to result in a
lower copy number of integrated vectors in the genome
and also a decreased genetic stability although still
very reasonable. The use of relatively small multicopy
integration vectors (1 3 kb) results in a high copy
number of integrated vectors but with a decreased
genetic stability. These results show that the optimal
length for a multicopy integration vector, resulting in
high copy number of integrated vectors and good genetic
stability, is approximately the length of a single
ribosomal DNA unit; for S. cerevisiae about 9 kb.
This example clearly demonstrates the feasibility of the
use of multicopy integration in S. cerevisiae for the
production of proteins. The high genetic stability of
the multicopy integrant confer an important advantage as
compared to the extrachromosomal plasmid-system where
cells have to be grown under selective pressure. The
multicopy integrants appeared to be very stable on YPD-
agar plates as well as during growth in YPD- and YPGal
culture medium for many generations. Considering the
high level of expression of the a-galactosidase enzyme
and the good mitotic stability of the integrated a-
galactosidase expression cassettes, this integrant-
system is a realistic option for large—scale production
of the a—galactosidase enzyme or any other protein.
EXAMPLE 9 MULTICOPY INTEGRATION IN SACCHAROHYCES
CEREVISIAE USING OTHER DEFICIENT SELECTION MARKERS.
We have found that for multicopy integration in yeast
there are two prerequisites for the multicopy integra-
tion vector; the multicopy integration vector should
contain ribosomal DNA sequences and a selection marker
with a specific degree of deficiency. In the previous
examples multicopy integration is obtained using a
multicopy integration vector with ribosomal DNA
sequences and the defective LEU2 gene (LEU2d) as a
selection marker. In this example the use of other (than
LEU2d) defective selection markers in order to obtain
multicopy integration in yeast is described. In this
example multicopy integration vectors are used with
either a deficient TRP1 or a deficient URA3 instead of
the LEU2d gene. The expression of both these genes was
severely curtailed by removal of a significant part of
their 5' flanking regions. Using these multicopy 7
integration vectors, multicopy integrants were obtained
in which approximately 200 copies of the vector
integrated. This example clearly demonstrates that
multicopy integration can also be effected by other
deficient selection markers. All standard DNA manipula-
tions were carried out as described in Maniatis (7).
. Construction and analysis of pMIRY plasmids
containing a deficient TRP1 gene as selection
marker.
In order to test the possibility that multicopy
integration into the genome can be obtained using
different types of selection pressure during transforma-
tion, series of pMIRY2.1-analogous plasmids (pMIRY2.l is
identical to pUR2770) were constructed, containing
deficient alleles of two genes commonly used as
selection markers: the TRP1 and URA3 genes of S.
cerevisiae. The TRP1 gene encodes the enzyme
N-(5’-phosphoribosyl-1)-anthranilate (PRA) isomerase,
which catalyzes the third step in the biosynthesis of
tryptophan (31). Transcription of the TRP1 gene is
initiated at multiple sites which are organized into two
clusters (Fig. 19), one at about position -200 relative
to the ATG start codon and the other just upstream of
this codon (32). Each of the two clusters is preceded by
putative TATA elements as well as (dA:dT)—rich regions
that could act as promoter elements (3). When the
upstream region of the TRP1 gene is deleted up to the
EcoRI site at position -102 (TAl), the first cluster of
transcription start sites is removed and the expression
level of the gene drops to only 20% to 25% of the value
of its wild-type counterpart (31). This particular
deficient TRP1 allele is currently used as selection
marker in several yeast vectors. We hypothesize that
this degree of deficiency was not high enough and
therefore the deletion in the 5’—flanking sequence was
extended to either position -30 (TA2) or -6 (TA3)
upstream of the ATG codon. The TA2 gene still contains
part of the downstream cluster of transcription
initiation sites. In the TA3 deletion mutant both
clusters as well as all poly dA:dT stretches and
putative TATA elements are deleted. These two mutant
TRP1 genes as well as the original TAl gene were used in
construction of the pMIRY6-T series of plasmids.
Construction of this series was carried out as follows
(Fig. 20): first, the 766 bp AccI—PstI (Fig. 19)
fragment, containing the TRP1 coding region plus 30 bp
of 5'-flanking sequence, was cloned between the SmaI and
PscI sites of pUC19, resulting in plasmid pUC19-TA2 (the
AccI site was made blunt by filling in the 3'-end using
T4 polymerase). Subsequently, the 3.5 kb SphI fragment
from a pUC18 subclone containing the Bg1II—B rDNA
fragment (27) was inserted into the SphI site of the
pUC19—TA2 polylinker giving plasmid pMIRY6-TA2. Plasmids.
pMIRY6-TA'l and pMIRY,6-TA3 are derivativesof pMIRY6-TA2.'
To obtain pMIRY6-‘I‘AlIwth‘e3 357 ;bp“E‘coRI-BgI1’I'TRPl
fragment was first cloned between the EcoRI and the
BamHI sites of pUC19, giving plasmid pUC19-TA1. In the
next step the 1.2 kb ScaI-EcoRV fragment of pMIRY6—TA2
which contains a portion of the pUC19 sequence as well
as part of the TRPl gene (Fig. 20), was replaced by the
1.3 kb ScaI—EcoRV fragment from pUC19-TA1 restoring the
length of the 5’ flanking sequence of the TRP1 gene to
102 bp. pMIRY6—TA3 was constructed in a similar way.
First, the 405 bp A1uI fragment from the TRPl gene was
cloned into the SmaI site of pUC19, giving pUC19-TA3.
Subsequently, the 1.2 kb ScaI-EcoRV fragment of
pMIRY6-TA2 was replaced by the 1.2 kb ScaI—EcoRV
fragment from pUC19-TA3, to give pMIRY6-TA3.
Plasmids, pMIRY6-TA1, pMIRY6-TA2 and pMIRY6~TA3 were
transformed into yeast after linearization with HpaI
within the rDNA sequence, in order to target integration
to the rDNA locus. In Fig. 21 a gel electrophoretic
analysis of total DNA is shown from two independently
isolated transformants of each type after digestion
with EcoRV in the case of pMIRY6-TA1 and sacI in the
cases of pMIRY6-TA2 and pMIRY6-TA3. In the case of the
pMIRY6-TA2 (lanes 3 and 4) and pMIRY6—TA3 (lanes 5 and
6) transformants, the rDNA band and the plasmid bands
are of comparable intensity. Thus, the copy number of
each of the two plasmids is as high as the number of
rDNA units per haploid genome, which is approximately
150 (33). The copy number of the pMIRY6-TA2 and -TA3
plasmids is of the same order. In contrast, transforma-
tion with pMIRY6—TA1 did not result in high-copy—number
transformants. As shown in Fig. 21 (lanes 1 and 2), no
6.9 kb band corresponding to the linearized pMIRY6-TA1
plasmid is visible upon sacI digestion of the total DNA
from pMIRY-TAl transformed cells. The results described
above clearly demonstrate that multicopy integration
into the yeast rDNA locus does not absolutely require
the presence of the LEU2d gene selection marker in the
vector. Instead, deficient TRP1 alleles can be used,
provided their expression falls below a critical level.
. Construction and analysis of a pMIRY plasmid with a
deficient URA3 gene as selection marker.
Next to the LEU2 and the TRP1 genes, the URA3 gene is
one of the most widely used selection markers in yeast
vectors (34). The URA3 gene encodes orotidine-5’-phos-
phate carboxylase (OMP decarboxylase). The expression of
this gene is controlled at the level of transcription by
the PPR1 gene product which acts as a positive regulator
(35). Deletion analysis suggests that the sequence
essential for PPR1 induction of URA3 is located in a 97
bp long region located just upstream of the ATG
translation start codon (36). In order to obtain a
promoter of the URA3 gene with the desired degree of
deficiency we have deleted most of this region, using a
PstI site located 16 bp upstream of the ATG start codon._
To that end a Bg1II linker was inserted in the Smal
site of pFL1 (36B) located in the 3' flanking region of
the URA3 gene at position +880 relative to the ATG
translational start signal, yielding pFL1- Bg1II. The
0.9 kb PstI-Bg1II fragment, comprising the URA3 coding
region together with its flanking 3' region abutted by
the Bg1II site and only 16 bp of its 5' flanking region
abutted by the PscI site, was cloned between the Pscl
and BamHI sites of pUC19, yielding pUC19-U (Fig. 22).
The 2.8 kb SacI-StuI IDNA fragment containing part of
the Bg1II-B rDNA fragment, was isolated from pUC-BR and
inserted between the Smal and SacI sites in pUC19-UA,
giving plasmid pMIRY7~UA. Copy number analysis of two
independently isolated pMIRY7-UA transformants is shown
in Fig. 23. The plasmid band and the rDNA band have
similar intensities which means that the plasmid is
integrated in about 200 copies per cell, a result
similar to that obtained with plasmids pMIRY6—TA2 and
pMIRY6-TA3. This example clearly demonstrates that
multicopy integration into the yeast ribosomal DNA locus
is also effected using genes other than LEU2d as
selection marker. Indeed, it seems likely that any gene
involved in the biosynthesis of a essential nutrient can
support this process, when employed as selection marker
in a pMIRY plasmid, provided that it is expressed but
its expression is below a critical level.
This means that surprisingly we have found that besides
the ribosomal DNA sequence a deficient, but essential
gene must be present on the multicopy integration vector
in order to obtain multicopy integration, in a S.
cerevisiae strain deficient for that essential gene, of
this multicopy integration vector and that the obtained
multicopy integrants can be stable for many
generations. so the principle of multicopy integration
can be extended to all S. cerevisiae auxotrophic strains
thus permitting a choice from a range of host strains
for the expression of any particular gene. Such a choice
is an important factor in the optimization of
heterologous gene expression in yeast. In particular Trp
auxotrophy is an attractive marker for use in an
industrial process since even poorly defined media can
easily be depleted of tryptophan by heat—sterilization.
EXAMPLE 10 STABIDITY OF THE MULTICOPY INTEGRANT SU5O
IN CONTINUOUS CULTURES.
The multicopy integrant is cultivated in a continuous
culture (chemostat) with a working volume of 800 ml at a
dilution rate of 0.1 h'1 (a mean residence time of 10
hours). The integrant SUSOB is a transformant of strain
Saccharomyces cerevisiae CBS 235.90 with the multicopy
integration vector pUR2774 (see example 8). The pH was
controlled at 5.0 using 10% NH4OH. Foaming was sup~
pressed using a silicon oil based antifoam (Rhodorsil
426 R Rhone-Poulenc) The feed composition used was A.
A steady state was maintained for 120 hours with a
stable expression of 360 mg/1 a-galactosidase at a
biomass dry weight concentration of 11.06 g/l. Similar
conditions were stable in other experiments for more
than 500 hours. The residual glucose concentration was
below the detection limit of 0.05 g/l. The residual
galactose concentration was 4.2 +/- 0.1 g/l. The inlet
contained 170 mg/1 leucine derived from the yeast
extract and DHW. This resulted in a steady state leucine
concentration of 2.0 +/- 0.4 mg/l as determined with a
amino acid analyzer. After a period of time, 50 mg/l
leucine was added to the feed A. Surprisingly the
residual leucine concentration in the culture dropped to
0.7 +/- 0.2 mg/l. This was accompanied by a considerable
decrease of a—galactosidase activity within 80 hours to
144 mg/1 (Fig. 24). In Fig. 25 the determination of the
copy number during various stages of the experiments is
shown. Samples were taken, chromosomal DNA isolated,
digested with BglII and subsequently southern blotting
was performed using the ribosomal DNA probe as described
in example 8. SUSOB 1 is a positive control grown in a
shake flask. Clearly can be seen that the copy number of
integrated vectors, by comparing the smaller hybridizing
DNA fragment (chromosomal rDNA units‘ 1 150 copies)
with the larger hybridizing DNA fragment (the integrated
vector), is about 100. This is the same for SU50B 2, a
southern blot of a sample taken before the adding of the
leucine. For SUSOB 3, a sample taken after the adding of
leucine and the drop in a-galactosidase expression the
copy number has decreased to about 10. This experiment
shows that the decrease in a—galactosidase expression is
accompanied by a decrease in copy number of the a-
galactosidase gene. The leucine uptake of the culture is
higher after addition of leucine.
The experiments described above show quite surprisingly
that the genetic stability of the integrated plasmids is
due to the fact that the intracellular production of
leucine is required for growth, in spite of the presence
of an appreciable amount of extracellular leucine. Due
to the inefficiency of the LEU2d promoter, production of
sufficient amounts of leucine is only possible when a
large number of LEU2d genes is present on the chromo-
some. .
Such a large number of integrated genes can be stably
maintained when the integration site is in, or directly
linked to the ribosomal DNA locus and under proper
growth rate conditions and medium composition.
Media composition g/l
Compound A B _ C D
NH4Cl 7.6 7.6 7.6
KHZPO4 2.8 4.0 4.0
MgS04.7aq 0.6 0.6 0.6
trace metals 10 10
yeast extract 5 10
(Difco)##
peptone 0.0 0.0 20
DHW (UF) 125 0.0
glucose 5.5 20 20
galactose 10 20 10
histidine 0.05 0.2 0.2
vitamin 2 1 1
solution
leucine 0.05
(added)
pH 5.0 5.0 5.0 5.0
DHW: de—proteinized hydrolysed whey ex DMV Netherlands.
UF : ultra filtrated molecular weight cutoff 10 kD.
## : yeast extract contains 8-9 %w/w leucine.
EXAMPLE ll PARAMETERS AFFECTING THE STABILITY OF
MULTICOPY INTEGRANT SUSOB
Strain SUSOB (as described in example 10) was cultivated
in shake flasks in media C and D. This gives an example
of two extreme media ranging from a complex, rich medium
to a minimal medium. Medium C (YPGAL) contains 524 mg/l
leucine. Surprisingly, the integrant was stable in YPGAL
media for many sub-cultivations (see example 8) . In
medium D and other minimal media with leucine the
expression decreased rapidly. The residual concentration
of leucine (derived from the yeast extract and peptone)
in the medium C decreased from 524 mg/l to 393 mg/1. The
leucine concentration in medium D reduced from 50 to
about 20 mg/1. The growth rate of the strain in minimal
media is about 0.1 h'1 while the growth rate on medium C
is 0.27 h'1. Addition of yeast extract increases the
growth rate up to 0.27 h'1 combined with an improved
stability of the a-galactosidase production.
The complex media not only increase the growth rate, but
also increase the a—galactosidase concentration in the
culture.
These experiments clearly show that the multicopy
integrant was stable at high growth rates in the
presence of leucine. Based on this finding an efficient
fermentation process.can.be developed meaning_a>“
substantial amount of protein per culture volume_per~
hour can be obtained.
EXAMPLE
EXPRESSION OF THE SYNTHETIC LIPASE GENES
IN HANSENULA POLYHORPHA.
The synthetic lipase genes were integrated in the H.
polymorpha genome using the following procedure (Fig.
26; in each figure of this example the used restriction
enzyme recognition sites are marked with an asterisk;
restriction recognition sites between brackets are
removed due to the cloning procedure):
Plasmid pUR6038 (Fig. 27) was digested to comple-
tion with the restriction enzymes EcoRI and EcoRV.
After separation of the fragments by agarose gel
electrophoresis the vector fragment was isolated as
described in Example 2.
Several different synthetic cassettes were
assembled as described in Example 3. These
cassettes encoded a number of amino acids necessary
for a correct joining of the invertase signal
sequence with different length of the pre—mature
lipase gene. This was done to establish the most
optimal construct with respect to expression,
processing and export of the lipase enzyme.
Furthermore, these cassettes had EcoRI and ECORV
ends.
Typical examples are given in Fig. 26.
The assembled cassettes were ligated in the vector
prepared under a.
The plasmids thus obtained (pUR6B50, 6851 and 6852
Fig. 28) were partially digested with the restric-
tion enzyme XhoI and the linearized plasmid was
isolated.
Plasmid pUR3501 (21, Fig. 29) was partially
digested with Xhol. After agarose gel electropho-
resis a DNA fragment of approximately 1500 bp was
isolated, containing the H. polymorpha methanol
oxidase (MOX) promoter followed by the first amino
acids of the S. cerevisiae invertase signal
sequence Xhol DNA fragment from position 0 to 1500
from pUR3501).
The 1.5 kb fragment from e. was ligated in the
vector fragments as prepared in d resulting in
plasmids UR6860, 6861, 6862 Fig. 30.
The ligation mixture was transformed to E- coli.
From single colonies, after cultivation, the
plasmid DNA was isolated and the correct plasmids,
as judged by restriction enzyme analysis, were
selected and isolated in large mounts.
signal sequence and synthetic lipase gene with a
length of approximately 2.5 kb were isolated out of
agarose gel.
i. Plasmid pUR3511 (the H. polymorpha methanol oxidase
(MOX) terminator cloned in the BamHI, HinCII
restriction sites of pEMBL9, Fig. 31) was digested
with SmaI and EcoRI, after which the vector was
isolated out of an agarose gel.
j. The pUR3511 vector and the 2.5 kb fragments,
obtained in h., were ligated and cloned in E. coli.
In the constructs obtained, the lipase gene is
followed by the MOX transcription terminator.
Typical examples of these constructs are pUR6870,
6871 and 6872 (Fig. 32).
k. These plasmids were digested with EcoRI and
HindIII, after which the fragments of approximately
3 kb. were isolated from an agarose gel. The sticky
ends were filled in with Klenow polymerase.
. Plasmid pUR3513; this is plasmid YEp13 (37) from
which the 2pm sequences have been deleted by
removal of a SalI fragment (Fig. 33) was digested
with PvuII.
m. The linear plasmid pUR3513 and the fragments
obtained in k. were ligated to obtain the final
constructs among which pUR6880, 6881 and 6882 (Fig.
).
Introduction of the expression cassettes in the H.
polymorpha genome.
Transformation of plasmid DNA to the Hansenula polymor-
pha strain A16 using selection for LEU+ phenotype can be
performed as described by (21, 38, 39).
Analysis of the integrants can be performed using the
Southern blot procedure (7).
PRODUCTION OF GUAR a-GALACTOSIDASE IN
USING MULTICOPY INTEGRATION.
EXAMPLE 13
HANSENULA POLYHORPHA
In this example the expression of a heterologous
protein, a-galactosidase from guar Cyamopsis tetrago-
noloba ), using multicopy integration in Hansenula
polymorpha, is described. The gene encoding a-galac-
tosidase was fused to homologous expression signals as
is described in Overbeeke (21) resulting in the
expression vector pUR3510. The a-galactosidase expres-
sion cassette of pUR3510 consists of the H. polymorpha
methanol oxidase promoter, the S. cerevisiae invertase
signal sequence, the a—galactosidase gene (encoding
mature a-galactosidase) and the R. polymorpha methanol
oxidase terminator. This expression cassette was
isolated and inserted in the multicopy integration
vector pUR2790 resulting in pUR3540. The multicopy
integration vector_pUR3S40 was transformed to H.
polymorpha and surprisingly multicopy integrants were
obtained. The obtained multicopy integrants expressed
and secreted the plant protein a-galactosidase. This
example clearly demonstrates that it is possible to
obtain multicopy integrants in H. polymorpha and these
multicopy integrants can be used for the production of
proteins. Also it appeared that multicopy integrants in
H. polymorpha were obtained using S. cerevisiae
ribosomal DNA sequences and a S. cerevisiae deficient
selection marker. All DNA manipulations were carried out
using standard techniques as described in Maniatis (7).
The plasmid pUR3510 (21) was digested with HindIII and
BamHI and the DNA fragment containing the a~ga1ac—
tosidase expression cassette was isolated. The multicopy
integration vector pUR2790 is derived from pUR2740 by
replacing the Bg1II-HindIII 500 bp fragment containing
8. oligorhiza DNA and a 100 bp S. cerevisiae ribosomal
DNA by a Bg1II-HindIII polylinker sequence containing
multiple cloning sites. The multicopy integration vector
pUR279O was partially digested with HindIII and
digested to completion with Bg1II and subsequently the
vector fragment was isolated. The BglII-HindIII vector
fragment and the HindIII-BamHI fragment, containing the
a—galactosidase expression cassette, were ligated
resulting in the multicopy integration vector pUR3540
(see also Fig. 35; all used restriction recognition
sites are marked with an asterisk). The ligation mixture
was transformed to E. coli . From single colonies, after
cultivation, the plasmid DNA was isolated and the
correct plasmids, as judged by restriction enzyme
analysis, were selected and isolated in large amounts.
The multicopy integration vector pUR3540 was linearized
with Smal and the linearized vector pUR3540 was
transformed to H. polymorpha A16 (LEU2') using the
procedure described by Roggenkamp et al (39). The LEU2+
colonies, being the multicopy integrants, were isolated
and used for further experiments. The multicopy
integrant and the parent strain A16 as a control were
grown under non—selective conditions (1% Yeast Extract,
2% Bacto-peptone, 2% glucose for 40 hours at 37 °C) and
chromosomal DNA was isolated as described by Janowicz et
al. (40). The total DNA was digested with HindIII and
the digested chromosomal DNA was analyzed by Southern
hybridization (7). An XhoI 878 bp fragment, containing
a part of the methanol oxidase promoter [position -1313
to position -435, Ledeboer (41)], was labelled with 32P
and used as a probe. The result of this hybridization
experiment is shown in Fig. 36, (lane 2 parent strain
and lane 1 multicopy integrant). In lane 2, the parent
strain, a DNA fragment of approximately 14 kb can be
seen to hybridize with the methanol oxidase promoter
probe, corresponding to a DNA fragment containing the
entire methanol oxidase gene.which_is present in a
single copy in the genome- In lane 1, the multicopy
integrant, an additional hybridization signal was:
obtained,corresponding to a DNA fragment of approximate-
ly 8 kb. This fragment is part of the integrated vector
pUR3540 being the HindIII fragment containing amongst
others the a-galactosidase expression cassette and the
methanol oxidase promoter. By comparison of the inten-
sities of the hybridization signals in lane 2 it can be
estimated that over 20 copies of the multicopy integra-
tion vector are integrated in the E. polymorpha genome.
The multicopy integrant was analyzed for a-galactosidase
expression as described by Overbeeke (21). Upon
induction with methanol a-galactosidase was detected in
the medium using the enzyme activity assay.
This example clearly demonstrates that multicopy
integration can be achieved in H. polymorpha and thus
the multicopy integration system can be used for the
production of (e.g. heterologous) proteins in H.
polymorpha . This example also demonstrates that it is
possible to obtain multicopy integration of an expres-
sion vector in the genome of a yeast (e.g. H. polymor-
pba) using the two prerequisites, ribosomal DNA
sequences and a deficient selection marker. Such a
selection marker can be homologous or originating from
another host (e.g. S. cerevisiae) as long as the
expression level of the deficient gene is below a
critical level.
EXAMPLE 14. MULTICOPY INTEGRATION IN KLUYVEROMYCES.
In this example a procedure is described to obtain
multicopy integration of a plasmid vector in the genome
of Kluyveromyces marxianus var. lactis . Multicopy
integration vectors were constructed containing
ribosomal DNA sequences originating from S. cerevisiae
and deficient selection markers origination from the
multicopy integration vectors (pMIRY6-TAl, pMIRY6-TA2
and pMIRY6-TA3). The multicopy integration vectors were
transformed to a TRP’ K. marxianus strain surprisingly
resulting in transformants having multiple copies of the
vector integrated in the genome of the Kluyveromyces
strain. Also this example clearly demonstrates that
multicopy integration can be obtained in yeasts using
either homologous or heterologous deficient selection
markers.
From the multicopy integration vectors pMIRY6-TA1,
pMIRY6-TA2 and pMIRY6-TA3 (see example 9) the S.
cerevisiae ribosomal DNA was removed by digestion with
SphI, isolation of the vector fragment followed by
ligation of the vector fragment. In the resulting vector
a 4400 bp EcoRI K. marxianus ribosomal DNA fragment
(42, Fig. 37) was cloned in the EcoRI site resulting in
the multicopy integration vectors pMIRK7AT1, pMIRK7AT2
and pMIRK7AT3~(Fig. 38). The multicopy integration
vectors, after linearization with SacI, were
transformed to the K. marxianus strain MSK 110 (3. URA"
A, TRP1::URA3), (43) using the LiAc procedure (44).
Transformants were selected for TRP+ phenotype. The
obtained integrants were grown under non-selective
conditions (0.67% Yeast Nitrogen Base with amino acids,
2% glucose, 30 °C), for 6-7, 30-35 and 60-70 genera-
tions. This was performed by growing the integrants to
OD 550 nm of 2 to 3, dilution in fresh non-selective
medium to CD 550 nm of 0.1 and followed by growth to OD
550 nm of 2 to 3. This cycle was repeated several times.
From these integrants total DNA was isolated (45),
digested with PstI and separated on a 0.8% agarose gel,
followed by Southern analysis using the EcoRI-PstI
fragment of the K. lactis ribosomal DNA (Fig. 37) as a
probe. In Fig. 39 the result obtained with the integrant
of pMIRK7AT1 are shown. In lane 5 the hybridisation of
the rDNA probe with the digested chromosomal DNA of the
host strain is shown, the rDNA probe hybridizes with the
1 150 repeated copies of the rDNA unit. In lane 1 the
pMIRK7AT1 integrant is shown. The hybridisation with
rDNA copies, as for the parent strain, can be seen but
in addition the repeated integrated copies of the
multicopy integration vector. As a control the hybri-
disation result of the linearized multicopy integration
vector with the rDNA probe (lane 6) is shown. The
relative intensity of the hybridization signal can be
used to estimate the copy number of integrated vector.
The hybridisation signal with the rDNA units corresponds
t 150 copies. Comparison of the intensity of hybridisa-
tion signal of the integrated copies of the vector with
the intensity of the hybridization signal with the rDNA
units the copy number can estimated to be at least 50.
This result shows that surprisingly multicopy integra-
tion can also be obtained in the yeast genus K1uyvero-
myces. In lane 2, 3 and 4 the integration pattern is
shown after non-selective growth of the multicopy
integrant, also used in lane 1, for 6-7, 30-35 and 60-70
generations respectively. It can clearly be seen that
the relative intensity of the hybridisation signals with
the integrated vector does not decrease. This surprising
finding proves that the multicopy integration is
completely stable even after prolonged growth under non-
selective conditions. Similar results were obtained
using the multicopy integrants of pMIRK7AT2 and
pMIRK7AT3 .
This example clearly demonstrates that it is possible to
obtain multicopy integration in Kluyveromyces using a
multicopy integration vector with the two prerequisites
ribosomal DNA sequences and a deficient selection
marker, in this example even a heterologous selection
marker. The multicopy integrants are stable for at least
60 generation under non-selective conditions. By
analogy with the examples 8 and 13, production of a
protein in Kluyveromyces using multicopy integrants can
be obtained by insertion of an expression cassette, with
a gene coding for a protein of commercial interest, in
the multicopy integration vector and_transformation of
the resulting vector including the expression cassette.
These multicopy integrants can be used for the produc-
tion of the protein of commercial interest. Because of
the unique properties of the multicopy integration
system, high copy number and high genetic stability,
these multicopy integration transformants can be used in
any known fermentation production process for the
production of a, commercially interesting, protein.
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Elements (ed J.A. Shapiro), Academic Press, 300-
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LEGENDS TO FIGURES
In the figures of the different plasmids the order of
length is given.
Figure 1 A schematic drawing of the plasmid
pUR6002 comprising the P. glumae lipase gene.
The complete nucleotide sequence of the
Figure 2
for details see text.
P. glumae lipase gene;
Figure 3 A schematic drawing of the construction
pUR6103; for details see text.
Figure 4 A schematic drawing of the construction
of the plasmids pUR6107 and pUR6108; for details see
text.
Figure 5
A. The complete nucleotide sequence of the
synthetic lipase gene in pUR6038.
B. The nucleotide sequence of the 3’
flanking region of the synthetic lipase gene in pUR6600.
Figure 6 An example of the construction of a
cassette in the synthetic lipase gene.
Figure 7 A schematic drawing of the construction
of plasmid pUR6131; for details see text.
Figure 8 An example of the improved resistance of
a mutant lipase in a detergent system.
Figure 9 A schematic drawing of the construction
of the plasmid pUR6801. Plasmid pUR6801 is a S.
cerevisiae/E. coli shuttle vector comprising the
synthetic lipase gene with yeast expression- and
secretion sequences.
Figure 10 A western analysis of lipase expression
in S. cerevisiae using pUR6801. The corresponding blot
was incubated with lipase specific antibodies.
Standards: 1 pg and 0.25 pg P. glumae lipase.
total intra—cellular protein of the
host strain SU10.
total intra—cellular protein of SU10
transformed with pUR6801.
total extracellular protein of SU10
transformed with pUR6801.
SU10:
TF17 cells:
TF17 supernatant:
Figure 11 A schematic drawing of the multicopy
integration vector pUR2790.
Figure 12 A schematic drawing of the construction
of pUR6803. Plasmid pUR6803 is a multicopy integration
vector comprising the lipase expression cassette.
Figure 13 A western analysis of lipase expression
of multicopy integrants. The multicopy integrants were
obtained by transforming S. cerevisiae strain SU50 with
the multicopy integration vector pUR6803: 7 independent
multicopy integrants are shown. The corresponding blot
was incubated with lipase specific antibodies.
Standards: 1 pg and 0.25 pg P. glumae lipase.
SU50: total intracellular protein of host strain
SU50.
— 7: total intra-cellular protein of 7 independent
multicopy integrants.
Figure 14 A schematic drawing of the multicopy
integration vector pUR2774 comprising the a—ga1acto-
sidase expression cassette.
Figure 15
A. A schematic drawing of the genetic orga-
nization of the ribosomal DNA locus of S. cerevisiae.
B. A schematic drawing of the genetic
organization of a multicopy integration of pUR2774 in
the ribosomal DNA locus of S. cerevisiae (multicopy
integrant SUSOB).
Figure 16 Ethidium bromide stained agarose gel of
undigested and BglII digested total DNA of the multicopy
integrants SUSOB and SUSOC.
Figure 17 southern blot of total DNA of multicopy
integrants using the a-galactosidase probe.
parent strain YT61 L (SU50) total DNA
digested with Bg1II.
SU50 * Bg1II:
C * Bg1II: total DNA of multicopy integrant SUSOC
digested with Bg1II.
B * Bg1II: total DNA of multicopy integrant SUSOB
digested with Bg1II.
C: undigested total DNA of multicopy
integrant SUSOC.
Figure 18 Southern blot of multicopy integrants
using the ribosomal DNA probe.
parent strain YT61 L (SU50) total DNA
digested with Bg1II.
SU50 * Bg1II:
C * Bg1II: total DNA of multicopy integrant SUSOC
digested with Bg1II.
B * Bg1II: total DNA of multicopy integrant SUSOB
‘digested with Bg1II. V
C: undigested total DNA of multicopy
integrant SUSOC.
Figure 19 Structure of the TRP1 gene from (32).
[AT]: poly(dA:dT) stretch, (UAS), partial general
control upstream activation site. The actual sequence is
indicated for the putative TATA elements. The TRP1
coding sequence is indicated by the black bar. The
various mRNA species are indicated by the arrows. The
scale is in base pairs. The restriction sites used to
construct the promoter deletions are indicated.
Figure 20 Construction of plasmids pMIRY6—Ti1,
pMIRY6—Ti2 and pMIRY6-Ti3 containing TRP1 alleles with
various promoter deletions. The coordinates indicated
for several of the restriction sites show their position
with respect to the ATG start codon (the A being
position +1). For each plasmid the position (-6, -30 or
-102) of the 5'-end of the TRP1 gene is indicated. A
more detailed map of the rDNA fragment present in the
various pMIRY6 plasmids is shown at top right. The
non—transcribed rDNA spacer is abbreviated as "N".
Figure 21 Plasmid copy number of pMIRY6-TA1 (lanes
1 and 2), pMIRY6-TA2 (lanes 3 and 4) and pMIRY6-TA3
(lanes 5 and 6) transformants. Total DNA was isolated
from the transformed cells and digested with EcoRV in
the case of pMIRY6-TAl and SacI in the case of
pMIRY6-TA2 and pMIRY6-TA3. The fragments were separated
by electrophoresis on an 0.8% gel. The DNA was stained
with EtBr. The plasmid and the rDNA bands are indicated.
Figure 22 Construction of pMIRY7—UA containing a
URA3 gene in which most of the promoter has been
deleted. The coordinates indicated for several of the
restriction sites refer to their positions with respect
to the ATG start codon (the A being position +1). The
position of the 5’-end of the URA3i is indicated (A16).
A more detailed map of the rDNA fragment present in
pMIRY7—UA is shown at top right. The non-transcribed
spacer is abbreviated as "N".
Figure 23 Plasmid copy number of pMIRY7-UA
transformants. Total DNA was isolated from the
transformed cells and digested with SacI. The fragments
were separated by electrophoresis on an 0.8% gel. The
9.1 kb rDNA band and the 6.4 kb plasmid band are
indicated.
Figure 24 Stability of multicopy integrant SUSOB in
continuous culture; for details see text.
Figure 25 Southern blot of total DNA digested with
BglII of multicopy integrant SUSOB isolated at different
stages of the continuous culture.
SUSOB grown in shake flask.
SUSOB at the start of the continuous culture.
SUSOB after the-addition of leucine.
SUSOB 1:
SUSOB 2:
SUSOB 3:
A schematic drawing of the construction
lipase expression vectors for H. polymorpha
and pUR6882. Each individual stage of
route is shown in a separate drawing
for details see text.
Figure 26
route of the
pUR6880, pUR6881
the construction
(Fig. 27 to 34):
Figure 27 A schematic drawing of pUR6038.
Figure 28 A schematic drawing of pUR6852.
Figure 29 A schematic drawing of pUR3501.
Figure 30 A schematic drawing of pUR6862.
Figure 31 A schematic drawing of pUR3511.
Figure 32 A schematic drawing of pUR6872.
Figure 33 A schematic drawing of pUR3513.
Figure 34 A schematic drawing of pUR6882.
Figure 35 A schematic drawing of the H. polymorpha
multicopy integration vector pUR3540 comprising the a-
galactosidase expression cassette. All used restriction
recognition enzyme sites are marked with an asterisk.
Figure 36 Southern analysis of total DNA digested
with HindIII of the H. polymorpha multicopy integrant
obtained using pUR3540.
lane 1: multicopy integrant.
lane 2: untransformed host strain.
Figure 37 The cloned ribosomal DNA of K. lactis is
shown (42). From this vector the indicated BamHI-SacI
fragment was subcloned in pTZl9U (46). From the
resulting vector the EcoRI fragment was used in the
construction of pMIRK7AT1, pMIRK7AT2 and pMIRK7AT3. The
EcoRI-PstI fragment was used as a probe in the
hybridization experiments.
Figure 38 A schematic drawing of the multicopy
integration vectors pMIRK7AT1, pMIRK7AT2 and pMIRK7AT3.
Figure 39 Hybridization of digested chromosomal DNA
of multicopy integrant after growth under non-selective
conditions with ribosomal DNA probe. Lane 1- 4:
multicopy integrant MIRK7AT1: lane 5: parent strain MSK
110, lane 6: linearized multicopy integration vector
pMIRK7AT1. Chromosomal DNA was isolated at the start of
the experiment (lane 1), after 6-7 generations (lane 2),
after 30-35 generations (lane 3) and after 60-70
generations (lane 4),
Claims (3)
1. In a process for preparing a protein by a fungus transformed by multicopy integration of an expression Vector in the ribosomal DNA of the fungus, - the expression vector including in addition to an expressible structural gene encoding the protein an expressible deficient selection marker gene needed for the production of an ingredient essential for growth of the fungus, _ - said fungus having been modified prior to transformation to inactivate the wild type gene corresponding to said expressible deficient selection marker, the improvement wherein fungal cells are maintained with high copy number integrants, and consequent improved production of the protein, by using the expression vector which has approximately the same length as one DNA sequence that codes for a ribosomal DNA. unit of the fungus, the latter being preferably between about 8.3 kb and about 11.1 kb, — whereby cells with high copy number integrants are preferentially maintained over cells with low copy number integrants, whereby the production of the protein is improved.
2. A process according to claim 1, in which the length of the expression vector is about 8-10 kb.
3. A process according to claim 1, in which the fungus is a Saccharomyces cerevisiae and the length of the expression vector is about 9 kb. F. R. KELLY & co., AGENTS FOR THE APPLICANTS.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBUNITEDKINGDOM07/07/19898915659.0 |
Publications (2)
Publication Number | Publication Date |
---|---|
IE19980989A1 IE19980989A1 (en) | 1991-01-07 |
IE83463B1 true IE83463B1 (en) | 2004-06-02 |
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