CQNSTITUTIVE PROMOTER
The invention refers to an isolated nucleic acid sequence comprising a strong
constitutive promoter and a method of producing a protein of interest in a eukaryotic
cell culture under the control of such a promoter.
BACKGROUND
Successful production of recombinant proteins has been accomplished with
eukaryotic hosts. The most prominent examples are yeasts like Saccharomyces
cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi like Aspergillus
awamori or Trichoderma reesei, or mammalian cells like e.g. CHO cells. While the
production of some proteins is readily achieved at high rates, many other proteins are
only obtained at comparatively low levels.
The heterologous expression of a gene in a host organism usually requires a
vector allowing stable transformation of the host organism. A vector would provide the
gene with a functional promoter adjacent to the 5' end of the coding sequence. The
transcription is thereby regulated and initiated by this promoter sequence. Most
promoters used up to date have been derived from genes that code for proteins that
are usually present at high concentrations in the cell.
EP0103409A2 discloses the use of yeast promoters associated with expression
of specific enzymes in the glycolytic pathway, i.e. promoters involved in expression of
pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, phosphorglycerate
mutase, hexokinase 1 and 2 , glucokinase, phosphofructose kinase, aldolase
and glycolytic regulation gene.
WO 97/44470 describes yeast promoters from Yarrowia lipolytica for the
translation elongation factor 1 (TEF1 ) protein and for the ribosomal protein S7 that are
suitable for heterologous expression of proteins in yeast, and EP1951877A1 describes
the use of the P. pastoris TEF1 promoter for the production of heterologous proteins.
WO2005003310 provides methods for the expression of a coding sequence of
interest in yeast using a promoter of the glyceraldehyde-3-phosphate dehydrogenase
or phosphoglycerate mutase from oleaginous yeast Yarrowia lipolytica.
Promoter sequences derived from genes involved in the methanol metabolic
pathway of Pichia pastoris are disclosed in US4808537 and US4855231 (alcohol
oxidase AOX1 , AOX2) and US6730499B1 (formaldehyde dehydrogenase FLD1 ) .
US20080153126A1 includes mutant promoter sequences based on the AOX1
promoter.
The AOX1 promoter is induced only in response to methanol and repressed by
other carbon sources, such as glucose or ethanol. Methanol has the disadvantage that
it is unsuitable for use in the production of certain products, since it is potentially
hazardous for its toxicity and flammability. Therefore, alternatives to the AOX1
promoter are sought.
Vassileva et al. (J. Biotechnol. (2001 ) 88: 2 1-35) describe the use of the GAP
promoter to express HBsAg in P. pastoris, using multicopy expression cassettes as an
alternative to the AOX1 promoter. The constitutive system was proposed for
continuous culture to permit maintenance of the cells in mid-exponential phase.
Promoters used in Pichia pastoris are either tightly regulated (like pAOX or
pFLD) being active on specific substrates such as methanol, or they are constitutively
active in many different conditions, media and substrates. Among the constitutive
ones, especially the GAP and the TEF promoters have been described to be strong,
and useful for recombinant protein production.
However, it was shown that the activity of both constitutive promoters is not
constantly strong during a fed -batch production process. Especially in the later phases
of the process, when cell growth rates are slow, also the activity of the promoters in
getting low, thus limiting expression levels of the gene of interest (GOI) and production
yields (Stadlmayr et al. 2010. J Biotechnol. 150: 519-529).
Selection of suitable promoters is not intuitive or rational, as even highly
abundant glycolytic enzymes such as enolase (ENO), those phosphate isomerase
(TP!) or glucose-6-phosphate isomerase (PGI) do not have promoters that are as
strong as pGAP and pTEF (Stadlmayr et al. 2010. J Biotechnol. 150: 519-529; Gasser
et al. 2010. Metabolic Engineering 12:573-580).
Qin et al. (Applied and Environmental Microbiology (201 1) 3600-3608) describe
a GAP promoter library and various mutants with varying activities.
WO2007/015178 A2 describes translational fusion partners capable of inducing
secretory production of recombinant proteins, and a P. pastoris cDNA library.
CN 102180954 A describes cell surface display system using the P. pastoris cell
wall protein GCW14 as an anchored protein.
t is desirable to provide improved recombinant eukaryotic cell Sines to produce
fermentation products that can be isolated with high yields. Therefore, it is the object of
the present invention to provide for alternative regulatory elements suitable for
recombinant production methods, which are simple and efficient.
SUMMARY OF THE INVENTION
The object is solved by the subject matter as claimed.
According to the invention there is provided an isolated nucleic acid sequence
comprising a promoter, which is a native sequence of Pichia pastoris comprising or
consisting of the pCS1 nucleic acid sequence of SEQ ID 1, or a functionally active
variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a
combination thereof.
According to a specific aspect, there is provided an isolated nucleic acid
sequence comprising a promoter, which is a native sequence of Pichia pastoris
comprising or consisting of the pCS1 nucleic acid sequence of SEQ ID 1, or a
functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID
1, or a combination thereof, wherein said functionally active variant exhibits
substantially the same activity as pCS1 , specifically the pCS1 nucleic acid sequence of
SEQ ID 1.
Specifically, the pCS1 nucleotide sequence is identical with the sequence of
SEQ ID 1.
Preferably, the nucleic acid sequence of the invention is not identical to the
nucleic acid of SEQ ID 87 as listed in WO2007/015178 A2 (i.e. SEQ ID 24 of this
application).
Specifically, the functionally active variant is
a) a size variant of pCS1 of SEQ ID 1, preferably consisting of or comprising the
nucleic acid sequence selected from the group consisting of SEQ ID 2, 3, 4 , 5, 6, 7 and
8;
b) a mutant of the pCS1 of SEQ ID 1, or a mutant of the size variant of a), which
mutant has at least 60% homology to the sequence SEQ ID 1 or to the size variant;
c) a hybrid comprising
- a sequence selected from the group consisting of pCS1 of SEQ ID 1, a size
variant of a), and a mutant of b); and
- a least one further sequence selected from the group consisting of pCS1 of
SEQ ID 1, a size variant of a), a mutant of b), and a heterologous sequence; or
d) a sequence which hybridizes under stringent conditions to any of the size
variant, or the mutant nucleic acid sequences of a), or b).
Preferably, the size variant of pCS1 of SEQ ID 1 consists of any of SEQ ID 2,
SEQ ID 3, SEQ ID 4 , SEQ ID 5 or SEQ ID 6
According to a specific embodiment, the functionally active variant is selected
from the group consisting of homologs with
i) at least about 60% nucleotide sequence identity;
ii) homologs obtainable by modifying the nucleotide sequence of pCS1 of SEQ
ID 1 or size variants thereof, by insertion, deletion or substitution of one or more
nucleotides within the sequence or at either or both of the distal ends of the sequence,
preferably with a nucleotide sequence of 80 bp to 1500 bp, or of 200 bp to 1500 bp,
more preferably at least 200 bp;and
iii) analogs derived from species other than Pichia pastoris.
Specifically the functionally active variant of the invention has substantially the
same promoter activity as pCS1 .
According to a specific embodiment, the nucleic acid sequence is operably
linked to a nucleotide sequence encoding a protein of interest (POI), which nucleic acid
is not natively associated with the nucleotide sequence encoding the POI.
According to a specific aspect, the nucleic acid sequence further comprises a
signal peptide gene enabling the secretion of the POI, preferably wherein the signal
peptide gene is located adjacent to the 5' end of the nucleotide sequence encoding the
POI.
Accordingly, the invention specifically refers to the nucleic acid sequence which
further comprises a nucleic acid sequence encoding a signal peptide enabling the
secretion of the POI, preferably wherein nucleic acid sequence encoding the signal
peptide is located adjacent to the 5' end of the nucleotide sequence encoding the POI.
The invention further provides for an expression construct comprising a nucleic
acid sequence of the invention, preferably an autonomously replicating vector or
plasmid, or one which integrates into the chromosomal DNA of a host cell.
The invention further provides for a recombinant host cell which comprises the
nucleic acid sequence of the invention or the expression construct of the invention,
preferably a eukaryotic cell, more preferably a yeast or filamentous fungal cell, more
preferably a yeast cell of the Saccharomyces or Pichia genus.
According to a specific aspect, the recombinant host cell comprises multiple
copies of the nucleic acid sequence, and/or multiple copies of the expression
construct. For example, the recombinant cell comprises 2 , 3, 4 , or more copies (gene
copy number, GCN).
Specifically, the recombinant host cell is selected from the group consisting of
mammalian, insect, yeast, filamentous fungi and plant cells, preferably a yeast,
preferably any of the P. pastoris strains CBS 704, CBS 2612, CBS 7435, CBS 9173-
9189, DSMZ 70877, X-33, GS1 15, KM71 and SMD1 168. In some embodiments, the
recombinant host cell is a P. pastoris strain other than X-33.
The invention further provides for a stable culture of a plurality of the cell of the
invention.
The invention further provides for a method of producing a POI by culturing a
recombinant host cell line comprising the nucleic acid sequence or the promoter of the
invention, or the expression construct of the invention, and a nucleic acid encoding the
POI under the transcriptional control of said promoter, comprising the steps of
a) cultivating the cell line under conditions to express said POI, and
b) recovering the POI.
Specifically, the POI is expressed under growth -limiting conditions, e.g. by
cultivating the cell line at a growth rate of less than the maximal growth rate, typically
less than 90%, preferably less than 80%, less than 70%, less than 60%, less than
50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less
than 3%, less than 2%, less than 1%, less than 0,5%, less than 0,4%, less than 0,3%,
or less than 0,2% of the maximum growth rate of the cells. Typically the maximum
growth rate is individually determined for a specific host cell.
According to a specific embodiment, the cell line is cultivated under batch, fedbatch
or continuous cultivation conditions, and/or in media containing limited carbon
substrate.
Specifically, the cultivation is performed in a bioreactor starting with a batch
phase followed by a fed-batch phase or a continuous cultivation phase.
Specifically, the host cells are grown in a carbon source rich medium during the
phase of high growth rate (e.g. at least 50%, or at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, at least 98%, at least 99%, or up to the maximum
growth rate) and producing the POI during a phase of low growth rate (e.g. less than
90%, preferably less than 80%, less than 70%, less than 60%, less than 50%, or less
than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%,
less than 2%, less than 1%, less than 0,5%, less than 0,4%, less than 0,3%, or less
than 0,2% of the maximum growth rate) e.g. while limiting the carbon source,
preferably by feeding a defined minimal medium. Specifically the defined minimal
medium does not comprise a transcription-inducing carbon source.
The POI is specifically a heterologous protein, preferably selected from
therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides,
protein antibiotics, toxin fusion proteins, carbohydrate - protein conjugates, structural
proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process
enzymes, growth factors, hormones and cytokines, or a metabolite of a POI,
specifically including a cell metabolite of the recombinant cell culture that expresses a
gene of interest under the transcriptional control of a promoter of the invention.
The invention further provides for a method to identify a constitutive promoter
from eukaryotic cells, comprising the steps of
a) cultivating eukaryotic cells at a high growth rate;
b) further cultivating the cells at a low growth rate;
c) providing samples of the cell culture of step a) and b),
d) performing transcription analysis in said samples and comparing the
transcript levels with the transcript levels of the native pGAP promoter of the cells; and
f ) selecting the constitutive promoter that has a higher transcription strength as
compared to the native pGAP promoter at high and low growth rates, preferably by
determining a transcript level of the identified constitutive promoter which is at least
1.1-fold higher as compared to the native pGAP promoter, preferably at least 1.2-fold,
preferably at least 1.3-fold, preferably at least 1.4-fold, preferably at least 1.5-fold,
preferably at least 1.6-fold, preferably at least 1.7-fold, preferably at least 1.8-fold,
preferably at least 1.9-fold, preferably at least 2-fold, preferably at least 3-fold,
preferably at least 4-fold, preferably at least 5-fold, preferably at least 10-fold, or at
least 15-fold higher.
Specifically, the transcript level is determined at a high growth rate and a low
growth rate within the range of 0,01 to 0,2 h , preferably within the range of 0,015 to
0,15 h , e.g. by examining at least two samples of a cell culture, a first one
representing a high growth rate, such as at least 0,05 h , preferably at least 0,06 h ,
or at least 0,07 h , at least 0,08 h , at least 0,09 h , at least 0,1 h , e.g. at a growth
rate of 0 , 5 h , and a second one representing a low growth rate, such as less than
the first one, e.g. less than 0,05 h , preferably less than 0,04 h , less than 0,03 h , or
less than 0,02 h , e.g. at a growth rate of 0,015 h . The transcription level is
specifically determined by analysis of the gene expression patterns using DNA
microarrays, e.g. according to Example 11 below.
The invention further provides for the use of an isolated nucleic acid sequence
comprising or consisting of a promoter which when operatively linked to a nucleotide
sequence encoding a POI directs the expression thereof in a host cell at an expression
level that is higher than under control of the native pGAP promoter at high and low
growth rates, preferably in a method of producing a POI by cultivating a host cell
transformed with the nucleic acid sequence, wherein the cultivation is performed in a
bioreactor starting with a batch phase followed by a fed -batch phase or a continuous
cultivation phase.
Specifically, the expression level is determined at a high growth rate and a low
growth rate within the range of 0,01 to 0,2 h , preferably within the range of 0,015 to
0,15 h , e.g. by examining at least two samples of a cell culture, a first one
representing a high growth rate, such as at least 0,05 h , preferably at least 0,06 h ,
or at least 0,07 h , at least 0,08 h , at least 0,09 h , at least 0,1 h , e.g. at a growth
rate of 0,1 5 h and a second one representing a low growth rate, such as less than
the first one, e.g. less than 0,05 h , preferably less than 0,04 h , less than 0,03 h , or
less than 0,02 h , e.g. at a growth rate of 0,015 h . The expression level is specifically
determined under growth-limited conditions. An example of determining the expression
strength in growth -limited conditions e.g. in a glucose-limited chemostat cultivation, at
high and low growth rates is provided in Example 10 below.
Specifically, the expression level is at least 1.1-fold higher compared to the
pGAP promoter, preferably at least 1.2-fold, preferably at least 1.3-fold, preferably at
least 1.4-fold, preferably at least 1.5-fold, preferably at least 1.6-fold, preferably at least
1.7-fold, preferably at least 1.8-fold, preferably at least 1.9-fold, preferably at least 2-
fold, preferably at least 3-fold, preferably at least 4-fold, preferably at least 5-fold,
preferably at least 10-fold, or at least 15-fold higher.
According to a specific aspect, the isolated nucleic acid sequence of the
invention or the expression construct of the invention is used in a method of producing
a POI by cultivating a host cell transformed with the nucleic acid sequence and/or the
expression construct, preferably wherein the cultivation is performed in a bioreactor
starting with a batch phase followed by a fed-batch phase or a continuous cultivation
phase.
FIGURES
Figure 1: Nucleic acid sequence pCS1 (985 bp, SEQ ID 1) of P. pastoris, and
promoter sequences which are DNA sequences including pCS1 and additional
nucleotides at the 5' end, or pCS1 fragments, promoting expression of CS1 in P.
pastoris, which comprise 1488bp (SEQ ID 2), 767bp (SEQ ID 3), 500bp (SEQ ID 4),
344bp (SEQ ID 5), 234bp (SEQ ID 6), 138bp (SEQ ID 7) and 85bp (SEQ ID8).
Figure 2 : Sequences of CS1 coding nucleotide sequence and amino acid
sequence
i) of strains GS1 15, CBS7435 and CBS2612 (PAS_chr1 -4_0586), coding
sequence (SEQ ID 9), translated sequence (XM_002490678.1 , SEQ ID 10); and
ii) of strain DSMZ70382 (PIPA02805), coding sequence (SEQ D 11) , translated
sequence (SEQ ID 12).
Figure 3: Native pGAP promoter sequence of P. pastoris (GS1 15) (SEQ ID 13).
DETAILED DESCRIPTION OF THE INVENTION
Specific terms as used throughout the specification have the following meaning.
The term "carbon source" or "carbon substrate" as used herein shall mean a
fermentable carbon substrate, typically a source carbohydrate, suitable as an energy
source for microorganisms, such as those capable of being metabolized by host
organisms or production cell lines, in particular sources selected from the group
consisting of monosaccharides, oligosaccharides, polysaccharides, alcohols including
glycerol, in the purified form, in minimal media or provided in raw materials, such as a
complex nutrient material. The carbon source may be used according to the invention
as a single carbon source or as a mixture of different carbon sources.
The term "carbon substrate rich conditions" as used herein specifically refers to
the type and amount of a carbon substrate suitable for cell growth, such as a nutrient
for eukaryotic cells. The carbon source may be provided in a medium, such as a basal
medium or complex medium, but also in a (chemically) defined medium containing a
purified carbon source. The carbon source for use in a growth phase of a celi
cultivation process is herein also called "basal carbon source" and typically provided in
an amount to provide for cell growth, for example to obtain cell densities of at least
5 g/L cell dry mass, preferably at least 10 g/L cell dry mass, or at least 15 g/L cell dry
mass, e.g. exhibiting viabilities of more than 90% during standard sub-culture steps,
preferably more than 95%.
In a growth phase, the carbon source is typically used in an excess or surplus
amount, which is understood as an excess providing energy to increase the biomass,
e.g. during the cultivation of a cell line with a high specific growth rate.
This surplus amount is particularly in excess of the limited amount of a carbon
source as used under growth-limited conditions, to achieve a residual concentration in
the fermentation broth that is measurable and typically at least 10-fold higher,
preferably at least 50-fold or at least 100-fold higher than during feeding the cell culture
with a medium containing limited carbon substrate.
The term "carbon substrate limited conditions" or "limited carbon source", herein
also referred to as "supplemental carbon source", such as used according to the
invention is herein understood to specifically refer to the type and amount of a carbon
substrate facilitating the production of fermentation products by production cell lines, in
particular in a cultivation process with controlled growth rates of less than the
maximum growth rate. The production phase specifically follows a growth phase, e.g.
in batch, fed-batch and continuous cultivation process.
In general, cell culture processes are classified into batch culture, continuous
culture, and fed-batch culture. Batch culture is a culture process by which a small
amount of a seed culture solution is added to a medium and cells are grown without
adding an additional medium or discharging a culture solution during culture.
Continuous culture is a culture process by which a medium is continuously added and
discharged during culture. The continuous culture also includes perfusion culture. Fedbatch
culture, which is an intermediate between the batch culture and the continuous
culture and also referred to as semi-batch culture, is a culture process by which a
medium is continuously or sequentially added during culture but, unlike the continuous
culture, a culture solution is not continuously discharged.
Specifically preferred is a fed-batch process which is based on feeding of a
growth limiting nutrient substrate to a culture. The fed -batch strategy, including single
fed-batch or repeated fed-batch fermentation, is typically used in bio-industrial
processes to reach a high cell density in the bioreactor. The controlled addition of the
carbon substrate directly affects the growth rate of the culture and helps to avoid
overflow metabolism or the formation of unwanted metabolic byproducts. Under carbon
source limited conditions, the carbon source specifically may be contained in the feed
of a fed-batch process. Thereby, the carbon substrate is provided in a limited amount.
Also in chemostat or continuous culture as described herein, the growth rate
can be tightly controlled.
A "limited amount" of a carbon source is herein understood as the amount of a
carbon source necessary to keep a production cell line under growth-limited
conditions, e.g. in a production phase or production mode. Such a limited amount may
be employed in a fed-batch process, where the carbon source is contained in a feed
medium and supplied to the culture at low feed rates for sustained energy delivery, e.g.
to produce a POI, while keeping the biomass at low specific growth rates. A feed
medium is typically added to a fermentation broth during the production phase of a cell
culture.
The limited amount of a carbon source may, for example, be determined by the
residual amount of the carbon source in the cell culture broth, which is below a
predetermined threshold or even below the detection limit as measured in a standard
(carbohydrate) assay. The residual amount typically would be determined in the
fermentation broth upon harvesting a fermentation product.
The limited amount of a carbon source may as well be determined by defining
the average feed rate of the carbon source to the fermenter, e.g. as determined by the
amount added over the full cultivation process, e.g. the fed -batch phase, per cultivation
time, to determine a calculated average amount per time. This average feed rate is
kept low to ensure complete usage of the supplemental carbon source by the cell
culture, e.g. between 0.6 g L h (g carbon source per L initial fermentation volume
and h time) and 25 g L h , preferably between 1.6 g L h and 20 g L h .
The limited amount of a carbon source may also be determined by measuring
the specific growth rate, which specific growth rate is kept low, e.g. lower than the
maximum specific growth rate, during the production phase, e.g. within a
predetermined range, such as in the range of 0.001 h 1 to 0.20 h , or 0.02 h to
0.20 h , preferably between 0.02 h and 0.15 h .
Any type of organic carbon suitable used for eukaryotic cell culture may be
used. According to a specific embodiment, the carbon source is a hexose such as
glucose, fructose, galactose or mannose, a disaccharide, such as saccharose, an
alcohol, such as glycerol or ethanol, or a mixture thereof.
According to a specifically preferred embodiment, the basal carbon source is
selected from the group consisting of glucose, glycerol, ethanol, or mixtures thereof,
and complex nutrient material. According to a preferred embodiment, the basal carbon
source is glycerol.
According to a further specific embodiment, a supplemental carbon source is a
hexose such as glucose, fructose, galactose and mannose, a disaccharide, such as
saccharose, an alcohol, such as glycerol or ethanol, or a mixture thereof. According to
a preferred embodiment, a supplemental carbon source is glucose.
Specifically, the method may employ glycerol as a basal carbon source and
glucose as a supplemental carbon source.
Specifically, a feed medium as used herein is chemically defined and methanolfree.
The term "chemically defined" or "defined" with respect to cell culture medium,
such as a minimal medium or feed medium in a fed -batch process, shall mean a
cultivation medium suitable for the in vitro cell culture of a production cell line, in which
all of the chemical components and (poly)peptides are known. Typically a chemically
defined medium is entirely free of animal-derived components and represents a pure
and consistent cell culture environment.
The term "cell line" as used herein refers to an established clone of a particular
cell type that has acquired the ability to proliferate over a prolonged period of time. The
term "host cell line" refers to a cell line as used for expressing an endogenous or
recombinant gene or products of a metabolic pathway to produce polypeptides or cell
metabolites mediated by such polypeptides. A "production host cell line" or "production
cell line" is commonly understood to be a cell line ready-to-use for cultivation in a
bioreactor to obtain the product of a production process, such as a POL The term
"eukaryotic host" or "eukaryotic cell line" shall mean any eukaryotic cell or organism,
which may be cultivated to produce a POI or a host cell metabolite. It is well
understood that the term does not include human beings.
According to specifically preferred eukaryotic host cells of the invention, the cell
or cell line is selected from the group consisting of mammalian, insect, yeast,
filamentous fungi and plant cell lines, preferably a yeast.
Specifically the yeast is selected from the group consisting of Pichia, Candida,
Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces, Saccharomyces,
Komagataella, preferably a methylotrophic yeast.
A specifically preferred yeast is Pichia pastoris, Komagataella pastoris, K.
phaffii, or K. pseudopastoris.
The term "cell culture" or "cultivation", also termed "fermentation", with respect
to a host cell line is meant the maintenance of cells in an artificial, e.g., an in vitro
environment, under conditions favoring growth, differentiation or continued viability, in
an active or quiescent state, of the cells, specifically in a controlled bioreactor
according to methods known in the industry.
When cultivating a cell culture using the culture media of the present invention,
the cell culture is brought into contact with the media in a culture vessel or with
substrate under conditions suitable to support cultivation of the cell culture. In certain
embodiments, a culture medium as described herein is used to culture cells according
to standard cell culture techniques that are well-known in the art. In various aspects of
the invention, a culture medium is provided that can be used for the growth of
eukaryotic cells, specifically yeast or filamentous fungi.
Cell culture media provide the nutrients necessary to maintain and grow cells in
a controlled, artificial and in vitro environment. Characteristics and compositions of the
cell culture media vary depending on the particular cellular requirements. Important
parameters include osmolality, pH, and nutrient formulations. Feeding of nutrients may
be done in a continuous or discontinuous mode according to methods known in the art.
The culture media used according to the invention are particularly useful for producing
recombinant proteins.
Whereas a batch process is a cultivation mode in which all the nutrients
necessary for cultivation of the cells are contained in the initial culture medium, without
additional supply of further nutrients during fermentation, in a fed -batch process, after
a batch phase, a feeding phase takes place in which one or more nutrients are
supplied to the culture by feeding. The purpose of nutrient feeding is to increase the
amount of biomass in order to increase the amount of recombinant protein as well.
Although in most cultivation processes the mode of feeding is critical and important,
the present invention employing the promoter of the invention is not restricted with
regard to a certain mode of cultivation.
ln certain embodiments, the method of the invention is a fed -batch process.
Specifically, a host cell transformed with a nucleic acid construct encoding a desired
recombinant POI, is cultured in a growth phase medium and transitioned to a
production phase medium in order to produce a desired recombinant POI.
The feed medium may be added to the culture medium in the liquid form or else
in an alternative form, such as a solid, e.g. as a tablet or other sustained release
means, or a gas, e.g. carbon dioxide. Yet, according to a preferred embodiment the
limited amount of a supplemental carbon source added to the cell culture medium, may
even be zero. Preferably, under conditions of a limited carbon substrate, the
concentration of a supplemental carbon source in the culture medium is 0-1 g/L,
preferably less than 0.6 g/L, more preferred less than 0.3 g/L, more preferred less than
0.1 g/L, preferably 1-50 mg/L, more preferred 1-10 mg/L, specifically preferred 1 mg/L
or even below, such as below the detection limit as measured with a suitable standard
assay, e.g. determined as a residual concentration in the culture medium upon
consumption by the growing cell culture.
In a preferred method, the limited amount of the carbon source provides for a
residual amount in the cell culture which is below the detection limit as determined in
the fermentation broth at the end of a production phase or in the output of a
fermentation process, preferably upon harvesting the fermentation product.
Preferably, the limited amount of a supplemental carbon source is growth
limiting to keep the specific growth rate lower than the maximum specific growth rate,
such as in the range of 0.001 h to 0.20 h , or 0.02 h to 0.20 h , preferably between
0.02 h and 0.15 h .
In another embodiment, host cells of the present invention are cultivated in
continuous mode, e.g. a chemostat. A continuous fermentation process is
characterized by a defined, constant and continuous rate of feeding of fresh culture
medium into the bioreactor, whereby culture broth is at the same time removed from
the bioreactor at the same defined, constant and continuous removal rate. By keeping
culture medium, feeding rate and removal rate at the same constant level, the
cultivation parameters and conditions in the bioreactor remain constant.
A stable cell culture as described herein is specifically understood to refer to a
cell culture maintaining the genetic properties, specifically keeping the POI production
level high, e.g. at least at a g level, even after about 20 generations of cultivation,
preferably at least 30 generations, more preferably at least 40 generations, most
preferred of at least 50 generations. Specifically, a stable recombinant host cell Sine is
provided which is considered a great advantage when used for industrial scale
production.
The cell culture of the invention is particularly advantageous for methods on a n
industrial manufacturing scale, e.g. with respect to both the volume and the technical
system, in combination with a cultivation mode that is based on feeding of nutrients, in
particular a fed-batch or batch process, or a continuous or semi-continuous process
(e.g. chemostat).
The term "expression" or "expression system" or "expression cassette" refers to
nucleic acid molecules containing a desired coding sequence and control sequences in
operable linkage, so that hosts transformed or transfected with these sequences are
capable of producing the encoded proteins or host cell metabolites. In order to effect
transformation, the expression system may be included in a vector; however, the re
levant DNA may also be integrated into the host chromosome. Expression may refer to
secreted or non-secreted expression products, including polypeptides or metabolites.
"Expression constructs" or "vectors" or "plasmid" used herein are defined as
DNA sequences that are required for the transcription of cloned recombinant
nucleotide sequences, i.e. of recombinant genes and the translation of their mRNA in a
suitable host organism. Expression vectors or plasmids usually comprise an origin for
autonomous replication in the host cells, selectable markers (e.g. an amino acid
synthesis gene or a gene conferring resistance to antibiotics such as zeocin,
kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a
suitable promoter sequence and a transcription terminator, which components are
operably linked together. The terms "plasmid" and "vector" as used herein include
autonomously replicating nucleotide sequences as well as genome integrating
nucleotide sequences.
The expression construct of the invention specifically comprises a promoter of
the invention, operably linked to a nucleotide sequence encoding a POI under the
transcriptional control of said promoter, which promoter is not natively associated with
the coding sequence of the POI.
The term "heterologous" as used herein with respect to a nucleotide or amino
acid sequence or protein, refers to a compound which is either foreign, i.e.
"exogenous", such as not found in nature, to a given host cell; or that is naturally found
in a given host cell, e.g., is "endogenous", however, in the context of a heterologous
construct, e.g. employing a heterologous nucleic acid. The heterologous nucleotide
sequence as found endogenously may also be produced in an unnatural, e.g. greater
than expected or greater than naturally found, amount in the cell. The heterologous
nucleotide sequence, or a nucleic acid comprising the heterologous nucleotide
sequence, possibly differs in sequence from the endogenous nucleotide sequence but
encodes the same protein as found endogenously. Specifically, heterologous
nucleotide sequences are those not found in the same relationship to a host cell in
nature. Any recombinant or artificial nucleotide sequence is understood to be
heterologous. An example of a heterologous polynucleotide is a nucleotide sequence
not natively associated with the promoter according to the invention, e.g. to obtain a
hybrid promoter, or operably linked to a coding sequence, as described herein. As a
result, a hybrid or chimeric polynucleotide may be obtained. A further example of a
heterologous compound is a POI encoding polynucleotide operably linked to a
transcriptional control element, e.g., a promoter of the invention, to which an
endogenous, naturally-occurring POI coding sequence is not normally operably linked.
The term "variant" as used herein in the context of the present invention shall
specifically refer to any sequence derived from a parent sequence, e.g. by size
variation, e.g. elongation or fragmentation, mutation, hybridization (including
combination of sequences), or with a specific degree of homology, or analogy.
The invention specifically provides for a promoter which is a wild -type promoter,
e.g. of P. pastoris, or a functionally active variant thereof, e.g. capable of controlling
the transcription of a specific gene in a wild-type or recombinant eukaryotic cell.
The term "native" as used herein in the context of the present invention shall
specifically refer to an individual structure or component of an organism, which is
naturally associated with its environment. It is, however, well understood, that native
structures or components may be isolated from the naturally associated environment,
and provided as isolated native structures or components. Such isolated native
structures or components may as well be of artificial or synthetic origin, and still have
the same characteristics as the ones of natural origin.
Though some embodiments of the present invention refer to native structures or
components, e.g. in the isolated form, it is well understood that the materials, methods
and uses of the invention, e.g. specifically referring to isolated nucleic acid sequences,
amino acid sequences, expression constructs, transformed host cells and recombinant
proteins, are "man-made" and are therefore not considered as a result of "law of
nature".
The functionally active variant promoter may e.g. be derived from the promoter
sequence pCS1 (SEQ ID 1) by mutagenesis, thus employing the pCS1 sequence as a
"parent" sequence, to produce sequences suitable for use as a promoter in
recombinant cell lines. Such variant promoter may be obtained from a (pCS1 ) library of
mutant sequences by selecting those library members with predetermined properties.
Variant promoters may have the same or even improved properties, e.g. improved in
promoter strength to support POI production, still with substantially the same promoter
function and strength at high and low growth rates, specifically understood herein as
"growth-rate independent function".
The variant promoter may also be derived from analogous sequences, e.g. from
eukaryotic species other than Pichia pastoris or from a genus other than Pichia, such
as from K. lactis, Z. rouxii, P. stipitis, H. polymorpha. Specifically, the analogous
promoter sequences natively associated with genes analogous to the corresponding P.
pastoris genes may be used as such or as parent sequences to produce functionally
active variants thereof. Specifically, a promoter analogous to pCS1 is characterised
that it is natively associated with a gene analogous to CS1 (see amino acid sequence
of SEQ ID 9 or 11) . The properties of such analogous promoter sequences or
functionally active variants thereof may be determined using standard techniques.
The "functionally active" variant of a nucleotide or promoter sequence as used
herein specifically means a mutant sequence, e.g. resulting from modification of a
parent sequence by insertion, deletion or substitution of one or more nucleotides within
the sequence or at either or both of the distal ends of the sequence, and which
modification does not affect (in particular impair) the activity of this sequence.
Specifically, the functionally active variant of the promoter sequence according
to the invention is selected from the group consisting of
- homologs with at least about 60% nucleotide sequence identity, preferably at
least 70%, at least 80%, or at least 90% degree of homology or sequence identity to
the parent sequence; and/or
- homologs obtainable by modifying the parent nucleotide sequence, such as
the pCS1 sequence or the sequence of a size variant used as a template to provide for
mutations, e.g. by insertion, deletion or substitution of one or more nucleotides within
the sequence or at either or both of the distal ends of the sequence, preferably with
(i.e. comprising or consisting of) a nucleotide sequence of 80 bp to 500 bp or of 200
bp to-1500 bp or 234 bp to 1488 bp, preferably at least 100 bp, at least 200 bp,
preferably at least 300 bp, more preferred at least 400 bp, at least 500 bp, at least 600
bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp; and
- analogs derived from species other than Pichia pastoris.
Specifically preferred functionally active variants are those derived from a
promoter according to the invention by modification, extension and/or fragments of the
promoter sequence, with (i.e. comprising or consisting of) a nucleotide sequence of at
least 80bp, preferably at least 100 bp, preferably at least 200 bp, preferably at least
250 bp, preferably at least 300 bp, more preferred at least 400 bp, at least 500 bp, at
least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp,
preferably up to 1500 bp.
A functionally active variant of a parent promoter sequences as described
herein may specifically obtained through mutagenesis methods. The term
"mutagenesis" as used in the context of the present invention shall refer to a method of
providing mutants of a nucleotide sequence, e.g. through insertion, deletion and/or
substitution of one or more nucleotides, so to obtain variants thereof with at least one
change in the non-coding or coding region. Mutagenesis may be through random,
semi-random or site directed mutation. Typically large randomized gene libraries are
produced with a high gene diversity, which may be selected according to a specifically
desired genotype or phenotype.
Some of the preferred functionally active variants of the promoter according to
the invention are size variants or specifically fragments of pCS1 , preferably those
including the 3' end of a promoter nucleotide sequence, e.g. a nucleotide sequence
derived from one of the promoter nucleotide sequences which has of a specific length
and insertions or a deletion of the 5' terminal region, e.g. an elongation or cut-off of the
nucleotide sequence at the 5' end, so to obtain a specific length with a range from the
3' end to a varying 5' end, such as with a length of the nucleotide sequence of at least
80bp, preferably at least 100 bp, preferably at least 200 bp, preferably at least 250 bp,
preferably at least 300 bp, more preferred at least 400 bp, at least 500 bp, at least 600
bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.
The elongated size variant of the invention preferably comprises additional one
or more nucleotide(s) at the 5' end of the pCS1 sequence, e.g. those which are
natively associated with the wild-type pCS1 sequence in the cell of origin.
For example, a functionally active variant of pCS1 may comprise a nucleotide
sequence or consist of a nucleotide sequence selected from the group consisting of
pCS a (SEQ D 2), pCS b (SEQ D 3), pCS1c (SEQ D 4), pCS1d (SEQ D 5), pCS1e
(SEQ ID 6), pCS1f (SEQ ID 7), and pCS1g (SEQ D 8), thus, a nucleotide sequence
within the range of 80-1500 bp. Preferably, the functionally active variant of pCS1
comprises or consists of a nucleotide sequence selected from the group consisting of
pCS1a (SEQ ID 2), pCS1 b (SEQ ID 3), pCS1c (SEQ ID 4), pCS1d (SEQ ID 5) and
pCS1e (SEQ ID 6).
The functionally active variant of a promoter of the invention is also understood
to encompass hybrids of the pCS1 or any of the functionally active variants thereof, in
particular any of the parent size variant or fragment sequences, e.g. resulting from
combination with one or more of any of the sequences that qualify as pCS1 or
functionally active variants thereof, e.g. at least two of such parent sequences, at least
3, at least 4 or at least 5 of the sequences, e.g. a combination of two or more of the
pCS1 elongated sequences or fragments selected from the group consisting of pCS1a,
pCS1 b, pCS1c, pCS1d, pCS1e, pCS1f, and pCS1 g; preferably, selected from the
group consisting of pCS1a, pCS1 b, pCS1c, pCS1d, and pCS1e. In another
embodiment, the hybrid is composed of at least one of the sequences selected from
pCS1 or any of the functionally active variants thereof, in particular any of the size
variant or fragment sequences, and a heterologous sequences which is e.g. not
natively associated with the pCS1 sequence in P. pastoris.
The functionally active variant of a promoter of the invention is further
understood to encompass a nucleotide sequence which hybridizes under stringent
conditions to the pCS1 promoter or any of the functionally active size variants or
fragments, mutants or hybrid nucleic acid sequences thereof.
As used in the present invention, the term "hybridization" or "hybridizing" is
intended to mean the process during which two nucleic acid sequences anneal to one
another with stable and specific hydrogen bonds so as to form a double strand under
appropriate conditions. The hybridization between two complementary sequences or
sufficiently complementary sequences depends on the operating conditions that are
used, and in particular the stringency. The stringency may be understood to denote the
degree of homology; the higher the stringency, the higher percent homology between
the sequences. The stringency may be defined in particular by the base composition of
the two nucleic sequences, and/or by the degree of mismatching between these two
nucleic sequences. By varying the conditions, e.g. salt concentration and temperature,
a given nucleic acid sequence may be allowed to hybridize only with its exact
complement (high stringency) or with any somewhat related sequences (low
stringency). Increasing the temperature or decreasing the salt concentration may tend
to increase the selectivity of a hybridization reaction.
As used in the present invention the phrase "hybridizing under stringent
hybridizing conditions" is preferably understood to refer to hybridizing under conditions
of certain stringency. In a preferred embodiment the "stringent hybridizing conditions"
are conditions where homology of the two nucleic acid sequences is at least 70%,
preferably at least 80%, preferably at least 90%, i.e. under conditions where
hybridization is only possible if the double strand obtained during this hybridization
comprises preferably at least 70%, preferably at least 80%, preferably at least 90% of
A-T bonds and C-G bonds.
The stringency may depend on the reaction parameters, such as the
concentration and the type of ionic species present in the hybridization solution, the
nature and the concentration of denaturing agents and/or the hybridization
temperature. The appropriate conditions can be determined by those skilled in the art,
e.g. as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, 1989).
The functionally active variant of the invention is specifically characterized by
exhibiting substantially the same activity as pCS1 .
The term "substantially the same activity " as used herein specifically refers to
the activity as indicated by substantially the same or improved promoter strength,
specifically the expression or transcriptional strength of the promoter, and its
substantially the same or improved characteristics with respect to the promoter
strength, specifically determined independent of the growth rate of the host cell such
as an expression or transcriptional strength substantially the same as pCS1 , e.g. +/-
20% or +/- 10%, and/or higher than the native pGAP promoter of the host cell, e.g. an
at least 1.1-fold increase, or an at least 1.2-fold increase, preferably at least 1.3-fold,
preferably at least 1.4-fold, preferably at least 1.5-fold, preferably at least 1.6-fold,
preferably at least 1.7-fold, preferably at least 1.8-fold, preferably at least 1.9-fold, and
preferably at least 2-fold increase relative to the pGAP promoter strength, or even
higher, e.g. at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least up
to a 15-fold activity, as determined in a suitable test system employing the same type
of a host cell, the same cultivation conditions and the same nucleic acid encoding an
expression product such as a PO .
The term "homology" indicates that two or more nucleotide sequences have the
same or conserved base pairs at a corresponding position, to a certain degree, up to a
degree close to 100%. A homologous sequence of the invention typically has at least
about 60% nucleotide sequence identity, preferably at least about 70% identity, more
preferably at least about 80% identity, more preferably at least about 90% identity,
more preferably at least about 95% identity, more preferably at least about 98% or
99% identity.
The homologous promoter sequence according to the invention preferably has a
certain homology to any of the pCS1 , pCS1a, pCS1b, pCS1c, pCS1d, pCS1e, pCS1f,
and pCS1g promoter nucleotide sequences of P. pastoris in at least specific parts of
the nucleotide sequence, such as including the 3' region of the respective promoter
nucleotide sequence, preferably a part with a specific length up to the 3' end of the
respective promoter nucleotide sequence, such as a part with a length of 80 bp to
1500 bp, preferably of 200 bp to 1500 bp, more preferably of 234 to 1488 bp;
preferably at least 100 bp, preferably at least 200 bp, preferably at least
300 bp, more preferred at least 400 bp, at least 500 bp, at least 600 bp, at least
700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp, and analogs derived from
species other than Pichia pastoris. Specifically at least those parts are preferably
homologous within the range of 300-1000 bp, including the 3' terminal sequence of the
respective promoter nucleotide sequence.
Analogous sequences are typically derived from other species or strains. It is
expressly understood that any of the analogous promoter sequences of the present
invention that are derived from species other than Pichia pastoris may comprise a
homologous sequence, i.e. a sequence with a certain homology as described herein.
Thus, the term "homologous" may also include analogous sequences. On the other
hand, it is understood that the invention also refers to analogous sequences and
homologs thereof that comprise a certain homology.
"Percent (%) identity" with respect to the nucleotide sequence of a gene is
defined as the percentage of nucleotides in a candidate DNA sequence that is identical
with the nucleotides in the DNA sequence, after aligning the sequence and introducing
gaps, if necessary, to achieve the maximum percent sequence identity, and not
considering any conservative substitutions as part of the sequence identity. Alignment
for purposes of determining percent nucleotide sequence identity can be achieved in
various ways that are within the skill in the art, for instance, using publicly available
computer software. Those skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve maximal alignment
over the full length of the sequences being compared.
The term "isolated" or "isolation" as used herein with respect to a nucleic acid, a
POI or other compound shall refer to such compound that has been sufficiently
separated from the environment with which it would naturally be associated, so as to
exist in "substantially pure" form. "Isolated" does not necessarily mean the exclusion of
artificial or synthetic mixtures with other compounds or materials, or the presence of
impurities that do not interfere with the fundamental activity, and that may be present,
for example, due to incomplete purification. In particular, isolated nucleic acid
molecules of the present invention are also meant to include those chemically
synthesized. With reference to nucleic acids of the invention, the term "isolated nucleic
acid" or "isolated nucleic acid sequence" is sometimes used. This term, when applied
to DNA, refers to a DNA molecule that is separated from sequences with which it is
immediately contiguous in the naturally occurring genome of the organism in which it
originated. For example, an "isolated nucleic acid" may comprise a DNA molecule
inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic
DNA of a prokaryotic or eukaryotic cell or host organism. Specifically, the term
"isolated nucleic acid" according to the invention excludes that the pCS1 sequence is
linked to a nucleic acid encoding the CS1 protein. An "isolated nucleic acid" (either
DNA or RNA) may further represent a molecule produced directly by biological or
synthetic means and separated from other components present during its production.
The term "operably linked" as used herein refers to the association of nucleotide
sequences on a single nucleic acid molecule, e.g. a vector, in a way such that the
function of one or more nucleotide sequences is affected by at least one other
nucleotide sequence present on said nucleic acid molecule. For example, a promoter
is operably linked with a coding sequence of a recombinant gene, when it is capable of
effecting the expression of that coding sequence. As a further example, a nucleic acid
encoding a signal peptide is operably linked to a nucleic acid sequence encoding a
POI, when it is capable of expressing a protein in the secreted form, such as a preform
of a mature protein or the mature protein. Specifically such nucleic acids operably
linked to each other may be immediately linked, i.e. without further elements or nucleic
acid sequences in between the nucleic acid encoding the signal peptide and the
nucleic acid sequence encoding a POI.
The term "promoter" as used herein refers to a DNA sequence capable of
controlling the expression of a coding sequence or functional RNA. Promoter activity
may be assessed by its transcriptional efficiency. This may be determined directly by
measurement of the amount of mRNA transcription from the promoter, e.g. by
Northern Blotting or indirectly by measurement of the amount of gene product
expressed from the promoter.
The promoter of the invention specifically initiates, regulates, or otherwise
mediates or controls the expression of a coding DNA. Promoter DNA and coding DNA
may be from the same gene or from different genes, and may be from the same or
different organisms.
The promoter of the invention is specifically understood as a constitutive
promoter, i.e. a promoter which controls expression without the need for induction, or
the possibility of repression. Therefore, there is continuous and steady expression at a
certain level. Because of the unique function of high promoter strength at all growth
phases or growth rates of the host cell, the constitutive promoter of the invention is
particularly useful in a fed -batch culture of the host cell line. Constitutive promoters of
the prior art had the disadvantage of low strength at growth -limited conditions, e.g. in
fed-batch processes, or were used in embodiments where the host cells were
maintained at a high growth rate, e.g. in the mid-exponential phase.
The strength of the promoter of the invention specifically refers to its
transcription strength, represented by the efficiency of initiation of transcription
occurring at that promoter with high or low frequency. The higher transcription strength
the more frequently transcription will occur at that promoter. Promoter strength is
important, because it determines how often a given mRNA sequence is transcribed,
effectively giving higher priority for transcription to some genes over others, leading to
a higher concentration of the transcript. A gene that codes for a protein that is required
in large quantities, for example, typically has a relatively strong promoter. The RNA
polymerase can only perform one transcription task at a time and so must prioritize its
work to be efficient. Differences in promoter strength are selected to allow for this
prioritization. According to the invention the promoter is relatively strong independent
of the metabolism or the growth rate of the host cell, e.g. both, during in phases of high
and low growth rates of a cell culture and specifically independent of the carbon
source, exhibiting a state of about maximal activity, specifically at about a constant
level.
The relative strength is commonly determined with respect to a standard
promoter, such as the respective pGAP promoter of the cell used as the host cell. The
frequency of transcription is commonly understood as the transcription rate, e.g. as
determined by the amount of a transcript in a suitable assay, e.g. RT-PCR or Northern
blotting. The strength of a promoter to express a gene of interest is commonly
understood as the expression strength or the capability of support a high expression
level/rate. For example, the expression and/or transcription strength of a promoter of
the invention is determined in the host cell which is P. pastoris and compared to the
native pGAP promoter of P. pastoris.
The transcription rate may be determined by the transcription strength on a
microarray, or with quantitative real time PGR (qRT-PCR) where microarray or qRTPCR
data show the difference of expression level between conditions with high growth
rate and conditions with low growth rate, or conditions employing different media
composition, and a high signal intensity as compared to the native pGAP promoter. A
suitable test system is specifically described in Example 11 below.
The promoter of the invention exerts a relatively high transcription strength,
reflected by a transcription rate or transcription strength of at least 110% as compared
to the native pGAP promoter in the host cell, sometimes called "homologous pGAP
promoter". Preferably the transcription rate or strength is at least 110%, preferably at
least 120%, or at least 130%, in specifically preferred cases at least 140%, at least
150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at
least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, or
at least about 15-fold, or even higher as compared to the native pGAP promoter, e.g.
determined in the eukaryotic cell selected as host cell for producing the POI, such as
determined under carbon substrate rich conditions, e.g. batch cultures, or under
carbon substrate limited conditions, e.g. chemostat or fed batch cultivations.
Preferably the transcription analysis is quantitative or semi-quantitative,
preferably employing qRT-PCR, DNA microarrays, RNA sequencing and transcriptome
analysis.
The expression rate may, for example, be determined by the amount of
expression of a reporter gene, such as eGFP, e.g. as described in the Example section
below, a test system is specifically described in Example 10. It could be shown that the
pCS1 promoter has a relatively high transcription rate of at least 1 0% as compared to
the native pGAP promoter, upon cultivating a clone in solution.
The promoter of the invention exerts relatively high expression strength,
reflected by an expression level of gene of interest, which is at least 110% as
compared to the native pGAP promoter in the host cell, sometimes called "homologous
pGAP promoter". Preferably the expression strength is at least 110%, preferably at
least 120%, or at least 130%, in specifically preferred cases at least 140%, at least
150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at
least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, or
at least about 15-fold, or even higher as compared to the native pGAP promoter, e.g.
determined in the eukaryotic cell selected as host cell for producing the POI, such as
determined under carbon substrate rich conditions, e.g. batch cultures, or under
carbon substrate limited conditions, e.g. chemostat or fed batch cultivations.
The native pGAP promoter initiates expression of the gap gene encoding
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a constitutive
promoter present in most living organisms. GAPDH (EC 1\2\1\12), a key enzyme of
glycolysis and gluconeogenesis, plays a crucial role in catabolic and anabolic
carbohydrate metabolism. Therefore, the pGAP promoter, though understood to be a
constitutive (such as non-inducible by feeding with specific carbon sources) promoter,
is a metabolic promoter that exerts increasing promoter strength with increasing
growth rate. Therefore, the pGAP promoter is hardly suitable in an efficient production
process during the cultivation of the host cell line in phases that are characterized by a
low growth rate.
In contrast, the promoter of the invention surprisingly maintains its high
promoter strength (essentially) at a constantly high transcript level at all growth phases
of a host cell culture.
The native pGAP promoter specifically is active in a recombinant eukaryotic cell
in a similar way as in a native eukaryotic cell of the same species or strain, including
the unmodified (non-recombinant) or recombinant eukaryotic cell. Such native pGAP
promoter is commonly understood to be an endogenous promoter, thus, homologous
to the eukaryotic cell, and serves as a standard or reference promoter for comparison
purposes.
For example, a native pGAP promoter of P. pastoris is the unmodified,
endogenous promoter sequence in P. pastoris, as used to control the expression of
GAPDH in P. pastoris, e.g. having the sequence shown in Figure 3: native pGAP
promoter sequence of P. pastoris (GS1 15) (SEQ ID 13). If P. pastoris is used as a host
for producing a POI according to the invention, the transcription strength or rate of the
promoter according to the invention is compared to such native pGAP promoter of P.
pastoris.
As another example, a native pGAP promoter of S. cerevisiae is the unmodified,
endogenous promoter sequence in S. cerevisiae, as used to control the expression of
GAPDH in S. cerevisiae. If S. cerevisiae is used as a host for producing a POI
according to the invention, the transcription strength or rate of the promoter according
to the invention is compared to such native pGAP promoter of S. cerevisiae.
Therefore, the relative expression or transcription strength of a promoter
according to the invention is usually compared to the native pGAP promoter of a cell of
the same species or strain that is used as a host for producing a POI.
It is specifically understood that the promoter of the invention is preferably not a
metabolic promoter such as for example a promoter naturally operably linked to a gene
encoding an abundant glycolytic enzyme or an enzyme of gluconeogenesis, a
ribosomal protein or an enzyme such as an intracellular protease or a protease being
secreted from the host cell.
Specifically preferred is a promoter of the invention, which has at least an
expression strength or transcription strength of pCS1 . The comparative promoter
strength employing the pGAP promoter as a reference may be determined by standard
means, such as by measuring the quantity of expression products or the quantity of
transcripts, e.g. employing a microarray, Northern Blot, RNA sequencing or qRT-PCR,
or else in a cell culture, such as by measuring the quantity of respective gene
expression products in recombinant cells. Exemplary tests are illustrated in the
Examples section.
The term "protein of interest (POI)" as used herein refers to a polypeptide or a
protein that is produced by means of recombinant technology in a host cell. More
specifically, the protein may either be a polypeptide not naturally occurring in the host
cell, i.e. a heterologous protein, or else may be native to the host cell, i.e. a
homologous protein to the host cell, but is produced, for example, by transformation
with a self-replicating vector containing the nucleic acid sequence encoding the POI, or
upon integration by recombinant techniques of one or more copies of the nucleic acid
sequence encoding the POI into the genome of the host cell, or by recombinant
modification of one or more regulatory sequences controlling the expression of the
gene encoding the POI, e.g. of the promoter sequence. In some cases the term POI as
used herein also refers to any metabolite product by the host cell as mediated by the
recombinantly expressed protein.
According to a specific embodiment, the term POI would exclude the CS1
protein of Pichia pastoris, e.g. characterized by any of the amino acid sequences of
Figure 2 , in particular, if the promoter of the invention is natively associated with any of
the nucleic acid sequences encoding such CS1 protein. .
Specifically, the POI as described herein is a eukaryotic protein, preferably a
mammalian protein, specifically a protein heterologous to the host cell.
A POI produced according to the invention may be a multimeric protein,
preferably a dimer or tetramer.
According to one aspect of the invention, the POI is a recombinant or hetero
logous protein, preferably selected from therapeutic proteins, including antibodies or
fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins,
carbohydrate - protein conjugates, structural proteins, regulatory proteins, vaccines
and vaccine like proteins or particles, process enzymes, growth factors, hormones and
cytokines, or a metabolite of a POI.
A specific POI is an antigen binding molecule such as an antibody, or a
fragment thereof. Among specific POIs are antibodies such as monoclonal antibodies
(mAbs), immunoglobulin (Ig) or immunoglobulin class G (IgG), heavy-chain antibodies
(HcAb's), or fragments thereof such as fragment-antigen binding (Fab), Fd, singlechain
variable fragment (scFv), or engineered variants thereof such as for example Fv
dimers (diabodies), Fv trimers (triabodies), Fv tetramers, or minibodies and singledomain
antibodies like VH or VHH or V-NAR.
According to a specific embodiment, a fermentation product is manufactured
using the POI, a metabolite or a derivative thereof.
The POI may specifically be recovered from the cell culture in the purified form,
e.g. substantially pure.
The term "substantially pure" or "purified" as used herein shall refer to a
preparation comprising at least 50% (w/w), preferably at least 60%, 70%, 80%, 90% or
95% of a compound, such as a nucleic acid molecule or a POI. Purity is measured by
methods appropriate for the compound (e.g. chromatographic methods,
polyacrylamide gel electrophoresis, HPLC analysis, and the like).
The term "recombinant" as used herein shall mean "being prepared by or the
result of genetic engineering". Thus, a "recombinant microorganism" comprises at least
one "recombinant nucleic acid". A recombinant microorganism specifically comprises
an expression vector or cloning vector, or it has been genetically engineered to contain
a recombinant nucleic acid sequence. A "recombinant protein" is produced by
expressing a respective recombinant nucleic acid in a host. A "recombinant promoter"
is a genetically engineered non-coding nucleotide sequence suitable for its use as a
functionally active promoter as described herein.
Therefore, a new promoter was identified with unique functional properties.
Unexpectedly, PAS_chr1-4 (herein called CS1 gene, SEQ ID 9) was identified by
analysis of transcription strength in production process conditions as the strongest
transcribed gene in P. pastoris. The encoded CS1 protein (SEQ D 10) is not a
glycolytic enzyme or protein, but predicted to be located on the cell surface via a GPIanchor.
Though the 9.43 Mbp genomic sequence of the GS1 15 strain of P. pastoris
has been determined and disclosed in US201 10021378A1 , the properties of individual
sequences, such as promoter sequences, have not been investigated in detail.
It was surprising that such promoter could be effectively used according to the
invention. Pichia promoters of the prior art such as used in industrial scale POI
production were mainly derived from the methanol metabolic pathway and needed the
addition of methanol to induce POI production, which is often not desired. The
promoter and method according to the invention has the advantage that it may provide
for an increased production by an enhanced expression, and has the reduced risk of
contamination due to the specific promoter regulation, in particular when using a
chemically defined medium, free of methanol.
It turned out that the promoter according to the invention would exert its
improved activity mainly independent of a suitable carbon substrate amount and
specific culture media. As an example, P. pastoris could be successfully cultivated
under conditions of an industrial production process. First a batch culture on a basal
carbon source, such as glycerol, was employed, followed by a fed batch with limited
feed of a supplemental carbon source, such as glucose. Samples were taken close to
the end of the first batch phase, and in limited growth conditions, e.g. using a limited
amount of supplemental carbon source. Transcriptome analysis with DNA microarrays
revealed specific genes that are strongly active on the supplemental carbon source
and in the presence of surplus carbon, i.e. the basal carbon source in excess amount.
The pCS1 promoter sequence was identified as surprisingly strong promoter at high
and low growth rates. The comparable pGAP promoter of the prior art was significantly
weaker.
The features of strong recombinant gene expression on the basal carbon
source, and strong expression on limited supplemental carbon source, could be
verified in fermentation processes.
The nucleotide sequences that could be used as constitutive promoter
sequences according to the invention, would provide for an improved recombinant
protein production, can be obtained from a variety of sources. The origin of the
promoter according to the invention is preferably from a yeast cell, most preferably
from methylotrophic yeast such as from the Pichia genus or from the P. pastoris
species, which promoter may then be used as a parent sequence to produce suitable
variants, e.g. mutants or analogs.
It is contemplated that a series of yeast cells, in particular of Pichia strains, may
be suitable to obtain respective promoter sequences or respective analogs in different
species.
Variants of the identified P. pastoris promoter, including functionally active
variants, such as homologs and analogs may be produced employing standard
techniques. The promoter may e.g. be modified to generate promoter variants with
altered expression levels and regulatory properties.
For instance, a promoter library may be prepared by mutagenesis of the
promoter sequences according to the invention, which may be used as parent
molecules, e.g. to fine-tune the gene expression in eukaryotic cells by analysing
variants for their expression under different fermentation strategies and selecting
suitable variants. A synthetic library of variants may be used, e.g. to select a promoter
matching the requirements for producing a selected POL Such variants may have
increased expression efficiency in eukaryotic host cells and high expression under
carbon source rich and limiting conditions.
The differential fermentation strategies would distinguish between a growth
phase and a production phase. Growth and/or production can suitably take place in
batch mode, fed-batch mode or continuous mode. Any suitable bioreactor can be
used, including batch, fed-batch, continuous, stirred tank reactor, or airlift reactor.
It is advantageous to provide for the fermentation process on a pilot or industrial
scale. The industrial process scale would preferably employ volumina of at least 10 L,
specifically at least 50 L, preferably at least 1 m3, preferably at least 10 m3, most
preferably at least 100 m3.
Production conditions in industrial scale are preferred, which refer to e.g. fed
batch cultivation in reactor volumes of 100 L to 10 m3 or larger, employing typical
process times of several days, or continuous processes in fermenter volumes of
approximately 50 - 1000 L or larger, with dilution rates of approximately 0.02 -
0.15 h .
The suitable cultivation techniques may encompass cultivation in a bioreactor
starting with a batch phase, followed by a short exponential fed batch phase at high
specific growth rate, further followed by a fed batch phase at a low specific growth rate.
Another suitable cultivation technique may encompass a batch phase followed by a
continuous cultivation phase at a low dilution rate.
A preferred embodiment of the invention includes a batch culture to provide
biomass followed by a fed -batch culture for high yields POI production.
It is preferred to cultivate the host cell line according to the invention in a
bioreactor under growth conditions to obtain a cell density of at least 1 g/L cell dry
weight, more preferably at least 10 g/L cell dry weight, preferably at least 20 g/L cell
dry weight. It is advantageous to provide for such yields of biomass production on a
pilot or industrial scale.
A growth medium allowing the accumulation of biomass, specifically a basal
growth medium, typically comprises a carbon source, a nitrogen source, a source for
sulphur and a source for phosphate. Typically, such a medium comprises furthermore
trace elements and vitamins, and may further comprise amino acids, peptone or yeast
extract.
Preferred nitrogen sources include NH4H2PO4, or NH3 or (NH4) S0 4
Preferred sulphur sources include MgS0 4, or(NH4) 2S0 4 or K2S0 4 ;
Preferred phosphate sources include NH4H2P0 , or H3PO4 or NaH P0 ,
KH2PO4, Na2HP0 or K2HP0 4
Further typical medium components include KCI, CaCI2, and Trace elements
such as: Fe, Co, Cu, Ni, Zn, Mo, Mn, I , B;
Preferably the medium is supplemented with vitamin B7;
A typical growth medium for P. pastoris comprises glycerol, sorbitol or glucose,
NH4H2PO4, MgS0 4, KCI, CaCI2, biotin, and trace elements.
ln the production phase a production medium is specifically used with only a
limited amount of a supplemental carbon source.
Preferably the host cell line is cultivated in a mineral medium with a suitable
carbon source, thereby further simplifying the isolation process significantly. An
example of a preferred mineral medium is one containing an utilizabie carbon source
(e.g. glucose, glycerol, sorbitol or methanol), salts containing the macro elements
(potassium, magnesium, calcium, ammonium, chloride, sulphate, phosphate) and trace
elements (copper, iodide, manganese, molybdate, cobalt, zinc, and iron salts, and
boric acid), and optionally vitamins or amino acids, e.g. to complement auxotrophies.
The cells are cultivated under conditions suitable to effect expression of the
desired POI, which can be purified from the cells or culture medium, depending on the
nature of the expression system and the expressed protein, e.g. whether the protein is
fused to a signal peptide and whether the protein is soluble or membrane-bound. As
will be understood by the skilled artisan, cultivation conditions will vary according to
factors that include the type of host cell and particular expression vector employed.
By selecting the suitable promoter sequence according to the invention,
optionally in combination with further preferred regulatory sequences, it is possible to
provide for, under comparable conditions, at least the same, or at least about an 1.1-
fold, or at least about 1.2-fold, at least about 1.5-fold, at least about 2-fold, at least
about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, or at
least up to about a 15-fold activity, e.g. under growth-limited conditions in a fed-batch
process, as represented by the promoter activity or transcription strength, or regulated
by the promoter strength relative to a GAP promoter that is homologous to the
production cell, a native pGAP, or isolated from P. pastoris.
A typical production medium comprises a supplemental carbon source, and
further NH H2P0 4, MgS0 4, KCI, CaCI2, biotin, and trace elements.
For example the feed of the supplemental carbon source added to the fermen
tation may comprise a carbon source with up to 50 wt % utilizabie sugars. The low
feed rate of the supplemental medium will limit the effects of product or byproduct
inhibition on the cell growth, thus a high product yield based on substrate provision will
be possible.
The fermentation preferably is carried out at a pH ranging from 3 to 7.5.
Typical fermentation times are about 24 to 120 hours with temperatures in the
range of 20 °C to 35°C, preferably 22-30°C.
ln general, the recombinant nucleic acids or organisms as referred to herein
may be produced by recombination techniques well known to a person skilled in the
art. In accordance with the present invention there may be employed conventional
molecular biology, microbiology, and recombinant DNA techniques within the skill of
the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch
& Sambrook, "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982).
According to a preferred embodiment of the present invention, a recombinant
construct is obtained by ligating the promoter and relevant genes into a vector or
expression construct. These genes can be stably integrated into the host cell genome
by transforming the host cell using such vectors or expression constructs.
Expression vectors may include but are not limited to cloning vectors, modified
cloning vectors and specifically designed plasmids. The preferred expression vector as
used in the invention may be any expression vector suitable for expression of a
recombinant gene in a host cell and is selected depending on the host organism. The
recombinant expression vector may be any vector which is capable of replicating in or
integrating into the genome of the host organisms, also called host vector.
Appropriate expression vectors typically comprise further regulatory sequences
suitable for expressing DNA encoding a POI in a eukaryotic host cell. Examples of
regulatory sequences include operators, enhancers, ribosomal binding sites, and
sequences that control transcription and translation initiation and termination. The
regulatory sequences may be operably linked to the DNA sequence to be expressed.
To allow expression of a recombinant nucleotide sequence in a host cell, the
expression vector may provide the promoter according to the invention adjacent to the
5' end of the coding sequence, e.g. upstream from the GOI or a signal peptide gene
enabling secretion of the POI. The transcription is thereby regulated and initiated by
this promoter sequence.
A signal peptide may be a heterologous signal peptide or a hybrid of a native
and a heterologous signal peptide, and may specifically be heterologous or homo
logous to the host organism producing the protein. The function of the signal peptide is
to allow the POI to be secreted to enter the endoplasmatic reticulum. It is usually a
short (3-60 amino acids long) peptide chain that directs the transport of a protein
outside the plasma membrane, thereby making it easy to separate and purify the
heterologous protein. Some signal peptides are cleaved from the protein by signal
peptidase after the proteins are transported.
Exemplary signal peptides are signal sequences from S. cerevisiae alphamating
factor prepro peptide and the signal peptide from the P. pastoris acid
phosphatase gene (PH01 ) .
A promoter sequence is understood to be operably linked to a coding sequence,
if the promoter controls the transcription of the coding sequence. If a promoter
sequence is not natively associated with the coding sequence, its transcription is either
not controlled by the promoter in native (wild-type) cells or the sequences are
recombined with different contiguous sequences.
To prove the function of the relevant sequences, expression vectors comprising
one or more of the regulatory elements may be constructed to drive expression of a
POI, and the expressed yield is compared to constructs with conventional regulatory
elements. A detailed description of the experimental procedure can be found in the
examples below. The identified genes may be amplified by PGR from P. pastoris using
specific nucleotide primers, cloned into an expression vector and transformed into a
eukaryotic cell line, e.g. using a yeast vector and a strain of P. pastoris, for high level
production of various different POI. To estimate the effect of the promoter according to
the invention on the amount of recombinant POI so produced, the eukaryotic cell line
may be cultured in shake flask experiments and fedbatch or chemostat fermentations
in comparison with strains comprising a conventional constitutive, e.g. a growthdependent
promoter, such as for example the standard pGAP promoter in the
respective cell. In particular, the choice of the promoter has a great impact on the
recombinant protein production.
The POI can be produced using the recombinant host cell line by culturing a
transformant, thus obtained in an appropriate medium, isolating the expressed product
or metabolite from the culture, and optionally purifying it by a suitable method.
Transformants according to the present invention can be obtained by intro
ducing such a vector DNA, e.g. plasmid DNA, into a host and selecting transformants
which express the POI or the host cell metabolite with high yields. Host cells are
treated to enable them to incorporate foreign DNA by methods conventionally used for
transformation of eukaryotic cells, such as the electric pulse method, the protoplast
method, the lithium acetate method, and modified methods thereof. P. pastoris is
preferably transformed by electroporation. Preferred methods of transformation for the
uptake of the recombinant DNA fragment by the microorganism include chemical
transformation, electroporation or transformation by protoplastation. Transformants
according to the present invention can be obtained by introducing such a vector DNA,
e.g. plasmid DNA, into a host and selecting transform ants which express the relevant
protein or host cell metabolite with high yields.
Several different approaches for the production of the POI according to the
method of the invention are preferred. Substances may be expressed, processed and
optionally secreted by transforming a eukaryotic host cell with an expression vector
harbouring recombinant DNA encoding a relevant protein and at least one of the
regulatory elements as described above, preparing a culture of the transformed cell,
growing the culture, inducing transcription and POI production, and recovering the
product of the fermentation process.
The host cell according to the invention is preferably tested for its expression
capacity or yield by the following test: ELISA, activity assay, HPLC, or other suitable
tests.
The POI is preferably expressed employing conditions to produce yields of at
least 1 mg/L, preferably at least 10 mg/L, preferably at least 100 mg/L, most preferred
at least 1 g/L.
It is understood that the methods disclosed herein may further include culti
vating said recombinant host cells under conditions permitting the expression of the
POI, preferably in the secreted form or else as intracellular product. A recombinantly
produced POI or a host cell metabolite can then be isolated from the cell culture
medium and further purified by techniques well known to a person skilled in the art.
The POI produced according to the invention typically can be isolated and
purified using state of the art techniques, including the increase of the concentration of
the desired POI and/or the decrease of the concentration of at least one impurity.
If the POI is secreted from the cells, it can be isolated and purified from the
culture medium using state of the art techniques. Secretion of the recombinant
expression products from the host cells is generally advantageous for reasons that
include facilitating the purification process, since the products are recovered from the
culture supernatant rather than from the complex mixture of proteins that results when
yeast cells are disrupted to release intracellular proteins.
The cultured transformant cells may also be ruptured sonically or mechanically,
enzymatically or chemically to obtain a cell extract containing the desired POI, from
which the POI is isolated and purified.
As isolation and purification methods for obtaining a recombinant polypeptide or
protein product, methods, such as methods utilizing difference in solubility, such as
salting out and solvent precipitation, methods utilizing difference in molecular weight,
such as ultrafiltration and gel electrophoresis, methods utilizing difference in electric
charge, such as ion-exchange chromatography, methods utilizing specific affinity, such
as affinity chromatography, methods utilizing difference in hydrophobicity, such as
reverse phase high performance liquid chromatography, and methods utilizing
difference in isoelectric point, such as isoelectric focusing may be used.
The highly purified product is essentially free from contaminating proteins, and
preferably has a purity of at least 90%, more preferred at least 95%, or even at least
98%, up to 100%. The purified products may be obtained by purification of the cell
culture supernatant or else from cellular debris.
As isolation and purification methods the following standard methods are
preferred: Cell disruption (if the POI is obtained intracellularly), cell (debris) separation
and wash by Microfiltration or Tangential Flow Filter (TFF) or centrifugation, POI
purification by precipitation or heat treatment, POI activation by enzymatic digest, POI
purification by chromatography, such as ion exchange ( EX), hydrophobic ointeraction
chromatography (HIC), Affinity chromatography, size exclusion (SEC) or HPLC
Chromatography, POI precipitation of concentration and washing by ultrafiltration
steps.
The isolated and purified POI can be identified by conventional methods such
as Western blot, HPLC, activity assay, or ELISA.
The POI can be any eukaryotic, prokaryotic or synthetic polypeptide. It can be a
secreted protein or an intracellular protein. The present invention also provides for the
recombinant production of functional homologs, functional equivalent variants,
derivatives and biologically active fragments of naturally occurring proteins. Functional
homologs are preferably identical with or correspond to and have the functional
characteristics of a sequence.
A POI referred to herein may be a product homologous to the eukaryotic host
cell or heterologous, preferably for therapeutic, prophylactic, diagnostic, analytic or
industrial use.
The POI is preferably a heterologous recombinant polypeptide or protein,
produced in a eukaryotic cell, preferably a yeast cell, preferably as secreted proteins.
Examples of preferably produced proteins are immunoglobulins, immunoglobulin
fragments, aprotinin, tissue factor pathway inhibitor or other protease inhibitors, and
insulin or insulin precursors, insulin analogues, growth hormones, interleukins, tissue
plasminogen activator, transforming growth factor a or b, glucagon, glucagon-like
peptide 1 (GLP-1 ) , glucagon-like peptide 2 (GLP-2), GRPP, Factor VII, Factor VIII,
Factor XIII, platelet-derived growth factorl , serum albumin, enzymes, such as lipases
or proteases, or a functional homolog, functional equivalent variant, derivative and
biologically active fragment with a similar function as the native protein. The POI may
be structurally similar to the native protein and may be derived from the native protein
by addition of one or more amino acids to either or both the C- and N-terminal end or
the side-chain of the native protein, substitution of one or more amino acids at one or a
number of different sites in the native amino acid sequence, deletion of one or more
amino acids at either or both ends of the native protein or at one or several sites in the
amino acid sequence, or insertion of one or more amino acids at one or more sites in
the native amino acid sequence. Such modifications are well known for several of the
proteins mentioned above.
A POI can also be selected from substrates, enzymes, inhibitors or cofactors
that provide for biochemical reactions in the host cell, with the aim to obtain the
product of said biochemical reaction or a cascade of several reactions, e.g. to obtain a
metabolite of the host cell. Examplary products can be vitamins, such as riboflavin,
organic acids, and alcohols, which can be obtained with increased yields following the
expression of a recombinant protein or a POI according to the invention.
In general, the host cell, which expresses a recombinant product, can be any
eukaryotic cell suitable for recombinant expression of a POI.
Examples of preferred mammalian cells are BHK, CHO (CHO-DG44, CHODUXB1
1, CHO-DUKX, CHO-K1 , CHOK1SV, CHO-S), HeLa, HEK293, MDCK,
NIH3T3, NSO, PER.C6, SP2/0 and VERO cells.
Examples of preferred yeast cells used as host cells according to the invention
include but are not limited to the Saccharomyces genus (e.g. Saccharomyces
cerevisiae), the Pichia genus (e.g. P. pastoris, or P. methanolica), the Komagataella
genus (K. pastoris, K pseudopastoris or K. phaffii), Hansenula polymorpha or
Kiuyveromyces lactis.
Newer literature divides and renames Pichia pastoris into Komagataella
pastoris, Komagataella phaffii and Komagataella pseudopastoris. Herein Pichia
pastoris is used synonymously for all, Komagataella pastoris, Komagataella phaffii and
Komagataella pseudopastoris.
The preferred yeast host cells are derived from methylotrophic yeast, such as
from Pichia or Komagataella, e.g. Pichia pastoris, or Komagataella pastoris, or K.
phaffii, or K pseudopastoris. Examples of the host include yeasts such as P. pastoris.
Examples of P. pastoris strains include CBS 704 (=NRRL Y-1603 = DSMZ 70382),
CBS 2612 (=NRRL Y-7556), CBS 7435 (=NRRL Y-1 1430), CBS 9173-9189 (CBS
strains: CBS-KNAW Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands), and DSMZ 70877 (German Collection of Microorganisms
and Cell Cultures), but also strains from Invitrogen, such as X-33, GS1 15,
KM71 and SMD1 168. Examples of S. cerevisiae strains include W303, CEN.PK and
the BY-series (EUROSCARF collection). All of the strains described above have been
successfully used to produce transformants and express heterologous genes.
A preferred yeast host cell according to the invention, such as a P. pastoris or S.
cerevisiae host cell, contains a heterologous or recombinant promoter sequences,
which may be derived from a P. pastoris or S. cerevisiae strain, different from the pro
duction host. In another specific embodiment the host cell according to the invention
comprises a recombinant expression construct according to the invention comprising
the promoter originating from the same genus, species or strain as the host cell.
The promoter of the invention is preferably derived from a gene encoding a
protein homologous to the host cell.
For example, a promoter according to the invention may be derived from yeast,
such as a S. cerevisiae strain, and be used to express a POI in a yeast. A specifically
preferred embodiment relates to a promoter according to the invention originating from
P. pastoris for use in a method to produce a recombinant POI in a P. pastoris producer
host cell line. The homologous origin of the nucleotide sequence facilitates its
incorporation into the host cell of the same genus or species, thus enabling stable
production of a POI, possibly with increased yields in industrial manufacturing
processes. Also, functionally active variants of the promoter from other suitable yeasts
or other fungi or from other organisms such as vertebrates or plants can be used.
If the POI is a protein homologous to the host cell, i.e. a protein which is
naturally occurring in the host cell, the expression of the POI in the host cell may be
modulated by the exchange of its native promoter sequence with a promoter sequence
according to the invention.
This purpose may be achieved e.g. by transformation of a host cell with a
recombinant DNA molecule comprising homologous sequences of the target gene to
allow site specific recombination, the promoter sequence and a selective marker
suitable for the host cell. The site specific recombination shall take place in order to
operably link the promoter sequence with the nucleotide sequence encoding the POI.
This results in the expression of the POI from the promoter sequence according to the
invention instead of from the native promoter sequence.
In a specifically preferred embodiment of the invention the promoter sequence
has an increased promoter activity relative to the native promoter sequence of the POI.
According to the invention it is preferred to provide a P. pastoris host cell line
comprising a promoter sequence according to the invention operably linked to the
nucleotide sequence coding for the POI.
According to the invention it is also possible to provide a wildcard vector or host
cell according to the invention, which comprises a promoter according to the invention,
and which is ready to incorporate a gene of interest encoding a POL The wildcard cell
line is, thus, a preformed host cell line, which is characterized for its expression
capacity. This follows an innovative "wildcard" platform strategy for the generation of
producer cell lines, for the POI production, e.g. using site-specific recombinasemediated
cassette exchange. Such a new host cell facilitates the cloning of a gene of
interest (GOI), e.g. into predetermined genomic expression hot spots within days in
order to get reproducible, highly efficient production cell lines.
According to a preferred embodiment the method according to the invention
employs a recombinant nucleotide sequence encoding the POI, which is provided on a
plasmid suitable for integration into the genome of the host cell, in a single copy or in
multiple copies per cell. The recombinant nucleotide sequence encoding the POI may
also be provided on an autonomously replicating plasmid in a single copy or in multiple
copies per cell.
The preferred method according to the invention employs a plasmid, which is a
eukaryotic expression vector, preferably a yeast expression vector. Expression vectors
may include but are not limited to cloning vectors, modified cloning vectors and
specifically designed plasmids. The preferred expression vector as used in the
invention may be any expression vector suitable for expression of a recombinant gene
in a host cell and is selected depending on the host organism. The recombinant
expression vector may be any vector which is capable of replicating in or integrating
into the genome of the host organisms, also called host vector, such as a yeast vector,
which carries a DNA construct according to the invention. A preferred yeast expression
vector is for expression in yeast selected from the group consisting of methylotrophic
yeasts represented by the genera Hansenula, Pichia, Candida and Torulopsis.
In the present invention, it is preferred to use plasmids derived from pPICZ,
pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis or pPUZZLE as the vector.
According to a preferred embodiment of the present invention, a recombinant
construct is obtained by ligating the relevant genes into a vector. These genes can be
stably integrated into the host cell genome by transforming the host cell using such
vectors. The polypeptides encoded by the genes can be produced using the recom
binant host cell line by culturing a transformant, thus obtained in an appropriate
medium, isolating the expressed POI from the culture, and purifying it by a method
appropriate for the expressed product, in particular to separate the POI from
contaminating proteins.
Expression vectors may comprise one or more phenotypic selectable markers,
e.g. a gene encoding a protein that confers antibiotic resistance or that supplies an
autotrophic requirement. Yeast vectors commonly contain an origin of replication from
a yeast plasmid, an autonomously replicating sequence (ARS), or alternatively, a
sequence used for integration into the host genome, a promoter region, sequences for
polyadenylation, sequences for transcription termination, and a selectable marker.
The procedures used to ligate the DNA sequences, e.g. coding for the
precursing sequence and/or the POI, the promoter and the terminator, respectively,
and to insert them into suitable vectors containing the information necessary for
integration or host replication, are well known to persons skilled in the art, e.g.
described by J. Sambrook et al., (A Laboratory Manual, Cold Spring Harbor, 1989).
It will be understood that the vector, which uses the regulatory elements
according to the invention and/or the POI as an integration target, may be constructed
either by first preparing a DNA construct containing the entire DNA sequence coding
for the regulatory elements and/or the POI and subsequently inserting this fragment
into a suitable expression vector, or by sequentially inserting DNA fragments
containing genetic information for the individual elements, followed by ligation.
Also multicloning vectors, which are vectors having a multicloning site, can be
used according to the invention, wherein a desired heterologous gene can be
incorporated at a multicloning site to provide an expression vector. In expression
vectors, the promoter is placed upstream of the gene of the POI and regulates the
expression of the gene. In the case of multicloning vectors, because the gene of the
POI is introduced at the multicloning site, the promoter is placed upstream of the
multicloning site.
The DNA construct as provided to obtain a recombinant host cell according to
the invention may be prepared synthetically by established standard methods, e.g. the
phosphoramidite method. The DNA construct may also be of genomic or cDNA origin,
for instance obtained by preparing a genomic or cDNA library and screening for DNA
sequences coding for all or part of the polypeptide of the invention by hybridization
using synthetic oligonucleotide probes in accordance with standard techniques
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989).
Finally, the DNA construct may be of mixed synthetic and genomic, mixed synthetic
and cDNA or mixed genomic and cDNA origin prepared by annealing fragments of
synthetic, genomic or cDNA origin, as appropriate, the fragments corresponding to
various parts of the entire DNA construct, in accordance with standard techniques.
In another preferred embodiment, the yeast expression vector is able to stably
integrate in the yeast genome, e. g. by homologous recombination.
A transformant host cell according to the invention obtained by transforming the
cell with the regulatory elements according to the invention and/or the POI genes may
preferably first be cultivated at conditions to grow efficiently to a large cell number.
When the cell line is prepared for the POI expression, cultivation techniques are
chosen to produce the expression product.
Specific examples relate to fed-batch fermentation of a recombinant production
P. pastoris cell line producing reporter proteins, employing a glycerol batch medium
and a glucose fed batch medium. Comparative promoter activity studies have proven
that the promoter according to the invention may be successfully used for recombinant
protein production.
According to a further example, human serum albumin (HSA) was produced as
a POI under the glucose-limit conditions, and the HSA yield and gene copy number
determined.
According to another example, fed-batch cultivation of P. pastoris strains
expressing HSA under the control of a promoter according to the invention was
performed.
Further examples refer to expressing a porcine carboxypeptidase B as mode!
protein under transcriptional control of the pCS1 promoter.
Yet, a further example refers to the expression of an antibody fragment under
the transcriptional control of pCS .
A further example refers to size or length variants of a promoter according to the
invention, such as the elongated pCS1 sequence pCS1a (SEQ ID 2) which comprises
the pCS1 sequence and an elongation at the 5' end, or fragments of pCS1 with a
length in the range of about 80bp to 800bp.
The foregoing description will be more fully understood with reference to the
following examples. Such examples are, however, merely representative of methods of
practicing one or more embodiments of the present invention and should not be read
as limiting the scope of invention.
EXAMPLES
Examples below illustrate the materials and methods used to identify a new
promoter and to analyze its expression properties in Pichia pastoris.
Example 1: Identification of a strongly expressed gene in P. pastoris
In order to identify a strong gene and its respective promoter of P. pastoris,
analysis of gene expression patterns was done using DNA microarrays. P. pastoris
cells grown in a glycerol batch and in glucose limit (chemostat) were analyzed.
a) Strain
A wild type P. pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands), which can
grow on minimal media without supplements, was used.
b) Cultivation of P. pastoris
Fermentations were performed with Minifors reactors (Infors-HT, Switzerland)
with a final working volume of 2.5 L.
Following media were used:
PTMi trace salts stock solution contained per liter
6.0 g CuS0 . 5H20 , 0.08 g Nal, 3.36 g MnS0 . H20 , 0.2 g Na2Mo0 4. 2H20 , 0.02
g H3BO3, 0.82 g CoCI , 20.0 g ZnCI2, 65.0 g FeS0 . 7H20 , 0.2 g biotin and 5.0 ml
H2S0 4 (95 %-98 %).
Glycerol Batch medium contained per liter
2 g Citric acid monohydrate 39.2 g Glycerol, 20.8 g NH4H2P0 4,
0.5 g MgS0 7H20 , 1.6 g KC , 0.022 g CaCI2'2H 20 , 0.8 g biotin and 4.6 ml PTM1
trace salts stock solution. HCI was added to set the pH to 5 .
Glycerol fed-batch medium contained per liter
632 g glycerol, 8 g MgS0 '7H 20 , 22 g KCI, and 0.058 g CaCI2'2H 20 .
Chemostat medium contained per liter
2 g Citric acid monohydrate (C6H807'H 20), 99.42 g glucose monohydrate, 22 g
NH4H2P0 , 1.3 g MgS0 7H20 , 3.4 g KCI, 0.02 g CaCI2 2H20 , 0.4 mg biotin and 3.2
ml PTM1 trace salts stock solution. HCI was added to set the pH to 5 .
The dissolved oxygen was controlled at DO = 20 % with the stirrer speed (500 -
1250 rpm). Aeration rate was 60 L h air, the temperature was controlled at 25°C and
the pH setpoint of 5 was controlled with addition of NH OH (25 %).
To start the fermentation, 1.5 L batch medium was sterile filtered into the
fermenter and P. pastoris was inoculated (from an overnight pre-culture in YPG, 180
rpm, 28°C) with a starting optical density (OD600) of 1. The batch phase of
approximately 25 h reached a dry biomass concentration of approximately 20 g/L, it
was followed by a 10 h exponential fed batch with glucose medium, leading to a dry
biomass concentration of approximately 50 g/L. Then, the volume was reduced to 1.5
L and the chemostat cultivation was started with a feed/harvest rate of 0.15 L h ,
resulting in a constant growth rate of m= 0.1 . The fermentation was terminated 50 h
after the chemostat start.
This fermentation has been performed three times to obtain the biological
replicates necessary for reliable microarray analysis.
Carbon limited conditions (no detectable residual glucose) during the chemostat
were verified by HPLC analysis of the culture supernatant
c) Sampling
Samples were taken at the end of the glycerol batch phase and in steady state
conditions of the glucose chemostat. Routine sampling as determination of optical
density or yeast dry mass, qualitative microscopic inspection and cell viability analysis
was done alongside during each fermentation. For microarray analysis, samples were
taken and treated as follows: For optimal quenching, 9 ml_ cell culture broth was
immediately mixed with 4.5 ml_ of ice cold 5% phenol (Sigma) solution (in Ethanol
abs.), and aliquoted. Each 2 ml_ were centrifuged (13200 rpm for 1 minute) in precooled
collection tubes (GE healthcare, NJ), supernatant was removed completely and
the tubes were stored at -80°C until RNA purification.
d) RNA purification and sample preparation for microarray hybridization
The RNA was isolated using TRI reagent according to the suppliers instructions
(Ambion, US). The cell pellets were resuspended in TRI reagent and homogenized
with glass beads using a FastPrep 24 (MP. Biomedicals, CA) at 5 m s for 40
seconds. After addition of chloroform, the samples were centrifuged and the total RNA
was precipitated from the aqueous phase by adding isopropanol. The pellet was
washed with 70% ethanol, dried and re-suspended in RNAse free water. RNA concentrations
were determined by measuring OD260 using a Nanodrop 1000 spectrophoto
meter (NanoDrop products, DE). Remaining DNA from the samples was removed
using the DNA free Kit (Ambion, CA). Sample volume equal to 10 g RNA was diluted
to 50 m I_ in RNAse free water, then DNAse buffer I and rDNAse I were added and
incubated at 37°C for 30 minutes. After addition of DNAse Inactivation Reagent, the
sample was centrifuged and the supernatant was transferred into a fresh tube. RNA
concentrations were determined again as described above. Additionally, RNA integrity
was analyzed using RNA nano chips (Agilent). To monitor the microarray workflow
from amplification and labelling to hybridisation of the samples, the Spike In Kit
(Agilent, Product Nr.: 5188-5279) was used as positive control. It contains 10 different
polyadenylated transcripts from an adenovirus, which are amplified, labelled and
cohybridised together with the own RNA samples. The samples were labelled with Cy
3 and Cy 5 using the Quick Amp Labelling Kit (Agilent, Prod. Nr.: 5190-0444). There
fore 500 ng of purified sample RNA were diluted in 8.3 m I_ RNAse free water, 2 m I_
Spike A or B, and 1.2 m I_ T7 promoter primer were added. The mixture was denatured
for 10 minutes at 65°C and kept on ice for 5 minutes. Then 8.5 m I_ cDNA mastermix
(per sample: 4 m I_ 5x first strand buffer, 2 m I_ 0.1 M DTT, 1 m I_ 10 m dNTP mix, 1 m I_
MMLV-RT, 0.5 m I_ RNAse out) were added, incubated at 40°C for 2 hours, then trans
ferred to 65°C for 15 minutes and put on ice for 5 minutes. The transcription mastermix
(per sample: 15.3 m _ nuclease free water, 20 m I_ transcription buffer, 6 m I_ 0.1 M DTT,
6.4 m I_ 50% PEG, 0.5 m I_ RNAse Inhibitor, 0.6 m I_ inorg. phosphatase, 0.8 m I_ T7 RNA
Polymerase, 2.4 m I_ Cyanin 3 or Cyanin 5) was prepared and added to each tube and
incubated at 40°C for 2 hours. In order to purify the obtained labelled cRNA, the
RNeasy Mini Kit (Qiagen, Cat. No. 74104) was used. Samples were stored at -80°C.
Quantification of the cRNA concentration and labelling efficiency was done at the
Nanodrop spectrophotometer.
e) Microarray analysis
The Gene Expression Hybridisation Kit (Agilent, Cat. No. 5188-5242) was used
for hybridisation of the labelled sample cRNAs. For the preparation of the hybridisation
samples each 300 ng cRNA (Cy3 and Cy 5) and 6 m I_ 10-fold blocking agent were
diluted with nuclease free water to a final volume of 24 m I_. After addition of 1 m I_ 25-
fold fragmentation buffer, the mixture was incubated at 60°C for 30 minutes. Then 25
m I_ GEx Hybridisation Buffer HI-RPM was added to stop the reaction. After centrifugation
for one minute with 13,200 rpm, the sample was chilled on ice and used for
hybridisation immediately. In-house designed P. pastoris specific oligonucleotide
arrays (AMAD-ID: 026594, 8x1 5K custom arrays, Agilent) were used. Microarray
hybridisation was done according to the Microarray Hybridisation Chamber User Guide
(Agilent G2534A). First, the gasket slide was uncovered and put onto the chamber
base, Agilent label facing up. The sample (40 m I_ per array) was loaded in the middle of
each of the eight squares. Then the microarray slide was carefully put onto the gasket
slide (Agilent label facing down) and the chamber cover was placed on and fixed with
the clamp. Hybridisation was done in the hybridisation oven for 17 hours at 65°C.
Before scanning, the microarray chip was washed. Therefore, the chamber was
dismantled, and the sandwich slides were detached from each other while submerged
in wash buffer 1. The microarray was directly transferred into another dish with wash
buffer 1, washed for 1 minute, transferred into wash buffer 2 (temperature at least
30°C) and washed for another minute. After drying of the microarray slide by touching
the slide edge with a tissue, it was put into the slide holder (Agilent label facing up).
The slide holder was put into the carousel and scanning was started.
f ) Data acquisition and statistical evaluation of microarray data
Images were scanned at a resolution of 50 nm with a G2565AA Microarray
scanner (Agilent) and were imported into the Agilent Feature Extraction 9.5 software.
Agilent Feature Extraction 9.5 was used for the quantification of the spot intensities.
The raw mean spot intensity data was then imported into the open source software R
for further normalisation and data analysis.
For data preprocessing and normalization the R packages limma, vsn and
marray were used. The intensity data was not background corrected and was
normalized with VSN.
The microarray data was browsed for entries with high signal intensity in both
states in order to identify strongly expressed constitutive genes. The most strongly
transcribed gene is shown in Table 1, with the signal intensity in both states. The data
of pGAP and pTEF are added as references. In average, pCS1 exceeded pGAP in
both conditions (glycerol batch and glucose-limited chemostat cultivation) by roughly
30%, while pTEF is about 2% weaker than pGAP.
% of pGAP % of pGAP
Promoter gene identifier Intensity1 intensity/ Intensity2 intensity/
transcription transcription
strength strength
pGAP PAS_chr2-1_0437 56235.2 100.0 4441 1.4 100.0
pTEF PAS_FragB_0052 47046.3 83.4 39956.2 89.9
pCS1 PAS_chr1-4_0586 83570.4 148.6 54723.2 122.7
Table 1: Microarray data of the genes, which promoters were selected for
further characterization and of pGAP and pTEF as controls
in glycerol batch phase (average of both channels)
2 in glucose-limited chemostat (average of both channels)
Example 2 : Comparative promoter activity studies of the newly identified
promoter pCS1 in P. pastoris using eGFP as intracellular^ expressed reporter gene
In order to analyze the properties of the newly identified promoter, shake flask
screenings were performed as follows: Pre-culture for 24 hours was done with rich
medium containing glycerol as carbon source - simulating the batch phase of the
process, which was followed by the main culture with minimal medium and glucose
feed beads - simulating the glucose-limited fed batch phase of the process. Green
fluorescence protein (eGFP) was used as intracellular reporter protein for promoter
activity.
a) Strain & expression vector
The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used
as host strain. Transformation of the strain was carried out with an in-house vector
named pPUZZLE (Stadlmayr et al. J . Biotechnol 2010;150(4):519-29), comprising of
an origin of replication for E. coli (pUC19), an antibiotic resistance cassette (Sh ble
gene conferring resistance to Zeocin) for selection in E. coli and yeast, an expression
cassette for the gene of interest (GO!) consisting of a multiple cloning site and the S.
cerevisiae CYC transcription terminator, and a locus for integration into the P.
pastoris genome (3 OC1 region).
b) Amplification and cloning of the newly identified promoter pCS1 into pPUZZLE
expression vector containing eGFP as GOI
The pCS1 promoter (SEQ ID) comprises 985bp of the 5'-non coding region of
the CS1 gene (see Example 1) up to the start codon ATG and was amplified by PGR
(Phusion Polymerase, New England Biolabs) from P. pastoris genomic DNA using the
primers shown in Table 2 . The sequence was cloned into the pPUZZLE expression
vector pPM1aZ10_eGFP, cut with Apal and Sbfl, resulting in pPM1aZ10_pCS1_eGFP.
Additionally, the vector pPM1aZ10_pGAP_eGFP, containing the commonly used
promoter of glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP of P.
pastoris, here SEQ ID 13) was used as reference. The promoters were inserted
upstream of the start codon of the eGFP gene using the Apal and the Sbfl restriction
sites (see Tables 2 and 3). The correctness of the promoter sequences was verified by
Sanger sequencing.
Table 2: Primers for PGR amplification of the promoters
Table 3: Amplification primers, cloning enzymes and the length of the cloned
promoter
c) Expression of eGFP in P. pastoris for ana!ysis of the promoter activity
All plasmids were linearized with Ascl within the 3 'AOX genome integration
region prior to electroporation (2 kV, 4 ms, GenePulser, BioRad) into electrocompetent
P. pastoris.
Positive transformants were selected on YPD plates (per liter: 10 g yeast
extract, 20 g peptone, 20 g glucose, 20 g agar-agar) plates containing 25 g/mL of
Zeocin (Invivogen, CA). Colony PGR was used to ensure the presence of the
transformed piasmid. Therefore, genomic DNA was gained by cooking and freezing of
P. pastoris colonies for 5 minutes each and directly applied for PGR with the appropriate
primers. For expression screening, a single colony was inoculated in liquid YPGZeo
medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 g glycerol and 25 mg
Zeocin) as pre-culture. After approximately 24h the pre-culture was used to inoculate
the main culture with an OD600 of 0.1 in 2 ml_ synthetic screening medium (per liter:
22 g glucose monohydrate, 22 g citric acid, 3.15 g (NH4)2HP0 , 0.027 g CaCI2
*2H20 ,
0.9 g KCI, 0.5 g MgS0 *7H20 , 2 ml_ 500 x biotin and 1.47 ml_ trace salts stock solution
[per liter: 6 g CuS0 4
*5H20 , 0.08 g Nal, 3 g nS0 4
*H20 , 0.2 g Na2Mo0 4
*2H20 , 0.02 g
H3BO3, 0.5 g CoCI2, 20 g ZnCI2, 5 g FeS0 4
*7 20 and 5 ml_ H2S0 ] ; pH set to 5 with
5M KOH; sterilized by filtration) and 2 glucose feed bead quarters (second feed bead
added after 24 hours; Kuhner, CH). Glucose- limiting growth conditions were achieved
due to the slow glucose release kinetics of these feed beads, which is described by the
following equation: (Glucose)=1 .63*t0.74 [mg/Disc]. Samples were taken at the end of
the pre-culture, and 24 and 48 hours after inoculation of the main culture. Cell density
was determined by measuring OD600, eGFP expression was analyzed by flow
cytometry as described in Stadlmayr et al. (J. Biotechnology 2010 Dec; 150(4):519-
29). For each sample 10,000 cells were analyzed. Auto-fluorescence of P. pastoris
was measured using untransformed P. pastoris wild type cells and subtracted from the
signal. Relative eGFP expression levels (fluorescence intensity related to cell size) are
shown as percentage of eGFP expression level of a clone expressing eGFP under the
control of the constitutive pGAP promoter.
The results are shown in Table 4 . The clone expressing under the control of the
pCS1 promoter exceeded pGAP by 38% at the pre-culture (batch) end, and had 4-fold
higher GFP expression levels at the main culture (fed batch) end.
Table 4 : Average GFP fluorescence per cell size of P. pastoris clones
expressing eGFP under the control of the novel pCS1 promoter. Data is shown relative
to pGAP at the same time point.
d) Determination of eGFP gene copy number (GCN) of the P. pastoris clones of
Example 2c
GCN stands for the number of reporter protein expression cassettes integrated
into the P. pastoris genome. GCN determination of clones expressing eGFP under the
control of pCS1 or pGAP was done as described in Example 5 further below. eGFP
expression levels were analysed as in Example 2c. Exemplarily, the results of one
clone of each promoter are shown in Table 5 . Clones expressing eGFP under the
control of the novel pCS1 promoter produced twofold amounts of eGFP in comparison
to clones expressing under the pGAP promoter with the same GCN in screening
cultures.
Table 5: eGFP expression in screening cultures under the control of pGAP or
pCS1 correlated to GCN. Per GCN, pCS1 clones express twice the amount of eGFP
as pGAP clones.
e) Analysis of pCS1 promoter strength in fed-batch fermentation
To assess pCS1 promoter activity in production process-like conditions, fed
batch cultivations of one pCS1 clone (pCS1_eGFP#4) and one pGAP clone
(pGAP_eGFP#2) each harboring one copy of the eGFP expression cassette (see
Example 2d) were performed.
Fed batch fermentations were performed in DASGIP reactors with a final
working volume of 1.0 L.
Following media were used:
P T -j trace salts stock solution contained per liter
6.0 g CuS0 . 5H20 , 0.08 g Nal, 3.36 g MnSO . H20 , 0.2 g Na2Mo0 . 2H20 , 0.02
g H3BO3, 0.82 g CoCI2, 20.0 g ZnCI 2, 65.0 g FeS0 4. 7H20 , 0.2 g biotin and 5.0 ml
H2SO4 (95 %-98 %).
Glycerol Batch medium contained per liter
2 g Citric acid monohydrate (C6H8O7' H2O), 39.2 g Glycerol, 12.6 g NH H2P0 ,
0.5 g MgS0 4 7H20 , 0.9 g KCI, 0.022 g CaCI2 2H20 , 0.4 mg biotin and 4.6 ml PTM1
trace salts stock solution. HCI was added to set the pH to 5 .
Glucose fed batch medium contained per liter
464 g glucose monohydrate, 5.2 g gS0 4
»7H20, 8.4 g KCI, 0.28 g CaCl 2*2H20 ,
0.34 mg biotin and 10.1 ml_ PTM1 trace salts stock solution.
The dissolved oxygen was controlled at DO = 20 % with the stirrer speed (400 -
1200 rpm). Aeration rate was 24 L h air, the temperature was controlled at 25°C and
the pH setpoint of 5 was controlled with addition of NH4OH (25 %).
To start the fermentation, 400 ml_ batch medium was sterile filtered into the
fermenter and P. pastoris clone pCS1_eGFP#1 was inoculated (from pre-culture) with
a starting optical density (OD600) of 1. The batch phase of approximately 25 h
(reaching a dry biomass concentration of approximately 20 g/L) was followed by a
glucose-limited fed batch starting with an exponential feed for 7 h and a constant feed
rate of 15 g/L for 13 h, leading to a final dry biomass concentration of approximately
110 g/L. Samples were taken during batch and fed batch phase, and analyzed for
eGFP expression using a plate reader (Infinite 200, Tecan, CH). Therefore, samples
were diluted to an optical density (OD600) of 5 . Fermentations were performed in
duplicates. Results are shown in Table 6 as relative fluorescence per bioreactor (FL/r).
The clone expressing under control of the pCS1 promoter had on average 4.2 fold
higher eGFP expression compared to pGAP during the whole fermentation process.
pGAP_eGFP#2 pCS1_eGFP#1 comparison FL/r
] FL/r STDEV FL/r STDEV pCS1/pGAP
-4.5 2.1 0.1 9.3 0.6 4.4
0.8 4.7 0.1 16.8 0.9 3.6
2.3 5.9 1.0 20.2 1.5 3.4
4.3 9.2 0.1 35.3 1.4 3.8
6.8 14.2 0.6 57.6 4.0 4.1
17.9 57.6 4.2 288.2 15.6 5.0
19.9 86.0 16.6 337.3 26.5 3.9
2 1.6 73.9 9.7 379.9 4 1.7 5.1
Table 6: Relative fluorescence per bioreactor of two different P. pastoris clones
expressing eGFP under the control of pGAP or pCS1 in an optimized fed batch
fermentation t indicates feed time.
f ) Promoter activity of pCS1 at different growth conditions and substrates
In order to gain more information about promoter activity of pCS1 on different
media compositions and growth conditions, strain pCS1_eGFP#4 was cultivated in YP
medium containing different carbon sources, at different pH values and in synthetic
minimal medium. Samples were taken 24 and 48 hours after inoculation of the main
culture and analyzed by flow cytometry as described in Example 2c.
A single colony of pCS1 -eGFP#4 or pGAP-eGFP#2 as reference was
inoculated in liquid YPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6
g glycerol and 25 mg Zeocin) as pre-culture. After approximately 24h the pre-culture
was used to inoculate the main culture with an OD600 of 0.1 in 2 mL main culture
medium. Main culture media were as follows: YP per liter: 20 g peptone, 10 g yeast
extract, pH 7.0-7.5; YPD: YP + 2% glucose, YPG: YP + 2% glycerol; YPM: YP + 1%
methanol; YPE: YP + 1% ethanol; YPfeed bead: YP + 1 glucose feed bead (Kuhner,
CH, diameter 6 mm); YPD ph4.5: YPD set to pH 4.5 with HCI; SCD: synthetic
screening medium (per liter: 22 g glucose monohydrate, 22 g citric acid, 3.15 g
(NH )2HP0 , 0.027 g CaCI2
*2H20 , 0.9 g KCI, 0.5 g MgS0 4
*7H20 , 2 mL 500 x biotin
and .47 mL trace salts stock solution [per liter: 6 g CuS0 *5H20 , 0.08 g Nal, 3 g
MnS0 *H20 , 0.2 g Na Mo0 4
*2H20 , 0.02 g H3B0 3, 0.5 g CoCI , 20 g ZnCI2, 5 g
FeS0 *7H20 and 5 mL H2S0 ] ; pH set to 5 with 5M KOH; sterilized by filtration). All
cultures except the one with the feed bead, were fed with 0.5 % of the respective
carbon source after 19 h and 43 h. Results of eGFP fluorescence per cell size are
shown in Table 7 .
On YPD, pCS1 expression levels exceed pGAP expression levels by 2-fold after
24 and 48 h, whereas on YPG pCS1 expression levels were 3.9 fold higher after 24 h
and 24 h. Expression levels under control of the novel pCS1 promoter were even
higher when using methanol or ethanol as carbon source, or when cultivated at pH 4.5
(Table 7).
Table 7: Relative eGFP fluorescence per cell size of P. pastoris clones
pGAP_eGFP#2 or pCS1 -eGFP#4 after 24 h and 48 h cultivation in different screening
media. Mean values and SD of 2 cultivations are given.
Example 3: Comparative promoter activity studies of the newly identified
promoter pCS1 in P. pastoris using human serum albumin (HSA) as extracellular
expressed reporter gene
In order to analyze the properties of the newly identified promoter for expression
of the secreted reporter protein HSA, shake flask screenings were performed as
follows: Pre-culture for 24 hours was done in rich medium containing glycerol as
carbon source - simulating the batch phase of the process, which was followed by the
main culture in buffered rich medium (2% glucose). The main culture was fed with
0.5% glucose every 12 hours
a) Strain & expression vector
The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used
as host strain. Transformation of the strain was carried out with an in-house vector
named pPUZZLE (Stadlmayr et ai. J. Biotechnol 2010 Dec; 150(4):519-29), selection
of positive transformants was based on the Zeocin resistance. For secretory
expression of human serum albumin (HSA) its native secretion leader was used.
b) Amplification and cloning of the newly identified promoter pCS1 into an in-house
expression vector
The promoter amplified in Example 2b was cloned into the pPUZZLE expression
vector pPM1aZ10_HSA, resulting in pPM1aZ10_pCS1_HSA. Additionally, the vector
pPM1aZ1 0_pGAP_HSA, containing the commonly used promoter of glyceraldehyde 3-
phosphate dehydrogenase promoter (pGAP) was used as reference. The promoters
were inserted upstream of the start codon of the HSA gene using the Apal and the Sbfl
restriction sites (see Table 3). The correctness of the promoter sequences was verified
by Sanger sequencing.
c) Expression of HSA in P. pastoris under control of the newly identified promoter
pCS1
All plasmids were linearized using Ascl restriction enzyme prior to
electroporation (using a standard transformation protocol for P. pastoris) into P.
pastoris. Selection of positive transformants was performed on YPD plates (per liter:
10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar) plates containing 25
g/mL of Zeocin. Colony PGR was used to ensure the presence of the transformed
plasmid as described in Example 2c.
For HSA expression screening, a single colony was inoculated in liquid YPGZeo
medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 g glycerol and 25 mg
Zeocin) as pre-culture. After approximately 24 h the pre-culture was used to inoculate
the main culture with an OD600 of 0.1 in BM medium (per liter: 20 g peptone, 10 g
yeast extract, 20 g glucose, 13.4 g yeast nitrogen base with ammonium sulfate, 0.4
mg biotin and 100 mM potassium phosphate buffer pH 6.0). The main culture was fed
with 0.5% glucose every 12 hours. Samples were taken at the end of the main culture.
Biomass concentration was determined by measuring OD600 or wet cell weight. HSA
concentration in the culture supernatant was quantified by the Human Albumin ELISA
Quantitation Set (Cat.No. E80-129, Bethyl Laboratories, TX, USA) following the
supplier ' s instruction manual. The HSA standard was used with a starting
concentration of 400 ng mL . Samples were diluted accordingly in sample diluent (50
mM Tris-HCI, 140 mM NaCI, 1% (w/v) BSA, 0.05% (v/v) Tween20, pH 8.0). HSA titers
from screening of clones expressing HSA under the control of pGAP ( 1 HSA gene
copy) and of 9 clones expressing eGFP under the control of pCS1 are presented in
Table 8 . All pCS -controlled clones secrete twice the amount of HSA as the pGAPcontrolled
clone with one gene copy. GCN of HSA were determined as described in
Example 5 . All analyzed clones under control of pCS1 harbored one copy of the
expression cassette ( GCN). Thus, all clones under the control of the novel pCS1
promoter had a two-fold higher HSA secretion yield (mg secreted HSA/ g biomass)
than pGAP clones with the same GCN in screening cultures.
Table 8: Quantification of secreted HSA levels in supernatants of P. pastoris
clones expressing HSA under the control of pGAP and pCS1 after 48 h screening
culture.
Example 4 : Fed-batch cultivation of P. pastoris strains expressing HSA under
control of the pCS1 promoter
To analyze the ability of pCS1 to drive expression of HSA in production process
like conditions, fed batch cultivations of one pCS1 clone (pCS1_HSA#1 ) harboring one
copy of the HSA expression cassette (see Example 3) were performed.
The fermentations were performed in DASGIP bioreactors with a final working
volume of 1.0 L. Two different P. pastoris strains expressing HSA under control of
pGAP (pGAP_HSA#3 having one HSA gene copy, and pGAP_HSA#4 having two HSA
gene copies, described in Prielhofer et al. 2013. Microb. Cell. Fact. 12:5) were
cultivated as reference.
Following media were used:
PTMi trace salts stock solution contained per liter
6.0 g CuS0 4. 5H20 , 0.08 g Nal, 3.36 g nS0 4. H20 , 0.2 g Na2Mo0 . 2H20 , 0.02
g H3BO3, 0.82 g CoCI2, 20.0 g ZnCI2, 65.0 g FeS0 . 7H20 , 0.2 g biotin and 5.0 ml
H2S0 4 (95 %-98 %).
Glycerol Batch medium contained per liter
39.2 g Glycerol, 27.9 g H3P0 4 (85%), 7.8 g MgSO 7H2O, 2.6 g KOH, 9.5 g
K2S0 , 0.6 g CaSO 2H2O, 0.4 mg biotin and 4.6 mL PTM1 trace salts stock solution.
The pH was adjusted to 5.85 after sterile filtering into the fermenter.
Glucose fed batch medium contained per liter
550 g glucose monohydrate, 6.5 g MgS0 7H20 , 10 g KCI, 0.35 g CaCI2 2H20 ,
0.4 mg biotin and 12 mL PTM1 trace salts stock solution.
The dissolved oxygen was controlled at DO = 20 % with the stirrer speed (400 -
1200 rpm). Aeration rate is 24 L h air, the temperature was controlled at 25°C and the
pH setpoint of 5.85 is controlled with addition of NH OH (25 %).
To start the fermentation, 400 mL batch medium was sterile filtered into the
fermenter and P. pastoris was inoculated (from pre-culture) with a starting optical
density (OD600) of 1. The batch phase of approximately 25 h reached a dry biomass
concentration of approximately 20 g/L and was followed by a fed batch with constant
feed of glucose medium (2 g L h ) for 100 hours, leading to a dry biomass
concentration of approximately 100 g/L. The pH was 5.85 during batch, and kept at
5.85 throughout the fermentation. Samples were taken during batch and fed batch
phase. HSA concentration was quantified using the Human Albumin ELISA
Quantitation Set (Bethyl, Cat. No. E80-129) as described in Example 3c.
As shown previously, the pGAP clone with 2 HSA copies secreted twice as
much as the pGAP clone with one copy (Prielhofer et al. 2013. Microb. Cell. Fact.
12:5). Two clones secreting HSA under control of pCS1 reached more than 4-fold
higher HSA titers at the end of batch and fed batch compared to a single copy pGAP
clone (results shown in Table 9). Correlated to biomass and GCN, the pCS1 clones
produced 390 % secreted HSA of the pGAP clone with the same gene copy number.
batch end fee batch end
HSA YDM HSA YDM [g % HSA/YDM
Promoter GCN m L-'l [g -1 m L-1l of PGAP
Pcsi # 1 1 38.1 22.2 377.7 119.9 390.7
PGAP 3 1 8.4 22.1 78.6 97.7 100.0
PGAP#4 2 17.4 24.4 167.7 121 .0 172.0
Table 9: Yeast dry mass concentrations and HSA titers in the supernatant at
batch end and fed batch end, and HSA titer per yeast dry mass at fed batch end of
bioreactor cultivations of P. pastoris clones expressing HSA under the control of pCS1
or pGAP.
Example 5: Determination of gene copy numbers (GCN) of selected clones
Expression strength is often correlated to the number o expression cassettes
integrated into the P. pastoris genome. Therefore the gene copy number of selected
clones was determined. Genomic DNA was isolated using the DNeasy Blood&Tissue
Kit (Quiagen, Cat. No. 69504). Gene copy numbers were determined using quantitative
PGR. Therefore, SensiMix SYBR Kit (Bioline, QT605-05) was used. The Sensi Mix
SYBR was mixed with the respective primers (given in Prielhofer et al. 2013. Microb.
Cell. Fact. 12:5) and the sample, and applied for real time analysis in a real-time PGR
cycler (Rotor Gene, Qiagen). All samples were analyzed in tri- or quadruplicates. Rotor
Gene software was used for data analysis.
Example 6: Comparative promoter activity studies of the newly identified
promoter pCS1 in P. pastoris using porcine carboxypeptidase B (CpB) as extracellular
expressed reporter gene
In order to analyze the properties of the newly identified promoter, shake flask
screenings are performed as follows: Pre-culture for 24 hours is done with rich medium
containing glycerol as carbon source, which is followed by the main culture with rich
media.
a) Strain & expression vector
The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) is used as
host strain. Transformation of the strain is carried out with an in-house vector named
pPUZZLE (Stadlmayr et al. J. Biotechnol 20 0 Dec; 150(4):519-29), selection of
positive transformants is based on the Zeocin resistance. For secretory expression of
porcine carboxypeptidase B (CpB) yeast alpha mating factor leader is used.
b) Amplification and cloning of the newly identified promoter pCS1 into an in-house
expression vector
The promoter amplified in Example 2b are cloned into the pPUZZLE expression
vector pP 1aZ30_a F_CpB, resulting in pPM1aZ30_pCS1_aMF_CpB. Additionally,
the vector pPM1dZ30_pGAP_CpB, containing the commonly used promoter of
glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP) is used as reference.
The promoters are inserted upstream of the start codon of the CpB gene using the
Apal and the Sbfl restriction sites The correctness of the promoter sequences is
verified by Sanger sequencing.
c) Expression of CpB in P. pastoris under control of the newly identified glucoselimit
induced promoters
Plasmids are linearized using Ascl restriction enzyme prior to electroporation
(using a standard transformation protocol for P. pastoris) into P. pastoris. Selection of
positive transformants is performed on YPD plates (per liter: 10 g yeast extract, 20 g
peptone, 20 g glucose, 20 g agar-agar) plates containing 25 g/mL of Zeocin. Colony
PGR is used to ensure the presence of the transformed plasmid as described in
Example 2c.
For CpB expression screening, a single colony is inoculated in liquid YPG-Zeo
medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 g glycerol and 25 mg Zeocin)
as pre-culture. After approximately 24h the pre-culture is used to inoculate the main
culture with an OD600 of 1 in YPD medium (per liter: 20 g peptone, 10 g yeast extract,
20 g glucose). Main culture is fed with 0.5% glucose every 12 hours. Samples are
taken at the end of the pre-culture, and 24 and 48 hours after inoculation of the main
culture. Biomass concentration is determined by measuring OD600 or wet cell weight.
CpB concentration in the culture supernatant is quantified by an enzymatic assay,
based on the conversion of hippuryl-L-arginine to hippuric acid by the CpB. Reaction
kinetics are measured by monitoring the absorption at 254nm at 25°C using a Hitachi
U-29 0 Spectrophotometer when the reaction is started. Samples and standards are
buffered with assay buffer (25 mM Tris, 100 mM HCI, pH 7.65) and are activated using
activation buffer (0.01mgl_-1 Trypsin, 300 mM Tris, 1mM ZnC , pH 7.65). Activation
buffer without trypsin is used instead of sample as negative control. The reaction is
started by adding the substrate solution (1mM hippuryl-L-arginine in assay buffer).
d) Fed-batch cultivation of P. pastoris strains expressing CpB under control of the
pCS1 promoter Fed batch fermentation is done as described in example 4 using
media as described in example 2d.
Example 7: Comparative promoter activity studies of the newly identified
promoter pCS1 in P. pastoris multicopy clones using human serum albumin (HSA) as
extracellular expressed reporter gene
To investigate if HSA production could be further enhanced by higher GCN,
Pcsi clones expressing HSA were amplified by the post-transformational vector
amplification method, as described by Marx et al., (2009). Vectors were generated that
integrate into the rDNA locus by homologous recombination. The amplification was
done by selection on stepwise increasing concentration of antibiotics.
a) Strain & expression vector
The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used
as host strain. Transformation of the strain was carried out with a variant of the vector
pPUZZLE containing the NTS region of the ribosomal DNA locus as integration site
(Marx et al. 2009. FEMS Yeast Res. 9(8): 1260-70.), selection of positive transformants
was based on the Zeocin resistance. For secretory expression of human serum
albumin (HSA) its native secretion leader was used.
The pCS1 promoter amplified in Example 2b was cloned into the pPUZZLE
expression vector pPM1 nZ30_HSA, resulting in pPM1nZ30_pCS1_HSA.
The promoter was inserted upstream of the start codon of the HSA gene using
the Apal and the Sbfl restriction sites. The correctness of the promoter sequences was
verified by Sanger sequencing.
b) Post-transformational vector amplification and expression of HSA in P.
pastoris under control of the newly identified promoter pCS1
Plasmids were linearized using Spel restriction enzyme prior to electroporation
(using a standard transformation protocol for P. pastoris) into P. pastoris. Initial
selection of positive transformants was performed on YPD p!ates (per !iter: 10 g yeast
extract, 20 g peptone, 20 g glucose, 20 g agar-agar) plates containing 25 g/mL of
Zeocin. Colony PGR was used to ensure the presence of the transformed plasmid as
described in Example 2c. Gene copy number amplification was done as described in
Marx et al. (FEMS Yeast Res. 2009 Dec;9(8):1 260-70) by repeated streaking of clones
on YPD agar plates containing higher Zeocin concentrations (50, 100 and 500 g/ L
Zeocin).
HSA expression screening and product quantification by HSA ELISA was
performed as described in Example 3c. GCN was determined of some of the clones
with the highest HSA secretion levels as described in Example 5. GCN-amplified PCs
clones and PGAP and Pcsi clones with known GCNs (one or two) as control were
cultivated in BM medium for 48 h.
By increasing HSA GCN, HSA secretion levels could also be increased (shown
in Table 10). Multicopy clones expressing HSA under the control of pCS1 with 3 HSA
gene copies produced two- to threefold higher amounts of HSA per WCW compared to
the single copy clone. A similar increase in productivity of the multicopy pCS1 clones
could also be expected in fed batch bioreactor cultivations as presented in Example 4 .
Table 10: Screening results of HSA expression with single and multicopy clones
under the control of pGAP or pCS1 . GCN and titers per GCN are shown.
Example 8: Comparative promoter activity studies of the new!y identified
promoter pCS1 in P. pastoris using antibody fragment (Fab) as extracellular expressed
reporter gene
In order to analyze the properties of the newly identified promoter, shake flask
screenings were performed as follows: Pre-culture for 24 hours was done with rich
medium containing glycerol as carbon source, which was followed by the main culture
with rich medium.
a) Strain & expression vector
The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity
Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used
as host strain. The pCS1 promoter amplified in Example 2b was cloned into the
pPUZZLE expression vector containing either LC or Fab-HC of the HyHEL antibody as
GOI. The promoter was inserted upstream of the start codon of the Fab genes using
the Apal and the Sbfl restriction sites. After sequence verification, the expression
cassettes for both chains were combined onto one vector by using the compatible
restriction enzymes Mrel and Agel.
b) Expression of Fab in P. pastoris under control of the newly identified promoter
pCS1
Plasmids were linearized using Ascl restriction enzyme prior to electroporation
(using a standard transformation protocol for P. pastoris) into P. pastoris. Selection of
positive transformants was performed on YPD plates (per liter: 10 g yeast extract, 20 g
peptone, 20 g glucose, 20 g agar-agar) plates containing 25 g/mL of Zeocin. Colony
PGR was used to ensure the presence of the transformed plasmid as described in
Example 2c.
Fab expression screening was performed similar to HSA expression screening
described in Example 3c. Quantification of intact Fab was done by ELISA using antihuman
IgG antibody (Abeam ab7497) as coating antibody ( 1: 1 ,000), and a goat anti-
Human Kappa Light Chains (Bound and Free) - alkaline phosphatase conjugated
antibody (Sigma A3813) as detection antibody ( 1 : 1 ,000). Human Fab/Kappa, IgG
fragment (Bethyl P80-1 15) was used as standard with a starting concentration of 50
ng/mL. Supernatant samples are diluted accordingly. Detection was done with pNPP
substrate (Sigma S0942). Coating-, Dilution- and Washing buffer were based on PBS
(2 mM KH2P0 , 10 mM Na2HP0 .2 H20 , 2.7 m g KCI, 8 mM NaCI, pH 7.4) and
completed with BSA (1% (w/v)) and/or Tween20 (0.1 % (v/v)) accordingly.
Genomic DNA from selected clones was isolated and GCNs of heavy chain
(HC) and light chain (LC) were determined as described in Example 5 . GCNs were
related to clone PCS1#5, for which HC and LC GCN was set to one. Fab expression
yields ( g Fab/g WCW), relative GCNs and Fab yield per GCN are shown in Table 11.
As for the other model proteins, approximately two-times higher Fab yield per GCN (on
average 35.4 g Fab/gWCW) were obtained for clones expressing under control of
pCS1 in comparison to clones expressing under control of pGAP.
Table 11: Screening results of Fab expression under the control of pGAP or
pCS1 . GCN and Fab yields per GCN are shown on the right.
c) Fed-batch cultivation of P. pastoris strains expressing Fab under control of the
pCS1 promoter.
Fed batch fermentations were done similar as described in example 4 using
media as described in example 2d.
In bioreactor cultivation Fab expression under the control of pCS1 resulted in
more than 3.0-fold higher specific productivities (qP) per GCN compared to pGAP (see
Table 12).
Fab Yield [mg qP g Fab Relative GCN related
clone Fab g 1 DCW] g DCW h 1] to pCS1_Fab#5 qP per GCN
Heavy Heavy Light
chain Light chain chain chain
pGAP_Fab#34 0.220 2.18 4 3 0.55 0.73
pCS1_Fab#4 0.262 ± 0.000 3.51 ± 0.14 2 2 1.76 1.76
pCS1_Fab#5 0.178 2.54 1 1 2.54 2.54
Table 12: Bioreactor cultivation results of Fab expression under the control of
pGAP or pCS1 . Fab yields at the end of the fed batch cultivation, mean specific
productivity qP (average Fab production rate per biomass (dry cell weight DCW) over
whole fed batch phase), relative GCN of the respective clones, and mean qP per GCN
are shown.
Example 9 : Comparison of variants of pCS1
Length variants of the pCS1 promoter are cloned as described in example 2a
and screened similar as described in example 2c. Clones expressing under the control
of pCS1 (standard length) and pGAP were used as controls. Forward primers and
lengths of pCS1 and its variants are listed in Table 13.
Table 13: pCS1 and its variants: forward primers and lengths of the pCS1 size
variants (SEQ D 1)
Screenings of clones expressing eGFP under the control of pGAP, pCS1 or
pCS1 revealed that pCS1 requires a minimal length of approximately 500 bp for
optimal activity. Shorter pCS1 variants loose activity with decreasing length (see Table
14).
Table 14: Screening results (relative fluorescence) of eGFP expression under
the control of pGAP, pCS1 or pCS1 variants.
Example 10: Verification of expression strength of a promoter in a clone
expressing eGFP under control of said promoter in growth-limited conditions at high
and low growth rates
a) Strain
A host strain (e.g. Pichia pastoris) expressing eGFP under control of a
"promoter of Interest" and a strain expressing eGFP under control of pGAP are used to
compare expression levels.
b) Cultivation of eGFP expressing strains for promoter comparison
These strains are cultivated in chemostat at two fixed specific growth rates (one
low, one high specific growth rate by setting the dilution rate), using DASGIP
bioreactors with a final working volume of 1.0 L.
Following media are used:
PTM1 trace salts stock solution contains per liter
6.0 g CuS0 4 5H20 , 0.08 g Nal, 3.36 g MnS0 4
» H20 , 0.2 g Na2Mo0 4 2H20 ,
0.02 g H3BO3, 0.82 g CoCI2, 20.0 g ZnCI2, 65.0 g FeS0 4* 7H20 , 0.2 g biotin and
5.0 ml H2S0 4 (95%-98%).
Glycerol Batch medium contains per liter
2 g Citric acid monohydrate (C6H80 7'H20), 39.2 g Glycerol, 12.6 g NH4H2P0 4,
0.5 g MgSO 7H2O, 0.9 g KCI, 0.022 g CaCI2 2H20 , 0.4 mg biotin and 4.6 ml
PTM1
trace salts stock solution. HCI is added to set the pH to 5 .
Chemostat medium contains per liter
2.5 g Citric acid monohydrate (C6H807'H 0), 55.0 g glucose monohydrate,
2 1.75 g (NH )2HP0 , 1.0 g MgSO 7H2O, 2.5 g KCI, 0.04 g CaCI2 2H20 , 0.4 mg biotin
and 2.43 mL PTM1 trace salts stock solution. HCI is added to set the pH to 5 .
The dissolved oxygen is controlled at DO = 20% with the stirrer speed (400 -
1200 rpm). Aeration rate is 24 L h-1 air, the temperature is controlled at 25°C and the
pH setpoint of 5 is controlled with addition of NH4OH (25%). To start the fermentation,
400 mL batch medium is sterile filtered into the fermenter and a P. pastoris clone is
inoculated (from pre-culture) with a starting optical density (OD600) of 1. The batch
phase of approximately 25 h (reaching a dry biomass concentration of approximately
20 g L-1 ) is followed by glucose-limited chemostat cultivation. The feed rate of
chemostat medium and the harvest rate are used to keep a constant specific growth
rate as desired. During this cultivation, culture broth volume is kept constant and cell
dry weight is determined in order to ensure a constant growth rate. Cells are ultivated
at a high and low growth rate of 0.15 and 0.015 h respectively. Therefore, the
feed/harvest rate is controlled at 150 mL h L (mL chemostat medium per liter culture
broth and hour) and 15 mL h 5 L , respectively
c) Sampling
Samples are taken in steady state conditions (after at least 5 volume
exchanges) and analyzed for eGFP expression using a plate reader (Infinite 200,
Tecan, CH). Therefore, samples are diluted to an optical density (OD600) of 5 .
Fermentations are performed in duplicates. Expression data is compared by
calculating relative fluorescence per bioreactor as described in example 2d).
Example 11: Identification of a P. pastoris promoter enabling high transcription
at high and low specific growth rates
In order to identify a promoter enabling high transcription at high and low growth
rates, analysis of gene expression patterns was done using DNA microarrays. Genes
displaying high transcription strength at high and low growth rates were selected from
the transcriptomics data. Therefore, P. pastoris cells were grown in chemostat
cultivation as described in example 10b) at high and low specific growth rates of 0.15
and 0.015 h , respectively. Sampling, RNA purification, sample preparation for
microarray hybridization, microarray analysis, data acquisition and statistical evaluation
are done as described in example 1c), 1d), 1e) and 1f). Genes and respective
promoters with high transcription strength at high and low growth rate were identified
by browsing the microarray data for genes with high signal intensities in both, high and
low growth rate conditions. As a second criterion, signal intensities should be higher
than those of the glyceraldehyde-3-phosphate dehydrogenase (GAP, synonyms
GAPDH and TDH3) gene in both conditions. To isolate the promoter, a nucleic acid
fragment of approximately 1000 bps upstream of the start codon ATG of the respective
gene was amplified.

CLAIMS
. An isolated nucleic acid sequence comprising a promoter, which is a native
sequence of Pichia pastoris comprising the pCS1 nucleic acid sequence of SEQ D 1,
or a functionally active variant thereof which is a size variant, a mutant or hybrid of
SEQ ID 1, or a combination thereof.
2 . The nucleic acid sequence according to claim 1, wherein the promoter is
consisting of the pCS1 nucleic acid sequence of SEQ ID 1, or a functionally active
variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a
combination thereof.
3 . The nucleic acid according to claim 1 or 2 , wherein said functionally active
variant exhibits substantially the same activity as the pCS1 nucleic acid sequence of
SEQ ID 1.
4 . The nucleic acid sequence according to claim 1 or 2 , wherein the functionally
active variant is
a) a size variant of pCS1 of SEQ ID 1, preferably comprising or consisting of the
nucleic acid sequence selected from the group consisting of SEQ ID 2 , 3, 4 , 5, 6, 7 and
8, most preferably the nucleic acid sequence consists of SEQ ID 2 , 3, 4 , 5, or 6;
b) a mutant of the pCS1 of SEQ ID 1, or a mutant of the size variant of a), which
mutant has at least 60% homology to the sequence SEQ ID 1 or to the size variant;
c) a hybrid comprising
- a sequence selected from the group consisting of pCS1 of SEQ ID 1, a
size variant of a), and a mutant of b); and
- at least one further sequence selected from the group consisting of
pCS1 of SEQ ID 1, a size variant of a), a mutant of b), and a heterologous
sequence; or
d) a sequence which hybridizes under stringent conditions to any of the size
variant, or the mutant nucleic acid sequences of a), or b).
5 . The nucleic acid sequence according to any of claims 1 to 4 , wherein the
functionally active variant is selected from the group consisting of homologs with
i) at least about 60% nucleotide sequence identity;
ii) homologs obtainable by modifying the nucleotide sequence of pCS1 of SEQ
D 1 or size variants thereof, by insertion, deletion or substitution of one or more
nucleotides within the sequence or at either or both of the distal ends of the sequence,
preferably with a nucleotide sequence of 80 bp to 1500 bp, more preferably at least
200 bp; and
iii) analogs derived from species other than Pichia pastoris.
6 . The nucleic acid sequence according to any of claims 1 to 5, which is
operably linked to a nucleotide sequence encoding a protein of interest (POl), which
nucleic acid is not natively associated with the nucleotide sequence encoding the POl.
7 . The nucleic acid sequence according to claim 6, which further comprises a
nucleic acid sequence encoding a signal peptide enabling the secretion of the POl,
preferably wherein nucleic acid sequence encoding the signal peptide is located
adjacent to the 5' end of the nucleotide sequence encoding the POl.
8 . An expression construct comprising the nucleic acid sequence according to
any of claims 1 to 7, preferably an autonomously replicating vector or plasmid, or one
which integrates into the chromosomal DNA of a host cell.
9 . A recombinant host cell which comprises a nucleic acid sequence according
to any of claims 1 to 7 or an expression construct according to claim 8, preferably a
eukaryotic cell, more preferably a yeast or filamentous fungal cell, more preferably a
yeast cell of the Saccharomyces or Pichia genus.
10. The recombinant host cell according to claim 9, comprising multiple copies
of the nucleic acid sequence, and/or multiple copies of the expression construct.
11. The recombinant host cell according to claim 9 or 10, which is selected from
the group consisting of mammalian, insect, yeast, filamentous fungi and plant cells,
preferably a yeast, preferably any of the P. pastoris strains CBS 704, CBS 2612, CBS
7435, CBS 9 173-91 89, DS Z 70877, X-33, GS1 15, KM71 and SMD1 168.
12. A stable culture of a plurality of the cell according to any of claim 9, 10 or 11.
13. A method of producing a POl by culturing a recombinant host cell line
comprising the promoter according to any of claims 1 to 7 or the expression construct
according to claim 8, and a nucleic acid encoding the POl under the transcriptional
control of said promoter, or the recombinant host cell according to any of claims 9 to
11, comprising the steps of
a) cultivating the cell line under conditions to express said POl, and
b) recovering the POl.
14. The method according to claim 13, wherein the POl is expressed under
growth-limiting conditions.
15. The method according to claim 3 or 14, wherein the cell line is cultivated
under batch, fed -batch or continuous cultivation conditions, and/or in media containing
limited carbon substrate.
16. The method according to claim 15, wherein the cultivation is performed in a
bioreactor starting with a batch phase followed by a fed-batch phase or a continuous
cultivation phase.
17. Method according to any of claims 13 to 16, wherein the POI is a
heterologous protein, preferably selected from therapeutic proteins, including
antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion
proteins, carbohydrate - protein conjugates, structural proteins, regulatory proteins,
vaccines and vaccine like proteins or particles, process enzymes, growth factors,
hormones and cytokines, or a metabolite of a POI.
18. A method to identify a constitutive promoter from eukaryotic cells,
comprising the steps of
a) cultivating eukaryotic cells at a high growth rate;
b) further cultivating the cells at a low growth rate;
c) providing samples of the cell culture of step a) and b),
d) performing transcription analysis in said samples and comparing the
transcript levels with the transcript levels of the native pGAP promoter of the cells; and
f ) selecting the constitutive promoter that has a higher transcription strength as
compared to the native pGAP promoter at high and low growth rates, preferably by
determining a transcript level of the identified constitutive promoter which is at least
1.1-fold higher as compared to the native pGAP promoter.
19. Use of an isolated nucleic acid sequence comprising a promoter which when
operatively linked to a nucleotide sequence encoding a protein of interest (POI) directs
the expression thereof in a host cell at an expression level that is higher than under
control of the native pGAP promoter at high and low growth rates, preferably in a
method of producing the POI by cultivating a host cell transformed with the nucleic acid
sequence, wherein the cultivation is performed in a bioreactor starting with a batch
phase followed by a fed-batch phase or a continuous cultivation phase.
20. Use according to claim 19, wherein the expression level is determined at a
high growth rate and a low growth rate within the range of 0,015 to 0,15 h .
2 1. Use according to claim 19 or 20, wherein the expression level is at least 1.1-
fold higher compared to the pGAP promoter.
22. Use of the isolated nucleic acid sequence of any of claims 1 to 7 or the
expression construct of claim 8 in a method of producing a POI by cultivating a host
cell transformed with the nucleic acid sequence and/or the expression construct,
preferably wherein the cultivation is performed in a bioreactor starting with a batch
phase followed by a fed-batch phase or a continuous cultivation phase.