Note: Descriptions are shown in the official language in which they were submitted.
BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 032 - Ma 76$
Dr. Lp/rd
Description
Optimized fermentation processes for the preparation of
foreign proteins in E.coli
The invention describes optimized fermentation processes
for the preparation of foreign proteins in E.coli using
the lac promoter or improved lac promoter ( for example
tac, trc). After the initial growth phase with glucose as
carbon source, induction of product formation is effected
(1) via IPTG with glucose limitation or (2) via lactose
or (3) via IPTG and lactose with lactose limitation. The
limitation of glucose or lactose is such that the oxygen
partial pressure remains above 10~.
The preparation of commercial quantities of many differ-
ent recombinant proteins in E.coli is well known in
principle. The expression of these proteins becomes
possible by cloning the coding cDNA into a multicopy
plasmid with the appropriate sequences.
Expression experiments on this are normally carried out
in shaken flasks. The yields of recombinant proteins in
this case are usually 50 -- 100 mg/l when cultures in
shaken flasks with volumes less than 100 ml are employed.
Although it is possible with these techniques success-
fully to prepare recombinant proteins, thexe is a need
for techniques with which the protein concentrations and
the preparable quantities are distinctly increased. One
approach which meets these requirements is the develop-
went of a fermentation process. The object of the inven-
tion is consequently the optimization of fermentation
processes for the expression of foreign proteins in
E.coli.
2~~2~~'~
2 _
Several such processes with which the said requirements
have at least partially been met have been described in
the literature. The use of the lac promoter in these
cases has meant that usually fusion proteins with an N-
terminal ~-galactosidase portion (~-Gal) have been
prepared. The yields in the fermentation are normally
between 0.1 and 2.0 g of fusion protein per liter. SaThen
the fused ~-Gal portion is taken into account, the actual
product concentration often decreases to 30~ of the said
value. Furthermore, elaborate purification processes are
required to remove the ,8-Gal portion from the product.
The present invention describes, inter alia, the expres-
sion of a mature product, i.e. 'the expression of a
product without a fusion portion which would subsequently
have to be eliminated again. Purification of the product
is made relatively straightforward by such processes.
However, in fermentation both the specific and the
volumetric yields of the pxoducts are normally consider-
ably lower. This is particularly true when the product of
the process is prepared in soluble and biologically
completely active form. In contrast to the formation of
protein which is inactive and stored as inclusion bodies,
the soluble and biologically active product may intervene
in the metabolism of the cells and cause drastic distur-
bances in the organism (E.coli) and may be degraded
considerably more easily by E.coli proteases. Despite
these problems, it has been possible in the processes to
date, in Which glucose was employed as carbon source and
isopropyl thiogalactoside (IPTG) was used for induction,
to obtain yields of 200 mg/1 of biologically completely
active product.
In order to optimize the fermentation, the invention
entailed improvements in the growth behavior end product
formation. Since the product is formed within the cells,
the specific product concentration (quantity of product
per cell) and the cell count are important. The product
of the two factors is the volumetric productivity of the
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process in grams per liter (g/1).
High cell density fermentations have frequently been
described in the literature for recombinant E.coli
strains. Cell densities up to 30 g of dry matter (DM) per
liter (1) are usually stated in this connection. It has
been possible, by a combination of several measures which
are known in principle, to develop a process in which the
recombinant E.coli K12 strain was fermented up to cell
densities of 50 g of DM/l, corresponding to 150 Assn. The
essential point here is that oxygen-enrichment of the
inlet air is not a condition of the process which is
described hereinafter. This has a beneficial effect on
the economics of the process because pure oxygen as
substrate results in high costs and, additionally, it is
possible to dispense with explosion-protection measures.
An important factor fox a process with a high volumetric
product yield is optimal induction of the promoter.
Induction with IPTG, which is carried out with the
abovementioned low cell densities, is described many
times in the literature.
It has been found that an improvement in the volumetric
yields by a factor of 5, from 0.2 g/1 to 1.0 g/1, is
achieved after induction by IpTG (1 mM to 10 mM, prefer-
ably 5 mM) and limitation of glucose as substrate in such
a way that the oxygen partial pressure is greater than or
equal to 10$.
A second embodiment of the invention comprises induction
of product formation in the case of growth with lactose
as carbon source and simultaneously natural inducer. The
oxygen partial pressure was likewise maintained at
greater than or equal to 10~ as above by controlling the
lactose addition. Induction by lactose is regarded in the
literature as suboptimal because this procedure is
alleged to be less efficient khan TPTG induction. To
date, no efficient fermentation processes in which
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lactose was employed as induces have been described. The
experimental approach according to the invention is based
on the consideration that a higher final concentration of
product can be achieved by a weaker induction and thus
slower product formation, because the slower pxoduct
formation has a less disturbing effect on the intrinsic
metabolism of E.coli. This approach has been confirmed in
appropriate experiments in which product concentrations
of 2 g/1 (+/-10~) were achieved, corresponding to a
' 10 doubling relative to above. The process differs from the
previous one induced by IPTG in that glucose was replaced
by lactose in the linear phase of growth. In the range of '
high metabolic activities at the end of the fermentation,
when addition of lactose was also limited in order to
maintain the partial pressure of oxygen above 10~, it was
possible in a third variant of the process to assist the
induction by lactose additionally by IPTG additions . This
additional TPTG induction is necessary only when the
power input of the specific chosen fermentation apparatus
is inadequate to supply the culture with oxygen. Since an
increased plasmid Loss is observed in the second proves s
described, on scale-up there is a crossing point as the
fermentation volumes increase, after which first the
second and then the first process is more economic,
because a slightly increased plasmid loss is observed on
induction with lactose.
The fermentation is tez-minated at the time when the
product concentration is at a maximum. Processes known to
those skilled in the art are used to concentrate (for
example in a separator) and disrupt (for example in a
high-gxessure homogenizes) the biomass, lifter sediments-
tion of the cell fragments, most of the product is
located in the clarified supernatant.
Accordingly, the invention relates to optimized ferments-
tion processes for the expression of foreign genes in
E.coli under the control of the lac promoter o~ optimized
lac promoter, with induction being effected at the end of
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the exponential phase of growth by
(1) IPTG with simultaneous substrate-limited glucose
addition, and the oxygen partial pressure is main
tained above 10~ by the limitation of the glucose
addition,
( 2 ) or by lactose as carbon source and simultaneously
natural inducer, with the oxygen partial pressure
being maintained above 10~ by the limitation of the
lactose addition,
( 3 ) or by lactose as carbon source and simultaneously
natural inducer and, in addition, IPTG, with the
oxygen partial pressure being maintained above 10~
by the limitation of the lactose addition.
In preferred variants of the process, in each case the
oxygen partial pressure is increased by one or more of
the following measuress
(a) Fermentation under superatmospheric pressure,
preferably up to 2 bar
(b) Controlled following of the power input (increasing
the stirrer speed) and of the aeration rate (up to
2 vvm)
(c) Reducing the temperature from 37°C to as far as 30°C
in order, via an improved Henry coefficient arid
reduced metabolic activity, to increase the oxygen
transfer rate and reduce the oxygen uptake rate
(necessary on scale-up above 1,000 l because the
specific power input decreases with increasing batch
size (= container)).
It is common to all the variants of the process that the
addition of sugar substrate as carbon source is con-
trolled to maximum values of 5 -- 10 g/l and the pH is
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controlled by addition of NH40H and H3P04 in the range
from pH 6.7 to pH 7.3 throughout the fermentation period.
The processes described above are preferably employed for
the genetically engineered preparation of the proteins
PP4 and PP4-x, which belong to the lipocortins,
(Grundmann et al., Proc. Natl. Acad. Sci. 85, (1985)
3708-3712) and the mutants and variants thereof.
The invention is further described in the examples and
patent claims.
Example:
The following example describes the fermentation of the
E.coli K12 strain W3110 lac IQ (Brent and Ptashne (1981)
Proc,Acad.Natl.Sci. USA 78, 4204-4208), this strain
having been transformed with the plasmid pTrc99A-PP4
(Amann et al. (1988) Gene 69, 301-315) or pTrc99A-PP4-X
(Grundmann et al. (1988) Behring Inst. Mitt. 82, 59-67).
Tab. 1 indicates a very suitable medium.
Tab. 1
Composition of an example of a growth medium;
(data in g or mg per liter)
Carbon source (sugar) as required
Yeast extract 20 g
NaH2P04 x HZO 1.2 g
Na2HP04 x 2 H20 8.5 g
KC1 1.0 g
MgS04 x 7 H20 2.0 g
Citric acid 0.25 g
NH4C1 5 . g
0
Thiamine 5.0 mg
H3B03 2.0 mg
( NH4 ) 6Mo~0z4x4 H20 0 . mg
8
CuSO,, x 5H20 0.16 mg
KI 0.4 mg
MnS04 x 7 H20 2.02 mg
ZnS04 x 7 H20 1.6 mg
Fermentation was carried out in a 10 1 Biostat E fermen
ter (manufacturers Braun Melsungen) with a feranentation
Volume Of 8 1.
During the fermentation no selection pressure was exerted
on plasmid-containing cells, i.e. the fermentation was
carried out without added antibiotics. The fermenter was
inoculated with an overnight shaken-flask preculture:
Glucose was employed as carbon source in the initial
phase o.f growth, the glucose being metered in sr~ as to
form less than 0.1 M acetic acid in this phase. Increased
acetate concentrations resulted in significantly lower
product yields . After 10--15 hours in the i~aitial phase ,of
growth, cell concentrations of about 50 ODfiso had been
achieved, and induction of product formation was effected
in three different alternative ways:
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(1) Metering-in of glucose continued after addition of
1-10 mM IPTG (preferably 5 mM IPTG)
At the end of the initial phase of growth, the product
formation was induced by adding 1 - 10 mM IPTG (prefer-
s ably 5 mM IPTG) while continuing to meter in glucose as
carbon source ("substrate"). In this case the rate of
product formation showed a distinct dependence on the
glucose concentration at the time. Glucose as actual
substrate and IPTG as apparent substrate appear as
competing substrates, with, according to the rules of
diauxia, glucose partially or completely suppressing the
activation of the lac operon. In this case yields of
1 g/1 PP4 or PP4-X were attained in the glucose-limited
system (glucose concentration less than 0.1 g/1).
The glucose limitation was carried out by setting the
pump or by means of on-line HPLC measurement. The growth
rate of the cells was not decreased by induction in non-
limited systems, while the growth rate of the cells
decreased as a function of the glucose concentration in
limited systems as expected. Cell densities between 100
and 150 OD6$o were reached, depending on the relevant
growth rates.,
(2) Metering in of lactose continued
At the end of the initial growth phase, the product
formation was induced by replacing glucose by lactose as
carbon source. Lactose is the physiological inducer of
the lac operon, but it brings about less complete induc-
tion than IPTG. During the induction phase, the cells
continued to grow to cell densities of 100 ODfiso~ The
product concentration reached values of l.5 g/1.
(3) Metering in of lactose continued, and additions of
1-10 mM IPTG (preferably 5 mM)
At the end of the initial growth phase, the product
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formation was induced by replacing glucose by lactose as
carbon source and additionally adding IPTG. In this case,
strong induction is brought about by IPTG and, at the
same time, the physiological substrate lactose is util-
ized. Accurate metering in of the carbon source is
unnecessary in this case, in contrast to glucose + IPTG.
An excess of up to 30 g/1 lactose has no adverse effect
on productivity. During induction the cells likewise
continue to grow up to cell densities of 100 ODsso ~ The
product concentration at the end of fermentation is
2.0 g/1.
The induction is carried cut by one of the processes as
a function of the particular fermentation batch size,
because the plasmid stability decreases from (1) to (3).
The fermentation parameters chosen in the described
experiments are summarized in Tab. 2.
Tab. 2: Fermentation parameters
pH: 7.0 (controlled by addition
of H3P04 and NH40H)
Aeration rate: 0.5 - 2.0 vvm
Number of revolutions: 1,500 rpm
Temperature: 37C (to 30C)
Gage pressure: to 2.0 bar
Substrate concentration: Glucose controlled at
less
than S.O g/1, limited
when
dissolved oxygen decreases;
lactose controlled in
subsequent metering in
( less than 30 g/l ) ,
limited
when dissolved oxygen
decreases.
Dissolved oxygen: greater thin 10~
