A METHOD FOR PRODUCING A POLYPEPTIDE IN YARROWIALIPOLYTICA
BACKGROUND OF THE INVENTION
THIS invention relates to a method for producing a polypeptide in Yarrowia lipolytica. In
particular, this invention relates to a method of optimizing the use of the hp4d promoter in
expressing a polypeptide of interest in Y. lipolytica, by manipulating the growth conditions
and hence the growth profile of the yeast.
Y. lipolytica is a non-conventional yeast which has been awarded Generally Regarded as
Safe (GRAS) status by the American Food and Drug Administration (FDA) for citric acid
production (Fickers et a/., 2005). A large number of molecular tools are available for
heterologous protein expression in Y. lipolytica as this yeast has a high secreting capacity.
Multi-copy vectors contain the ura3d4 marker, which is required in multiple copies to
complement, allowing for selection of transformants with multiple inserts (Madzak et a/.,
2004). The ura3d4 selection marker ensures selection of transformants with 10 - 13 copies
of the integrated cassette (Juretzek et a/., 2001).
The hp4d promoter is the most popular promoter used for expressing heterologous
polypeptides in Y. lipolytica (Madzak e a/., 2004). This promoter consists of four tandem
repeat copies of the upstream activating sequence 1 of the XPR2 promoter, and expression
is not significantly affected by environmental conditions. However, its regulation is unknown
and it is reported to be quasi constitutive with production of proteins under its regulation only
occurring during early stationary growth phase.
A need thus exists to gain a greater understanding of the hp4d promoter to determine
whether this promoter will find application in manipulating, and ultimately increasing, protein
expression in a host cell.
SUMMARY OF THE INVENTION
According to the invention, there is provided a method of expressing a polypeptide in
Yarowia lipolytica, the method comprising the steps of:
fermenting Y. lipolytica which has been transformed with a polynucleotide encoding
the polypeptide under the control of a hp4d promoter; and
limiting the growth rate of the Y. lipolytica during fermentation to below 0.045 h .
The growth rate may be limited to from about 0.023 h" to about 0.040 h , and more
preferably from about 0.035 h to about 0.039 h . Even more preferably, the growth rate
may be limited to about 0.035h 1 .
The growth rate may be limited by controlling the amount of a food source, such as a carbon
and/or nitrogen source that is fed to the fermentation solution containing the Y. lipolytica.
The carbon source may be glucose and the nitrogen source may be a yeast extract.
The polypeptide may be a protein such as an enzyme, for example, a lipase or mannanase.
The fermentation may be batch fermentation, fed batch fermentation, repeated fed batch
fermentation or a continuous fermentation process.
Preferably, the method increases polypeptide production in comparison to a control Y.
lipolytica whose growth rate was not limited.
In a further embodiment of the invention, there is provided Y. lipolytica, which has been
transformed with a polynucleotide, encoding a polypeptide under the control of a hp4d
promoter, for use in a method as described above.
In another embodiment of the invention, there is provided a kit comprising Y. lipolytica as
described above for performing a method as described above.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: shows the effect of a glucose spike on the p0 2, residual glucose and biomass
concentrations of steady state Y. lipolytica Po1f 413-5 fermentation.
Figure 2: shows the effect of dilution rate on biomass, residual glucose and volumetric
enzyme activity of . Iipolytica Po1f 413-5 in steady state continuous
fermentation.
shows the effect of dilution rate on the magnitude of response in lipase
production by Y. Iipolytica Po1f 413-5 in steady state continuous fermentation.
Growth rates are given as data labels.
shows the effect of dilution rate on volumetric and specific rate of lipase
production by Y. Iipolytica Po1f 413-5 in steady state continuous fermentation.
shows the growth of Y. Iipolytica Po1f 413-5 in duplicate batch fermentation.
shows lipase production by Y. Iipolytica Po1f 413-5 in duplicate batch
fermentation.
shows the effect of exponential full medium feed on the growth of . Iipolytica
Po1f 413-5. Full medium was fed at exponential feed rates of 0.029 h (open
symbols) and 0.041 h (closed symbols).
Figure 8: shows the effect of exponential full medium feed on lipase production by Y.
Iipolytica Po1f 413-5. Full medium was fed at exponential feed rates of 0.029
h 1 (open symbols) and 0.041 h (closed symbols).
Figure 9: shows the effect of exponential full medium feed on the rate of lipase
production by Y. Iipolytica Po1f 413-5. Full medium was fed at exponential
feed rates of 0.029 h 1 (open symbols) and 0.041 h 1 (closed symbols).
Figure 0: shows growth of Y. Iipolytica ManA:HmA (Roth et a/., 2009) in duplicate batch
fermentation.
Figure 11: shows the effect of exponential full medium feed on the growth of Y. Iipolytica
ManA:HmA. Full medium was fed at exponential feed rates of 0.035 h (open
symbols) and 0.045 h (closed symbols).
Figure 2: shows the effect of exponential full medium feed on the mannanase
production by Y lipolytica ManA:HmA. Full medium was fed at exponential
feed rates of 0.035 h (open symbols) and 0.045 h 1 (closed symbols)
Figure 13: shows the effect of exponential full medium feed on the rate of mannanase
production by Y lipolytica ManA:HmA. Full medium was fed at exponential
feed rates of 0.035 h (open symbols) and 0.045 h (closed symbols).
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described more fully hereinafter with reference to the
accompanying drawings, in which some, but not all embodiments of the invention are shown.
A method of expressing a polypeptide in Yarowia lipolytica is disclosed herein, wherein Y.
lipolytica, which has been transformed with a polynucleotide encoding the polypeptide under
the control of a hp4d promoter, is fermented and the growth rate of the . lipolytica is limited
during fermentation to below 0.045 h .
The growth rate can be limited to from about 0.023 h to about 0.040 h , and more
preferably from about 0.035 h to about 0.039 h . For example, the growth rate can be
limited to about 0.023, 0.024, 0.027, 0.029, 0.035 or 0.039 h 1.
The growth rate can be limited by controlling the amount of a carbon, nitrogen and/or other
source, such as glucose or yeast extract, that is fed to the fermentation solution.
The fermentation may be batch fermentation, batch fed fermentation or a continuous
fermentation process.
The polypeptide can be a heterologous or homologous polypeptide, protein or enzyme. In
the examples described below, lipase and mannanase were used as exemplary enzymes
expressed by Y. lipolytica.
Production of enzymes by Y. lipolytica under regulation of the quasi-constitutive hp4d
promoter occurs from the beginning of the stationary growth phase. Continuous
fermentation under glucose limited conditions was used to determine the effect of growth
rate on lipase produced under regulation of the hp4d promoter.
The Lip2 gene, encoding an extracellular lipase in Y. lipolytica, and endo-1 ,4-- -
mannanase (-mannanase) from Aspergillus aculeatu were over-expressed in lipolytica
Polf (MatA, Leu2-207, ura3-302, xpr2-322, axp-2) with a multi-copy expression cassette of
LIP2 under the quasi-constitutive hp4d promoter.
The highest volumetric lipase production of 13 014 nkat.ml was at a growth rate of
0.024 h , the slowest growth rate evaluated. However, the maximum rate of lipase
production was obtained at growth rates above 0.035 h . The critical growth rate for lipase
production was found to be between 0.035 h 1 and 0.039 h . The specific rate of lipase
production of 28 nkat.mg .h in continuous fermentation was 4 fold higher than the specific
rate of lipase production of 7 nkat.mg .h in batch fermentation, indicating that continuous
fermentation may be a feasible option for enzyme production by Y. lipolytica. Utilizing the
data obtained from the continuous fermentation, a fed batch strategy for protein production
by Y. lipolytica under regulation of the hp4d promoter was developed and evaluated for the
production of lipase and mannanase.
A maximum lipase titre of 22 508 (±4 219) nkat.ml 1 was obtained when the growth rate
during the fed batch phase of the fermentation was 0.027 h compared to 8 374 (±671)
nkat.ml 1 obtained at the higher growth rate of 0.040 h and 5 910 (±524) nkat.ml 1 in batch
fermentation. By limiting the growth rate of Y lipolytica we were able to achieve
simultaneous biomass and enzyme production, thereby increasing the productivity of the
fermentation. The volumetric lipase productivity was 357 nkat.ml 1 .h during the slower
growth rate compared to 133 nKat.mi .h" 1 during an exponential growth rate of 0.040 h .
A maximum mannanase titre of 40 835 (± 2 536) nkat.ml 1 was obtained when the medium
was fed exponentially at 0.035 h compared to 3 1 479 (± 1 819) nkat.ml 1 when the medium
was fed at an exponential feed rate of 0.045 h and 14 253 (± 2 807) nkat.ml 1 in batch
fermentation. The exponential feed strategy allowed for combined biomass and enzyme
production, thereby increasing the productivity of the fermentation. The volumetric enzyme
productivity was 913 nkat.ml 1 .h during the slower feed rate compared to 850 nkat.ml 1 .h
and 346 nkat.ml 1 .h 1 during an exponential feed rate of 0.045 h and batch production
respectively. This feeding strategy was evaluated using a carbon feed as an example.
The invention as described should not to be limited to the specific embodiments disclosed
and modifications and other embodiments are intended to be included within the scope of
the invention.
Although specific terms are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation. The term "protein" for example, should be
read to include "peptide" and "polypeptide" and 'ce versa. Furthermore, by definition
protein includes "enzymes".
Examples
Materials and methods
Organism maintenance and inoculum strains
Y. lipolytica Po1f 413-5 (MatA, Leu-2-207, ura3-302, xpr2-322, axp-2) and Y lipolytica
ManA:HmA (Roth et al., 2009) were cryo-preserved and stored at -80°C. The inoculum for
fermentation was prepared by sterilising 100 ml medium consisting of 15 g.l yeast extract,
8.9 g.l 1 malt extract and 6.67 g.l 1 glucose in 1 L Erlenmeyer flasks at 121°C for 15 min. The
pH was be adjusted to 5.5 with either 25% m.v NH OH or 25% m.v H2S0 before
sterilisation. The content of single cryovials was used to inoculate the flasks. The flasks
were incubated at 30°C on an orbital shaker at 180 rpm for 18h.
Continuous fermentation of Y. lipolytica
A 2 L continuous fermenter (BioFlo 3000, New Brunswick, USA,) containing 1.5 L modified
CSIRman medium consisting of 20 g.l"1 yeast extract and 20 g.l 1 glucose was inoculated
with 100 ml inoculum. The pH was controlled at pH 6.8 with NH4OH (25% m.v 1) or H2S0 4
(25% m.v"1) . The temperature was controlled at 28°C, aeration at 3 simp and agitation at 800
rpm. Cal Biomass and optical density (OD) were measured by taking 5 ml samples after
every retention time to determine steady state. Once steady state was reached as indicated
by constant OD and biomass for three retention times, a sample was taken and biomass,
OD, enzyme activity and residual glucose concentration was measured.
Batch and fed-batch Lipase fermentations
Duplicate batch fermentations were run in Labfors (Infors AG-Bottmingen/Switzerland)
bioreactors with a working volume of 2 L containing 1.5 L medium consisting of 20g.l yeast
extract and 40 g.l 1 glucose. Fed-batch fermenters were run in the same fermenters with an
initial charge volume of 1.3 L consisting of 10 g.l 1 yeast extract and 24 g.l 1 glucose. The
fermenters were inoculated with 100 ml inoculum. The pH was controlled at pH 6.8 with
25% m.v-1 NH OH or 20% m.v H2S0 . The temperature was controlled at 28°C and the
aeration set to 1 v.v .m 1 . The starting agitation was 500 rpm and ramped up manually to
control the p0 2 above 30% saturation. The feed consisted of 83.6 g.T glucose and 40 g.l 1
yeast extract. The feed was started at depletion of the initial charge glucose as determined
by Accutrend (Boehringer Mannheim). The starting feed rate was 1.1 g.h and increased
every ten seconds at an exponential rate of 0.029 h and 0.041 h , respectively.
Analysis
Growth rate, biomass, enzyme production and glucose utilization were determined by taking
10 g samples at 3 hourly intervals. Growth was measured by determining the OD at 660 nm
and the residual glucose was measured using Accutrend (Boehringer Mannheim). Triplicate
samples of 2 ml aliquots were centrifuged and the supernatants stored at -20°C for analyzes
of extracellular lipase activity. The pellets were used for dry cell weight determination by
drying to constant weight at 10°C.
The substrate for lipase assay was prepared by drop wise addition of 1 ml 8 mM p-
Nitrophenylpalmitate (pNPP) prepared in isopropanol to 9 ml of 100 mM phosphate or Tris-
HCI buffer, pH 8.0. The reaction was initiated by adding 25 - 50 of the enzyme sample
and the release of pNP was monitored at 410 nm at 37°C. The activity of the enzyme was
calculated as:
U.ml = (V /v e x d) X A min
where V = final volume,
v = sample volume,
= Extinction coefficient of p-NP at 410 nm,
pH 8.0 (15 Mol x cm" 1 = ml x 1 x cm 1) ,
d = light path of cuvette and
A.min = change of absorbance per minute.
The activity of the mannanase enzyme produced was determined by using 0.25%
galactomannan (Sigma) in 0.05 M citrate phosphate buffer, as described by Bailey et al.,
(1992). The amount of reducing sugars released during the degradation of mannan was
determined by the dinitrosalicylic acid method using mannose as standard (Miller ei a/.,
1960). One unit of enzyme was defined as the activity producing 1 mmol reducing sugar per
minute in mannose equivalents under the optimal assay conditions. Volumetric enzyme
activity was reported as unit of enzyme per ml fermentation broth while the specific enzyme
activity was reported as enzyme unit per mg dry cell weight in the fermentation broth.
Results and discussion
Continuous fermentation
The effect of growth rates between 0.024 h and 0.058 h 1 on the production of biomass and
lipase enzyme were evaluated in continuous fermentation. Glucose was determined to be
the growth limiting nutrient by spiking the fermenter with a concentrated glucose solution
(50% m/m) to obtain a final glucose concentration in the fermenter of 5 g.l . The p02
decreased immediately in response to the glucose spike and the biomass increased from 9.9
g.l 1 to 10.8 g.l over 1.5 hours (Fig. 1).
A constant biomass of 11.4 (±0.4) g.l 1 was maintained at the dilution rates evaluated and
the residual glucose remained below detection levels (Fig. 2). The highest volumetric
enzyme activity of 13 014 nkat.ml 1 was obtained at the slowest dilution rate of 0.024 h .
The highest dilution rate evaluated was 0.058 h 1 with a volumetric enzyme of 493 nkat.ml 1 .
The magnitude of the response in lipase production as a result of increasing growth rate was
calculated by dividing the fold decrease in enzyme activity by the fold increase in dilution
rate. The effect of increased growth rate on lipase production was the highest when the
growth rate was increased from 0.039 h to 0.044 h , resulting in a magnitude of response
of 4 indicating a critical growth rate for lipase production under regulation of the hp4d
promoter by Y. lipolytica Po1f 413-5 (Fig. 3).
Both the volumetric and specific rate of lipase production were at their highest at dilution
rates of 0.024 h and 0.035 h' at of 314 nkat.ml 1 .h 1 and 28 nkat.mg .h respectively
(Fig. 4). Increasing the dilution rate above 0.035 h 1 resulted in a decrease in the rate of
lipase production, with a sharp drop in the rate of production when the dilution rate was
increased above 0.039 h . At a dilution rate of 0.044 h , the volumetric rate of lipase
production was 1 463 nkat.ml 1 .h and the specific rate of production was 6 nkat.mg .h .
This drop in lipase productivity at growth rates above 0.039 h supports the results
indicating that the hp4d promoter is regulated by growth rate and that it is fully expressed at
growth rates slower than 0.035 h with 0.039 h being the critical growth rate for regulation
of the hp4d promoter.
The maximum specific rate of lipase production in continuous fermentation was
28 nkat.mg 1 .h compared to 7 nkat.mg 1 .h 1 in batch fermentation, making continuous
fermentation a feasible option for enzyme production.
To utilise the effect of growth rate on lipase production, a fed batch strategy from lipase
production by Y. lipolytics Po1f 413-5 was determined. The yield of biomass on glucose was
determined to be 0.59 g.g . From the data, a glucose feed rate for enzyme production in
fed-batch fermentations was calculated using the following formula:
((dcwto + (dcwto x ((2/td)))-dcw t0) x Yx/s) / dcwt
where dcw 0 = starting dew
td = doubling time
Yx/s = yield
At a growth rate of 0.039 h , the calculated glucose feed rate was 0.07 g.g .h 1 .
This feed rate was tested in fed-batch fermentations.
Batch fermentation
Lipase fermentation
Batch fermentations were run to determine the production of the lipase enzyme under
regulation of the hp4d promoter by Y. lipolytica Po1f 413-5 at maximum growth rates.
Y. lipolytica Po1f 413-5 grew at a maximum growth rate of 0.14 h and reached a biomass
concentration of 16 (±0.36) g.l 1 after 26 h (Fig. 5). The yield of biomass on glucose
consumed was 0.67 g.g 1.
The volumetric lipase produced by the end of the exponential growth phase was 1 274
(±377) nkat.ml 1 and increased 4.6 fold to a maximum lipase activity of 5 910 (±524) nkat.ml 1
after 38 hours (Fig. 6). The specific lipase production followed a similar trend and at the end
of exponential growth the specific lipase was 75 (+44) nkat.mg 1 but increased 5 fold over the
next 24 hours to 390 (±35) nkat.mg 1 .
A fed-batch strategy consisting of a full medium feed was used to limit the growth rate. The
feed was started after glucose depletion. The maximum growth rate during the batch phase
was 0.13 (±0.01) h (Fig. 7).
The exponential growth rates after feed start were 0.027 h and 0.40 h for medium fed at
exponential feed rates of 0.029 h and 0.041 h , respectively. During the batch phase of
the fermentation, 724 (±13) nKat.ml 1 lipase was produced. The majority of the lipase was
produced during the exponential feed period, with 7 545 (±246) nKat.ml 1 lipase produced
during growth at 0.40 h compared to 17 152 (±410) nKat.ml 1 lipase during growth at 0.027
h (Fig. 8).
The maximum volumetric lipase activity achieved during growth at the slower growth rate
was 2.7 fold higher at 22 508 (±4219) nKat.ml 1 than the maximum lipase activity of and
8 374 (±671) nKat.ml 1 obtained at the higher growth rate. The response of the specific
lipase activity to the growth rate was similar to that of the volumetric lipase activity, with a 2.8
fold higher activity obtained at the lower growth rate. The maximum specific lipase activity
was 1 281 (±31 1) nkat.mg 1 at a growth rate of 0.027h 1 compared to 452 (±352) nkat.mg 1 at
a growth rate of 0.040h 1 .
The productivity was not influenced by the slower growth rate, and maximum volumetric and
specific productivities of 357 nkat.ml .h 1 and 20.3 nkat.mg 1 .h were obtained at a growth
rate of 0.027 h 1. At a growth rate of 0.040h 1 , the volumetric productivity was 133
nKat.ml 1 .h and specific productivity was 7.2 nkat.mg 1 .h (Fig. 9).
Mannanase fermentation
Batch fermentations were run to determine the production of the mannanase enzyme under
regulation of the hp4d promoter by Y lipolytica at maximum growth rates. Y lipolytica grew at
a maximum growth rate of 0.23 h and reached a biomass concentration of 27 (±0.74) g.l
after 25 h at a yield of 0.68 g.g" glucose (Figure 10). Mannanase activity can only be
determined after glucose depletion since residual glucose interferes with the enzyme assay.
The mannanase was therefore determined at the point of glucose depletion and again 16
hours later. However, the activity at the point of glucose depletion is the most important, as
it provides an indication of the amount of enzyme that is produced during unlimited growth
rate. The volumetric and specific mannanase activity was 10 427 (± 967) nkat.ml 1 and 386
(±13) nkat.mg 1 , respectively, at glucose depletion but increased to 14 253 (± 2 807)
nkat.ml 1 and 527.9 (±88) nkat.mg 1 during the next sixteen hours.
Using a fed-batch strategy to limit the maximum growth rate, a full medium feed, with
glucose as the limiting nutrient as determined in continuous fermentation, was started at
glucose depletion (after 16 h) at a rate of 0.1 g(glucose).l .h and increased at exponential
rates of 0.035 and 0.045 h 1 , respectively (Fig. 1 ) . The growth rate during the batch phase
was 0.24 h 1 reaching a biomass of 15 (±0.3) g.l at a yield of 0.63 g.g . The exponential
feed rates employed limited the growth rates during the fed-batch phase of the fermentations
to 0.033 h and 0.044 h 1, respectively, and a final biomass of 28 (±0.6) g.l 1 was obtained at
the end of the fed batch phase.
The average volumetric and specific mannanase activities, for duplicate fermentations with
triplicate assays, at the end of the batch phase were 5 2 11 (± 602) nkat.ml and 436 (± 47)
nkat.mg 1 , respectively (Fig. 12). The maximum volumetric enzyme activity produced when
Y. lipolytica was fed medium at an exponential rate of 0.035 h was 40 480 (± 1 268)
nkat.ml 1 compared to 3 1 479 (± 1 819) nkat.ml 1 when the medium was fed at an exponential
rate of 0.045 h .
The specific enzyme activity increased 1.4 fold, from 1 109 (±60) nkat.mg 1 when the
medium was fed at an exponential feed rate of 0.045 h 1 to 1 533 (±83) nkat.mg 1 when the
fermenter was fed at an exponential rate of 0.035 h . The slower feed rate did not result in
slower productivity of the mannanase and the enzyme was produced at 913 nkat.ml 1.h at
exponential feed rates of 0.035 h" and 0.045 h (Fig. 13). This was 2.6 fold higher than the
productivity of 346 nKat.ml .h 1 achieved in batch fermentation.
Conclusion
This is the first reported data on the heterologous production of enzymes by Y. lipolytica in
continuous fermentation. The critical growth rate for enzyme production was found to be
between 0.035 h and 0.039 h , and from this data a glucose feed rate for fed-batch
fermentations could be calculated. This feed strategy was evaluated in fed batch
fermentation for production of enzymes under regulation of the hp4d promoter in Y.
lipolytica.
To evaluate the effect of growth rate on lipase production, two fed batch fermentations were
run with a full medium exponential feed based on the glucose concentration of the feed at
rates of 0.029 h 1 and 0.041 h , resulting in growth rates of 0.027 h and 0.040 h ,
respectively. The volumetric and specific enzyme activities were 3.8 and 3.3 fold higher
when the growth rate was limited 0.027 h 1 compared to batch fermentation. The volumetric
lipase activity at a growth rate of 0.027 h was 1.7 fold higher than that obtained at a growth
rate of 0.040 h . This compares favourably with the 1.9 fold increase in volumetric lipase
activity obtained in continuous fermentation when the growth rate was decreased from 0.039
h to 0.024 h .
The volumetric and specific mannanase activities were 2.7 and 2.9 fold higher when the
growth rate was limited by the exponential feed of 0.035 compared to batch fermentation.
The ability to maximize the specific enzyme activity utilizing an exponential feed rate below
0.045 h can be exploited by decreasing the feed rate from 0.056 h to 0.035 h once a high
biomass concentration has been reached. This will allow for high specific and volumetric
enzyme production under regulation of the hp4d promoter in Y. lipolytica.
The data presented in this report shows that the production of enzymes under regulation of
the hp4d promoter can be switched on during biomass production by limiting the growth rate
using an exponential feed strategy.
References
Bailey J, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of
xylanase activity. J Biotechnol 23: 257-270
Fickers P, Benetti P-H, Wache Y, Marty A , Mauersberger S, Smit MS, Nicaud J-M (2005)
Hydrophobic substrate utilization by the yeast Yarrowia lipolytica, and its potential applications.
FEMS Yeast Res 5: 527-543.
Juretzek T, Le Dall M, Mauersberger S, Gaillardin C, Barth G, Nicaud J-M (2001) Vectors for
the expression and amplification in the yeast Yarrowia lipolytica. Yeast 18, 97-1 13.
Madzak C, Gaillardin C, Beckerich J-M (2004) Heterologous protein expression and
secretion in the non-conventional yeast Yarrowia lipolytica. J Biotechnol 109: 63-81.
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activity. Anal Biochem 2: 127-132
Roth R, Moodley V and P van Zyl. (2009) Heterologous Expression and Optimized
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CLAIMS
. A method of expressing a polypeptide in Yarrowia lipolytica, the method comprising
the steps of:
fermenting Y. lipolytica which has been transformed with a polynucleotide, encoding
the polypeptide, under the control of a hp4d promoter; and
limiting the growth rate of the Y. lipolytica during fermentation to below 0.045 h .
2. The method of claim 1, wherein the growth rate is limited in the range of about
0.023 h to about 0.040 h .
3 . The method of claim 2 wherein the growth rate is limited in the range of about 0.035 h
to about 0.039 h .
4 . The method of claim 3 , wherein the growth rate is limited to about 0.035 h 1.
5 . The method of any one of claims 1 to 4, wherein the growth rate is limited by
controlling the amount of a food source that is fed to a fermentation solution containing
the . lipolytica.
6. The method of claim 5 , wherein the food source is a carbon and/or nitrogen source.
7. The method of any one of claims 1 to 6, wherein fermentation is carried out via any
one of batch fermentation, fed batch fermentation, repeated fed batch fermentation or
continuous fermentation.
8 . The method of claim 7, wherein the fermentation is fed batch fermentation.
9. The method of claim 7, wherein the fermentation is repeated fed batch fermentation.
10. The method of claim 7, wherein the fermentation is continuous fermentation.
11. The method of any one of claims 1 to 10, in which the level of polypeptide production
is increased in comparison to a control Y. lipolytica whose growth rate was not limited.
. lipolytica, which has been transformed with a polynucleotide, encoding a
polypeptide under the control of a hp4d promoter, for use in a method according any
one of claims 1 to 11.
A kit comprising Y. lipolytica according to claim 12 for performing a method according
to any one of claims 1 to 1 .