Table Of ContentPrielhoferetal.MicrobialCellFactories2013,12:5
http://www.microbialcellfactories.com/content/12/1/5
RESEARCH Open Access
Induction without methanol: novel regulated
Pichia
promoters enable high-level expression in
pastoris
Roland Prielhofer1, Michael Maurer2,3, Joachim Klein4, Jana Wenger4, Christoph Kiziak4, Brigitte Gasser1,2*
and Diethard Mattanovich1,2
Abstract
Background: Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris.
Therefore, novelregulatedpromotersare desired, ideally repressing heterologous gene expression during initial
growth and enabling it intheproduction phase. In a typical large scale fed-batch culture repression is desired
during the batch phase where cells grow ona surplus ofe.g. glycerol, while heterologous gene expression should
be active inthe feed phase under carbon(e.g. glucose) limitation.
Results: DNA microarray analysis of P. pastoris wild type cells growing inglycerol-basedbatch and glucose-based
fed batch was used for theidentification of genes withboth, strong repression on glycerol and high-level
expression in the feedphase. Six novel glucose-limitinducible promoterswere successfully applied to express the
intracellular reporter eGFP. The highest expression levels together withstrong repression inpre-culture were
achieved with the novel promoters P and P .
G1 G6
Human serum albumin (HSA) was used to characterize thepromoters with an industrially relevant secreted protein.
A P clone withtwo gene copies reached about 230% ofthebiomass specific HSA titer inglucose-based fed
G1
batch fermentation compared to a P clone withidenticalgene copy number, while P onlyachieved 39%. Two
GAP G6
clones each carrying elevengene copies,expressing HSA under control of P and P respectively were generated
G1 G6
bypost-transformational vector amplification. They producedabout 1.0and 0.7g L-1 HSA respectively inequalfed
batch processes. The suitability inproduction processes was also verified with HyHEL antibody Fab fragment for P
G1
and with porcine carboxypeptidase Bfor P .Moreover, themolecular function of thegene under the control of
G6
P was determined to encode a high-affinity glucose transporter and named GTH1.
G1
Conclusions: A set of novel regulated promoters, enablinginductionwithout methanol,was successfully identified
byusing DNA microarrays and shown to be suitable for high level expression ofrecombinant proteins in
glucose-based protein production processes.
Keywords: Pichia pastoris,Heterologous protein production, Glucose-limited fed batch cultivation, Inducible
promoter, High-affinity glucose transporter
*Correspondence:[email protected]
1UniversityofNaturalResourcesandLifeSciences,Departmentof
Biotechnology,Muthgasse18,Vienna1190,Austria
2AustrianCentreofIndustrialBiotechnology(ACIBGmbH),Muthgasse11,
Vienna1190,Austria
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Prielhoferetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Background On the other hand, constitutive promoters might be
The methylotrophic yeast Pichia pastoris is widely used advantageous for the over-expression of genes or to
as a production platform for heterologous proteins. co-express helper factors and marker genes. The
Latest developments in strain engineering for improved widely-used P controls the expression of glyceral-
GAP
proteinfolding and secretion and glyco-engineering have dehyde-3-phosphate dehydrogenase at a high basal
recently beenreviewed byDamascenoet al.[1]. level [9]. Its productivity can be influenced by control-
Another important target for strain development is ling the growth rate at the optimal activity of P
GAP
thepromoterdrivingexpressionoftheheterologousgene. [10], and by a decrease of available O levels [11]. The
2
A summary of the most important promoters of non- promoter of the translational elongation factor EF-1
methylotrophic and methylotrophic yeasts is provided alpha gene, P , was analyzed and showed a tighter
TEF1
by Mattanovich et al. [2]. While production of recombin- growth-associatedregulationthanP [12].
GAP
ant proteins in P. pastoris has been successfully achieved A promoter library of P was generated, leading to
AOX1
under control of the constitutive glyceraldehyde-3- a few variants that were slightly stronger than wild type
phosphate dehydrogenase promoter (P ), regulated P , and a number of variants with altered regulatory
GAP AOX1
promoters have several advantages: they enable initial properties, some of them being active without methanol
biomassgainwithoutproductformationandallowtuning [13]. Another library approach was done for P by
GAP
oftheproductionprocess.Additionally,apotentialimpact mutation and clones expressing yeast-enhanced green
ofproductaccumulationongrowthorviabilityofthecells fluorescent protein (yEGFP) under the control of
can be prevented by decoupling growth from the obtainedvariantsproduced8to218%offluorescencein-
productionphase. tensity compared to the wild type promoter [14].
However, today’s available regulated promoters of Potential promoter libraries can also be deduced from
P. pastoris have drawbacks. Many of them derive from microarray data and rational considerations. Focussing
methanol utilization pathway genes, which are generally on highly transcribed genes in general, 15 promoters
repressed by glucose and/or ethanol and strongly induced were selected for characterization and the promoter of
by methanol. P induces high-level expression of its thethiaminebiosynthesis geneP ,whichisregulated
AOX1 THI11
encoded alcohol oxidase 1, which catalyzes the oxidation by the availability of thiamine in the growth medium,
of methanol to formaldehyde [3]. Its weaker homolog wasdiscovered[15].
P has been used for protein production as well [4]. As described above, the number of strong promoters
AOX2
AnotherstrongpromoterofthispathwayisP ,itsgene with advantageous properties for protein production is
FLD1
formaldehydedehydrogenase is either induced bymethyl- limited in P. pastoris. This work was designated to
amineormethanol[5].Thepromoterofdihydroxyacetone identify novel promoters with both, high expression and
synthase, P ,wasreportedtobesimilarlyregulatedand an optimal regulation in production process conditions.
DAS
even stronger than P [3], however it is not com- Equally important, the addition of inducers was to be
AOX1
monly used for protein production. Methanol and avoided, because their use is often associated with extra
methylamine are both highly flammable and hazardous costs and safety precautions in large scale fermentation
to health, so safety precautions are required for their processes.
industrial use. In addition to that, methanol consump- A typical production process under the control of
tion is technically disadvantageous because it causes P uses glycerol in the batch phase, and a constant
GAP
high heat evolution and an increased oxygen demand glucose fed batch for 100 hours to reach more than 100
during the fed batch phase [6]. g L-1 cell dry weight [16]. In order to identify potential
TheP promotercontrolstheexpressionofisocitrate inducible promoters in the course of this process, we
ICL1
lyase and is regulated by the carbon source used for cell used DNA microarray analysis to compare gene expres-
growth. No detectable promoter activity is present when sion patterns of glycerol-excess (=batch growth phase)
cellsaregrowingonglucose,whileitgetsturnedonwhen and glucose-limited (=fed batch production phase) con-
cells are stationary or growing on ethanol [7]. Hence, this ditions. The expression capacity of selected promoter
promoter might be an alternative for some applications, targets was characterized with model proteins and veri-
but its regulatory properties are poor. PHO89 is a regu- fiedinfedbatchprocesses.
lated sodium phosphate symporter and its promoter was
investigatedandshowntoproducereasonableamountsof Results and discussion
protein [8]. Cells must be phosphate-limited for the full Identificationofnovelpromoterswithdesiredinduction
activation of P , and an increase in product titer was properties
PHO89
even shown in phosphate-limited stationary phase. Add- A typical P. pastoris protein production process avoiding
itionally, an impact on growth was reported and reduced methanol induction starts with a glycerol batch (surplus
cellularfitnesscanbeassumedintheseconditions. of carbon source) which is followed by a glucose fed
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batch (limit of carbon source) [10]. DNA microarrays Table1Identifiedpromotercandidates
were used to analyze gene expression patterns and to Promoter Gene P.pastorisgeneidentifier(strainGS115)
identify potential promoters for this cultivation strategy.
P GAP PAS_chr2-1_0437
GAP
In order to eliminate growth rate related effects, glucose-
P G1 PAS_chr1-3_0011
G1
limited conditions were analyzed in chemostat cultivation
P G3 PAS_chr4_0550
where the growth rate, similar to that in the batch phase, G3
wasfixedbycontrollingthedilutionrateat0.1h-1. PG4 G4 PAS_chr4_0043
The microarray data was mined for genes with both, PG6 G6 PAS_chr2-1_0853
high difference in expression level between repressed P G7 PAS_chr1-4_0570
G7
and induced state (fold change) as well as high signal in-
P G8 PAS_chr1-3_0165
G8
tensity in the induced state to identify potent promoters
forinduciblehigh-levelproteinproductioninP.pastoris.
Six potential promoters (abbreviated as P , P , P , controlled expression of eGFP (normalized to GCN)
G1 G3 G4
P ,P andP , seeFigure 1andTable 1)were consid- were induced from almost zero in batch phase to about
G6 G7 G8
eredforfurthercharacterization. 150% and 100% after 48 h screening culture, respect-
ively. The other promoters P , P , P , and P still
G3 G4 G7 G8
Verificationofpromoterstrengthandregulation showed a good regulation and induction strength suit-
At first, the strength and regulation of the novel promo- able for inducible protein expression, with expression
ters were assayed with the intracellular reporter protein strengths spanning a spectrum of about 20% to 120%
eGFP in small scale screening cultures. Both, repressive relative to P (Figure 2A). The next step was to inves-
GAP
conditions in pre-culture (glycerol excess) and induced tigate the induction behaviour of the novel promoters in
ones during main culture (glucose limit) were analyzed more detail.
during the screening. In order to simulate fed batch like
conditions in screenings, we had to adapt the screening Analysisoftheglucosedependentregulation
strategy. Instead of usual feedings with certain amounts The induction behaviour of the novel promoters was
of glucose which lead to repeated batch phases, we used characterized in screenings with eGFP producing clones
slow glucose releasing polymer particles (12mm feed in YP media containing different amounts of glucose
beads, Kuhner, CH), liberating glucose at a non-linear (ranging from 20 to 0.002 g L-1). The cells were culti-
rate of 1.63 ∙ t0.74 mg per disc (t = time [h]), which vated for 5–6 hours and eGFP expression was analyzed
equals to28.6mgper discafter48hours. byflowcytometry.
As shown in Figure 2, P and P had superior prop- Promoters P and P showed a flat induction course
G1 G6 G1 G7
erties in terms of both, regulation and induction leading to full activity only with less than 0.05 g L-1 glu-
strength. In order to visualize gene dosage effects, gen- cose. That is clearly different to P , P and P ´s stee-
G3 G4 G6
omic DNA of several clones was isolated and analyzed per regulation pattern which reach their top activity
by real-time PCR to determine the gene copy number already at around 4 g L-1 glucose (Figure 3). In other
(GCN) of eGFP. Compared to a P clone with one words,P isnotonlythestrongestbutalsomosttightly
GAP G1
gene copy, the specific fluorescence of P and P repressedbyglucoseamongthepromoterstestedhere.
G1 G6
Based on these regulatory features we intended to
characterize the functions of the genes under control
of the P promoters. At the time of their identifica-
G
tion, no or only putative functions were assigned to
the underlying genes aside from G1. Therefore, we
used NCBI Conserved Domain search to analyze the
protein sequences in order to identify putative gene
functions.
The gene under the control of P was previously
G1
functionally clustered with K. lactis high-affinity glucose
transporter HGT1 [17]. It contains two major facilitator
superfamily domains, same as the G7 gene, which is
Figure1Microarraydata(redchannel)ofidentifiedtarget
genesincomparisontoGAP.Barsrepresentrelativeexpression therefore assumed to be a glucose transporter too. For
levelsinglycerolexcess(batchphase,bluebarsontheleftside)and Saccharomyces cerevisiae, it was reported that hexose
inglucoselimit(chemostatcultivation,redbarsontherightside). transporters underlie complex regulation patterns and
Numbersintherightcolumnrepresentthefoldchangeofsignal
are expressed in dependence of glucose concentration
intensitybetweenglucoselimitandglycerolexcessconditions.
[18]. The regulation pattern exhibited by the promoters
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Thereby we could show that the P promoter remains
G1
repressed during the batch phase and that its induction
during fed batch clearly exceeds the strength of P .
GAP
Relative eGFP expression (fluorescence related to the
culture volume) and OD over the feed time are shown
600
inFigure2B.
Expressionofsecretedhumanserumalbumin
P and P were further selected to assay the produc-
G1 G6
tion of a secreted protein under their control. Human
serum albumin (HSA) is efficiently produced in P. pas-
toris. Therefore it can be regarded as an industrially
relevant secreted reporter protein. Its expression under
the control of P , P and P was screened in shake
G1 G6 GAP
flasks with glucose-limited conditions (through the use
of feed beads) in the main culture. During the glycerol
(batch) pre-culture, both P and P promoters remained
G1 G6
wellrepressed.Asseenbefore,duringmaincultureexpres-
sion under P was stronger than under P and, in rela-
G1 G6
tion to gene copy number, reached around 77% of the
biomass specific HSA yield of cells expressing under P ,
GAP
while P produced about 22% of P (Figure 4A). How-
G6 GAP
ever, the novel promoters might not show their full poten-
tialinshakeflaskscreenings,sincetheseconditionsarenot
Figure2ExpressionofeGFPundercontrolofthenovel strictlyglucoselimitedduringtheentireproductionphase.
promotersPG1,PG3,PG4,PG6,PG7andPG8.(A)SpecificeGFP To exploit the full potential of the novel promoters, glu-
fluorescenceinshakeflaskscreeningsrelatedtoPGAPandtoeGFP cose limited fed batch cultivations were performed. Based
genecopynumber.(B)Fedbatchcultivationsofsinglegenecopy
clonesexpressingeGFPunderthecontrolofP andP .Relative
GAP G1
eGFPexpression(solidlines)andOD (dashedlines)areshown
600
overthefeedtime.
P , P , P and P might be associated with a role in
G3 G4 G6 G8
central metabolism. An AKR (aldo keto reductase) do-
main was found in the gene expressed under P . The
G3
genes controlled by P and P are both putative
G4 G6
aldehyde dehydrogenases, predicted to be localized in
the cytosol and in the mitochondria, respectively. The
Gti1/Pac2 family domain found in G8 plays a role in
gluconate uptake upon glucose starvation and in sexual
developmentinSchizosaccharomycespombe.
We could show the need of an explicit glucose limit
for full activity of the novel promoters, which is most
pronounced for P . This demonstrates that the micro-
G1 Figure3Inductionbehaviourofthenovelpromoters.Specific
array data-based promoter selection is excellently suited eGFPfluorescenceofclonesexpressingeGFPunderthecontrolof
to select for promoters with features relevant for biopro- P ,P ,P ,P andP inmediacontainingdifferentamountsof
G1 G3 G4 G6 G7
cesses, and secondly indicates the novel promoter´s glucose.DataisrelatedtoPGAP,normalizedto1.0atthehighest
glucoseconcentrationof20gL-1andplottedagainstthe
advantagesinfedbatch fermentation.
logarithmicglucoseconcentration(trendlinecalculation:four
Toprovethisstatement,theapplicationofthestrongest
parameterlogisticcurve).Theglucoseconcentrationgivenonthex-
and most promising promoter PG1 was tested in a fed axisreferstotheglucosesetpointatthebeginningofthe
batchfermentationwheretrulyglucose-limitedconditions cultivation(serialdilutionsrangingfrom20to0.002gL-1).This
are present [10]. A single gene copy clone expressing screeningsetupanddataprocessingpointsoutrelativepromoter
activities,therebyshowingthekineticsofinduction,butdoesnot
eGFP was chosen for comparison to an equivalent single
allowcomparisonofpromoterstrength.
gene copy clone of eGFP under the control of P .
GAP
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Figure4ExpressionofsecretedHSAusingthenovelpromotersP andP inshakeflaskandfedbatchcultivations.(A)HSA
G1 G6
expressioninshakeflaskscreeningsrelatedtoP andtothegenecopynumber.(B)Drycellweightand(C)HSAtiterinfedbatchcultivations
GAP
ofdoubleandsinglegenecopyclonesexpressingunderthecontrolofP (circle,twocopies),P (diamond,onecopy)andP (blacksquare,
G1 G6 GAP
twocopiesandblack-and-white,onecopy).(D)Detailof(C)showinglatebatchandearlyfedbatchphase,highlightingthedifferentregulation
propertiesofthepromoters.ExceptforthesinglegenecopyP clone,allfermentationswereperformedinduplicates.
GAP
on the screening results, one clone each expressing suffi- PGAP-driven expression was already active in the batch
cient amounts of HSA under the control of P and P phase, and more than 5% of the final HSA amount was
G1 G6
was selected. Those clones, harbouring two and one gene alreadypresentatthebatchendforbothclonesexpressing
copies respectively, were compared with their respective underitscontrol.WhilethisisnotanissueincaseofHSA,
gene copy equivalent P clones (Figure 4B, 4C and 4D). it is a clear disadvantage compared to inducible promoters
GAP
Dry cell weight (DCW) and HSA titers are summarized in suchasP incaseoftoxicordifficulttoexpressproducts.
G1
Table 2. HSA titers of P clones correlate with their re- Figure 4C shows HSA titer over the feed time, and the
GAP
spective gene copy number. Again, the P clone showed unique repression/induction efficiency of P is clearly
G1 G1
superiorproperties-itclearlyoutperformedtheP clone pointed out in the first hours (Figure 4D). Both, P and
GAP G1
with the same gene copy number and produced about P showedgoodrepressioninthebatchphaseandinduc-
G6
230% of the biomass specific product yield compared to tionbytheglucoselimitedfeed.
P . P produced about 39% of the biomass specific To verify that the novel promoters also exhibit their
GAP G6
HSA yield of its gene copy equivalent P clone. Besides, superior regulatory properties and expression capacity
GAP
Table2SummaryoffedbatchcultivationsofP.pastorisexpressingHSAunderthecontrolofP ,P andP
G1 G6 GAP
Batchend Fedbatchend
Promoter GCN DCW HSA HSA/DCW DCW HSA HSA/DCW %HSA/DCW
[gL-1] [mgL-1] [mgg-1] [gL-1] [mgL-1] [mgg-1] ofP
GAP
P 2 24.3 0.5 0.0 126.9 303.1 2.4 231.2
G1*
P 1 23.9 0.3 0.0 127.1 24.3 0.2 38.9
G6*
P 2 23.8 10.1 0.4 123.7 128.2 1.0
GAP*
P 1 24.2 5.0 0.2 117.7 57.8 0.5
GAP
*performedinduplicates.
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Table3SummaryoffedbatchcultivationsofGCNamplifiedHSAexpressingclonesunderthecontrolofP andP
G1 G6
Batchend Fedbatchend
Promoter GCN DCW HSA HSA/DCW DCW HSA HSA/DCW
[gL-1] [mgL-1] [mgg-1] [gL-1] [mgL-1] [mgg-1]
P 11 18.9 0.2 0.0 114.0 1060.8 9.3
G1
P 11 22.5 0.3 0.0 110.8 728.7 6.6
G6*
*performedinduplicates.
in industrially relevant conditions, we elevated HSA KnockoutofG1
gene copy number by post-transformational vector Furthermore,wedecidedtoclarifythefunctionofthegene
amplification as described previously [19]. Thereby, PAS_chr1-3_0011, which underlies the control of the
we were able to produce more than 1 g L-1 HSA promoter P . It contains 12 transmembrane domains
G1
under the control of P with a clone harbouring 11 (predicted by TMHMM Server v. 2.0), two Major Facilita-
G1
gene copies, which corresponds to the 3.4-fold titer tor Superfamily (MFS) and other transporter domains.
of its two copy clone (Table 3). Again, P outper- Based on the sequence homology to the K. lactis high-
G1
formed a comparable clone with the same gene copy affinity glucose transporter HGT1, the gene controlled by
number under the control of the P promoter, P was expected to have a function in glucose transport
GAP G1
which produced 607 mg L-1 HSA in a similar fermen- [17],[20].Strongactivityofitspromoteratverylowglucose
tation [19]. High level HSA production was also concentrations further strengthened this assumption. For
achieved with an amplified clone expressing HSA (11 further verification, the gene was disrupted using the split
gene copies as well) under the control of the weaker marker cassette technique (primers given in Additional file
P promoter, which produced more than 720 mg L-1 1: Table S1) as described by Heiss et al. [21]. Similar as
G6
HSA (Table 3). This titer is approximately 30-fold described by Jørgensen and his colleagues [22], we com-
higher than the titer reached with the P single copy pared the glucose uptake of the wild type and a G1 knock
G6
clone (24 mg L-1), thus indicating that multiple copies out clone in glucose-limited chemostat cultivations at dif-
of expression cassettes under control of a weaker pro- ferent growth rates. The glucose saturation constants were
moter can also lead to high productivities. One calculated from the residual glucose concentrations
possibleexplanationofthiseffectcouldbethattheratio (Table 4) and a K of 9.7, 23.1 and 69.3 μM was obtained
S
of a repressing protein to promoter copy number and for three different dilution rates (μ=0.14, 0.1 and 0.05 h-1)
thus repressor binding sites is decreased in the ampli- for the wild type. Changing K values are observed for the
S
fiedclones,thereforeleadingtohighertranscription. wholecellindifferentconditions,whichisduetothediffer-
Additionally, the porcine enzyme carboxypeptidase ential regulation of its several transporters. A reduced
B(CpB)thatisusedforhumaninsulinproduction,andan
antibody Fab fragment were produced under the
control of P and P respectively, and exceeded the
G1 G6
production levels compared to P . Thereby we verified
GAP
again the suitability of these promoters in standard
glucosebasedproductionprocesses.
P activitydependsonspecificgrowthrate
G1
Weelucidated the expression activity ofP at different
G1
growth rates using an HSA clone with two gene copies
under its control. It was cultivated in chemostat with
different dilution rates and the highest specific product
formation was found at a growth rate of about 0.07 h-1
(Figure 5). This clearly differs to the profile obtained
with P in [10], where the highest specific product
GAP Figure5Correlationofspecificproductivitytospecificgrowth
formation was obtained only at higher growth rates. rateusingP .Specificproductformationrate(q )observedin
G1 p
Growth rate dependency may be utilized to optimize chemostatcultivationatdifferentdilutionratesofacloneexpressing
space-time yield or other parameters in the production HSAunderthecontrolofPG1aswellastherespectivetrendcurve
(splinecurve).
processes[10].
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Table4Glucosesubstratesaturationconstantsofa CBS2612 which can grow on minimal media supple-
wildtypeandaG1knockoutclone mented with biotin, was used for protein expression in
G1k.o.,μ =0.18h-1 Wildtype,μ =0.18h-1 this work.
max max
D[h-1] S[μM] X[gL-1] K [μM] S[μM] X[gL-1] K [μM] ThemaincultureforscreeningswaseitherdonewithYP
S S
orBMmediaandglucosefeedbeads(12mm,Kuhner,CH)
0.14 316.4 29.5 90.4 33.9 30.5 9.7
whichprovidedthecarbonsource.
0.10 123.8 31.3 99.0 28.9 32.1 23.1
YP media contained 20 g L-1 peptone and 10 g L-1
0.05 79.9 31.3 207.8 26.6 30.8 69.3
yeast extract, which can be supplemented with 12.6 g
Residualglucose(S),drycellweight(X)andsubstratesaturationconstant(KS) glycerol or 20 g glucose to obtain YPG and YPD,
ofawildtypeandaG1knockout(k.o.)cloneatdifferentdilutionratesin
respectively. For cultivation on plates, 5 g L-1 agar-agar
chemostatcultivations.
was added to the liquid medium. BM media was based
capacity of glucose uptake at low specific growth rates has on YP, supplemented with 13.4 g L-1 yeast nitrogen base
been reported before [22]. The G1 knock out clone (Cat.No. 291940, Becton Dickinson, FR) with ammo-
appearedtohavemuchhighersaturationconstantsof90.4, nium sulfate, 0.4 mg L-1 biotin and 100 mM potassium
99.0 and 207.8 μM at the same dilution rates, which was phosphatebufferpH6.0.
also described for the high-affinity glucose transporter dis-
ruptioninAspergillusniger[22].Theknockoutclonedoes Identificationofnovelinduciblepromoters
not display the low K values of the wild type, so that the
S
gene PAS_chr1-3_0011 was clearly identified as a high- a)Bioreactorcultivations
affinity glucose transporter. As the short name HGT1 is Fermentations for the identification of promoter
usedasanaliasforapeptidetransporterinS.cerevisiaewe candidates were done in 3.5 L working volume
suggest the short name GTH1 (glucose transporter with bioreactors (Minifors, Infors, CH) in three biological
highaffinity)forthisP.pastorisgene. replicates. Cells were grown for about 24 h in batch on
glycerol medium, followed by an exponential feed phase
onglycerolfedbatchmediumcalculatedasdescribedby
Conclusions
Resinaetal.[23]withaspecificgrowthrateofμ=0.1h-1
Efficient regulated promoters cannot necessarily be
and a substrate yield coefficient of Y of 0.5 g g-1.
found by classical batch screening approaches. Simulat- X/S
Sequentially, chemostat cultivation (D = μ = 0.1 h-1)
ing production conditions in lab scale and searching the
withhighdensityglucosemediumwasperformed.
promoter space offers a new target oriented approach.
Glycerol batch medium contained per liter: 2 g citric
We could show here that the cultivation of P. pastoris in
acid monohydrate, 39.2 g glycerol, 20.8 g NH H PO ,
conditions where repression or induction are desired, 4 2 4
0.5 g MgSO ∙ 7H O, 1.6 g KCl, 0.022 g CaCl ∙ 2H O,
followed by the analysis of transcript levels with DNA 4 2 2 2
0.8 mg biotin and 4.6 mL PTM1 trace salts stock
microarrays offers a potent opportunity to find new,
solution.HClwasaddedtosetthepHto5.0.
strongandregulatedpromoters.
Glycerol fed-batchmedium contained perliter:632 g
Six novel promoters wereidentified and further charac-
glycerol, 8 g MgSO ∙ 7H O, 22 g KCl, and 0.058 g
terized. All of them are activated by carbon source deple- 4 2
CaCl ∙2H O.
tion. The new promoters provide a tool box for 2 2
High-densitychemostatmediumcontainedperliter:2g
expression of recombinant genes and are thus well suit-
citric acid monohydrate, 99.42 g glucose monohydrate,
able for protein production processes. P had the most
G1 22gNH H PO ,1.3gMgSO ∙7H O,3.4gKCl,0.02g
favourable repression kinetics and exceeded the expres- 4 2 4 4 2
CaCl ∙ 2H O, 0.4 mg biotin and 3.2 mL PTM1 trace
sion levels of the well-established constitutive GAP pro- 2 2
saltsstocksolution.HClwasaddedtosetthepHto5.0.
moter in glucose limited fed batch cultures by more than
PTM trace salts stock solution contained per liter:
twofold. The molecular function of the gene under its 1
6.0 g CuSO ∙ 5H O, 0.08 g NaI, 3.36 g MnSO ∙
control was identified as high-affinity glucose transporter 4 2 4
H O, 0.2 g Na MoO ∙ 2H O, 0.02 g H BO , 0.82 g
and named GTH1. 2 2 4 2 3 3
CoCl , 20.0 g ZnCl , 65.0 g FeSO ∙ 7H O, 0.2 g
2 2 4 2
biotin and5.0mLH SO (95%-98%).
2 4
Materials and methods b)Microarrayhybridization
Strainsandcultivation RNApurificationandsamplepreparationaswellas
Escherichia coli DH10B (Invitrogen) was used for microarrayhybridization(in-housedesignedP.pastoris
subcloning. It was routinely cultivated in petri dishes or specificoligonucleotidearrays,AMAD-ID:034821,
shake flasks using LB media supplemented with 25 μg 8x15Kcustomarrays,Agilent)anddataanalysiswere
mL-1 Zeocin. A wild type Pichia pastoris strain doneasdescribedbyGrafetal.[24].
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Characterizationofpromoterstrengthandregulation the pre-culture was used to inoculate the main culture
with an optical density (OD ) of 0.1 (for eGFP) or 1
600
a)Cloning (for HSA, CpB and HyHEL Fab) in 10 mL YP or BM
Cloning and transformation was done using the in- medium, respectively. Glucose feed beads (12 mm,
house vector pPuzzle [15], which contains a Zeocin Kuhner, CH) were used to generate glucose-limiting
resistance cassette for selection in both E. coli and growthconditions.ExpressionofeGFPwasmeasuredat
yeast, an expression cassette for the gene of interest the end of pre-culture and at 24 and 48 hours of the
(GOI) consisting of a multiple cloning site and the main culture. Culture supernatant of clones expressing
S. cerevisiae CYC1 transcription terminator, and a secretedproteinwasharvestedfromthepre-cultureand
locus for integration into the P. pastoris genome after 48 hours and cell density was determined by
(3´ AOX1 region or rDNA locus). Promoter measuringwetcellweightorOD .
600
sequences (up to 1000 bps upstream of the start
codon of their respective genes) were PCR-amplified c)Comparativeanalysis ofP.pastorispromoteractivity
from P. pastoris genomic DNA (primer sequences In order to analyze relative transcription strength of
see Additional file 1: Table S1). The promoters were theP promotersatdifferentglucoseconcentrations,a
G
ligatedintopPuzzleinfrontofthestartcodonsofthe comparative promoter activity study using various
model proteins, using the ApaI and the SbfI glucoseconcentrations(rangingfrom20to0.002gL-1
restriction sites of the multiple cloning site of the glucose) was performed with eGFP expressing clones
vector.Vectorsexpressingtherespectivemodelprotein in24-wellplates (Cat.No.7701–5110,Whatman,UK)
under control of P were used as controls covered with breath seal membranes (Cat. No. B-100,
GAP
throughout the study. For the expression of ExcelScientific,CA).Glucoseconcentrationsof20,10,
heterodimeric HyHEL antibody Fab fragment (HyHEL 5,2.5,1.25,0.63,0.31,0.16,0.08,0.04,0.02,0.01,0.005,
Fab), the expression cassettes of light chain and Fab 0.002 g L-1 were obtained by serial dilution in YP
heavychain(eachundercontrolofP )werecombined media, and represent the inital setpoints. The main
G1
intoonevector(usingthestrategydescribedin[27]). culture was inoculated from YPG-Zeocin pre-culture
HSA was secreted by its native secretion leader, with an OD of 0.01 and samples were taken after
600
while for CpB and HyHEL Fab the S. cerevisiae alpha 5–6 hours and analyzed by flow cytometry.
mating factor signal sequence was used. To avoid
positional effects on reporter gene expression levels, d)Fedbatch cultivation
genome integration of the expression plasmids was Allfedbatchfermentationsweredonein1.0Lworking
targeted to either the 3´flanking region of the AOX1 volume bioreactors (SR0700ODLS, DASGIP, DE). The
gene or the ribosomal DNA locus (rDNA, for dissolved oxygen wascontrolled at DO = 20% with the
multicopyintegration)ofP.pastoris,respectively. stirrer speed (400 –1200rpm). Aeration rate was18 L
Plasmidswerelinearizedwithinthegenomeintegration h-1 air, the temperature was controlled at 25°C and the
regionpriortoelectroporation(2kV,4ms,GenePulser, pH was controlled at 5.85 for HSA [25] or pH 5.0 for
BioRad) into electrocompetent P. pastoris. Multicopy the other proteins [10] with addition of ammonium
integration of HSA expressing clones was done as hydroxide (25%). To start the fermentation, 300 mL
described by Marx et al. [19] and selected at higher batch medium was sterile filtered into the fermenter
Zeocinconcentrations(upto1000μgmL-1). and a P. pastoris clone was inoculated from an
P. pastoris cells were first selected and cultivated in overnight pre-culture with a starting optical density
petri dishes on YPD agar and then inoculated in an (OD ) of 1. For the cultivation of clones expressing
600
YPG medium as pre-culture for screenings and eGFP, the batch phase of approximately 25 h was
fermentations. Antibiotic selection by Zeocin was followed by a fed batch phase with a feeding rate
applied on plates and in pre-culture at a concentration optimized according to [10]. HSA expressing strains
of25μgmL-1orhigher. were cultivated as described by Marx et al. [19], where
the batch phase was followed by a constant feed of 2 g
b)Expressionscreening h-1 fed batch medium for 100 h, Carboxypeptidase B
Expression of intracellular eGFP and the secreted and HyHEL Fab expressing clones were cultivated
proteins HSA, CpB and HyHEL Fab with the novel similarly. Samples were taken during batch and fed
promotersincomparisontoP wasevaluatedinshake batchphase,andanalyzedforexpression.
GAP
flask screenings. All screenings were performed at 25°C GlycerolbatchandglucosefedbatchmediaforeGFP,
and with shaking at 180 rpm. Single colonies were HyHEL Fab and Carboxypeptidase B expressing
inoculated in YPG medium with selection pressure clones were exactly asdescribed in [10],whileforthe
(Zeocin) for pre-culture. After approximately 24 hours, productionofHSAthemediawasdescribedin[19].
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e)Chemostatcultivation CpB was quantified using an enzymatic assay based
A strain expressing HSA (2 GCN) under control of on the cleavage of hippuryl-L-arginine (Cat. No.
P was tested for its growth rate dependent H2508,Sigma, MO). Generation of hippuric acid was
G1
expressionbehaviour inchemostatatdifferentdilution monitored at its absorbance maximum of 254 nm.
rates(D=0.03,0.06,0.10,0.13)[10]. Prior to the measurement, the samples were desalted
Forcharacterizationofglucoseuptakecharacteristics, with Zeba Spin columns (Thermo Fisher Scientific,
the P. pastoris wild type strain and the strain deleted IL) and activated with trypsin (Cat. No. T8345,
for PAS_chr1-3_0011 were cultivated in glucose Sigma,MO).
limited chemostats at D = 0.05, 0.10, and 0.14 h-1.
Samplesweretakenrapidlyasdescribedbelow. c)Determinationofresidualglucose
The D-Glucose Assay - GOPOD-Format (Megazymes,
Analyticalmethods IE)wasusedtodetermineresidualglucoseofchemostat
samples. Supernatant sampling was done by pumping
a)Copynumberdeterminationwithreal-timePCR culture broth out of the bioreactor by producing an
Genomic DNA was isolated using the DNeasy overpressure, and its direct sterile filtration using a
Blood&Tissue Kit (Cat. No. 69504, Quiagen, DE). vacuum filter unit (Cat. No. 5141178, Whatman, UK).
Genecopynumbersweredeterminedwithquantitative Glucose-limited cultivations usually go along with very
PCR using the SensiMix SYBR Kit (QT605-05, Bioline low residual glucose concentrations in the supernatant,
reagents, UK). Theprimers(supplementary Additional so the manufacturer’s protocol wasadapted for glucose
file 1: Table S1) and sample were mixed with the concentrations from 10 to 100 mg L-1. Briefly, the
SensiMix and applied for real time analysis in a real- ratio of reaction buffer to sample was changed from
time PCR cycler (Rotor Gene, Qiagen, DE). All 30:1 to 3:1.
samples were analyzed in tri- or quadruplicates. Data
analysis was performed with the two standard curve Additional file
method of the Rotor Gene software. The actin gene
ACT1wasusedascalibrator. Additionalfile1:TableS1.PrimerSequences.Sequencesof
oligonucleotidesusedforamplificationofpromoters,determinationof
genecopynumbersofthemodelproteinexpressioncassettes,and
b)Determinationofproteinexpressionlevels
generationofG1disruptioncassette(includingverificationofpositive
A plate reader (Infinite 200,Tecan, CH) was used to knock-outs).
determine eGFP fluorescence in fermentation
samples.Therefore,samplesweredilutedtoanOD
600 Competinginterests
of 5 and fluorescence intensity was then related to Theauthorsdeclarethattheyhavenocompetinginterests.
theculturevolume.
Authors’contributions
Expression of eGFP in screenings was analyzed by
RPperformedtheexperimentalwork,dataanalysis,contributedtostudy
flow cytometry as described before [15]. Specific designanddraftedthemanuscript.MMcontributedtostudydesignand
eGFP fluorescence referred to in this study is the planningofbioreactorcultivations.JKandJWconceivedandprovided
industriallyrelevantscreeningconditions.CKsupporteddatainterpretation
fluorescence intensity related to the cell volume for
andplanningofpromotercharacterization.BGplannedandsupervisedthe
each data point as described by Hohenblum et al. experimentalwork,andcontributedtodataanalysisanddraftingthe
[26]. Then the geometric mean of the population´s manuscript.DMcoordinatedtheprojectandcontributedtodraftingthe
manuscript.BG,MMandDMconceivedofthestudy.Allauthorsreadand
specific fluorescence was normalized by subtracting
approvedthefinalmanuscript.
background signal (of non-producing P. pastoris wild
type cells) and related to expression under the Acknowledgements
controlofP . TheauthorsthankAlexandraB.Graffortheprocessingofthemicroarray
GAP
data.SpecialthanksgotoLisa-SophieHandler,ChristineRuschitzkaand
For quantification of HSA in shake flask and
ChristineSanystrafortheirhelpwithpromotercharacterizationduringtheir
fermentation supernatants, the Human Albumin bachelorthesesorinternship.Thisprojectwasfinanciallysupportedby
ELISA Quantitation Set (Cat. No. E80-129, Bethyl LONZA(Visp,CH).
Laboratories, TX) was used. The HSA standard was
Authordetails
applied with a starting concentration of 400 ng mL−1. 1UniversityofNaturalResourcesandLifeSciences,Departmentof
Dilution-,Blocking-andWashingbufferwerebasedon Biotechnology,Muthgasse18,Vienna1190,Austria.2AustrianCentreof
TBS (50 mM Tris–HCl, 140 mM NaCl, pH 8.0) and IndustrialBiotechnology(ACIBGmbH),Muthgasse11,Vienna1190,Austria.
3UniversityofAppliedSciencesFH-CampusVienna,SchoolofBioengineering,
completed with BSA (1% (w/v)) and/or Tween20 Muthgasse18,Vienna1190,Austria.4LonzaAG,Rottenstraße6,Visp3930,
(0.05%(v/v))accordingly. Switzerland.
HyHELFabwasdeterminedwith ELISAasdescribed
Received:10December2012Accepted:23January2013
previously[27]. Published:24January2013
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