Biotechnology and Bioprocess Engineering 27: 572-585 (2022) pISSN 1226-8372

DOI 10.1007/s12257-022-0006-z eISSN 1976-3816

RESEARCH PAPER

Auto-induction Screening Protocol for Ranking Clonal Libraries of

Pichia pastoris MutS Strains
David Wollborn, Rebecca Luise Müller, Lara Pauline Munkler, Rebekka Horstmann, Andrea Germer,
Lars Mathias Blank, and Jochen Büchs

Received: 18 January 2022 / Revised: 14 March 2022 / Accepted: 24 March 2022

© The Korean Society for Biotechnology and Bioengineering and Springer 2022

Abstract Screening Pichia pastoris (Komagataella phaffii) of high producers during primary screening. It is significantly
strain libraries utilizing the methanol inducible alcohol less time consuming and labor intensive. Nonetheless, it
oxidase 1 (AOX1) promoter requires addition of methanol leads to the same conclusion as the conventional screening
as an inducer during cultivation. As the AOX1 promoter is with manual methanol additions.
repressed in the presence of glucose or glycerol, common
screening procedures include long incubation times before Keywords: auto-induction, small-scale cultivation, high-
methanol induction by successive multiple additions. In throughput screening, bioprocess development, Pichia
this study, a P. pastoris MutS strain, secreting a green pastoris MutS, pAOX1
fluorescent protein (GFP), was used to characterize a
standard screening protocol in complex medium. Based on
these results, an auto-induction screening method for P. 1. Introduction
pastoris strains was developed using mineral SYN6
medium, where methanol was added from the beginning. The methylotrophic yeast Pichia pastoris (reclassified as
To gain more insight into growth and production behavior, Komagataella phaffii [1]) is a well-established cell factory
the cultures were online-monitored, using the Respiration for recombinant proteins. Benefits of using P. pastoris
Activity Monitoring System and the BioLector system. include an efficient protein secretion system, the ability to
Monitoring the oxygen transfer rate indicated that prolonged grow to high cell densities on simple mineral media and
cultivation phases before induction can be avoided and that the capability for post-translational modifications [2-4].
high glucose concentrations hinder inducible protein Altogether, these have led to the successful industrial
production. The results show the effects of using 20 g/L production of multiple proteins such as enzymes and
glucose, and the resulting ethanol formation on the screening antibody fragments [5]. The increased interest in P. pastoris
process. The newly developed auto-induction method was as a cell factory has also lead to well-established tools for
then used to screen and rank a strain library of high, genetic engineering [6-8], giving rise to large clonal libraries.
medium, and low GFP producers. These results demonstrate These libraries are heterogenic in their composition and
that the auto-induction method facilitates the identification require extensive screening as the clones differ from each
other in terms of genomic integration locus, gene copy
number, and the orientation of the expression cassette [9].
David Wollborn, Rebecca Luise Müller, Lara Pauline Munkler, Rebekka
Horstmann, Jochen Büchs* As a result, a primary screening step is required to test the
AVT - Biochemical Engineering, RWTH Aachen University, Aachen performance of every single clone. This screening is
52074, Germany
Tel: +49-241-80-24633; Fax: +49-241-80-22635 crucial for bioprocess development, as the selection of the
E-mail: jochen.buechs@avt.rwth-aachen.de best performing strain has a major impact on the economic
feasibility of the final production process [10]. As P. pastoris
Andrea Germer, Lars Mathias Blank
iAMB - Institute of Applied Microbiology, RWTH Aachen University, is a frequently used production host, there are many
Aachen 52074, Germany screening protocols available. They differ in operation
Auto-induction Medium for P. pastoris MutS Srains 573

conditions, used medium, and methanol induction method. simplifies the screening process, as no medium exchange
A common approach is the use of complex medium or or methanol addition is needed. The same approach is
complex components (e.g., yeast extract and peptone), as it described by Kenzom et al. [20] and leads to a screening
is reported to enhance productivity and facilitate product result comparable to the standard method where methanol
stability [11]. Additionally, some protocols suggest a fixed is manually added. Besides methanol addition from the
duration for the glucose growth phase, to ensure complete beginning, another variable factor is the used amount of
glucose consumption, as this carbon source represses the carbon source. Lee et al. [19] investigated the impact of
alcohol oxidase 1 (AOX1) promoter. Afterwards, methanol varying glycerol concentrations on the screening process
is added multiple times, to maintain inducing conditions. and found, that low glycerol concentrations lead to the
To enable high throughput screening at reasonable costs highest protein production rates. Lowering the amount of
and with sufficient sample volume, small-scale cultivation carbon source is a common method to reduce the oxygen
systems such as shake flasks and microtiter plates (MTPs) demand [10], as it leads to lower cell densities. However,
have become the option of choice [12,13]. The simple and this approach is not desirable, as lower cell densities often
functional design of MTPs specially allow a high degree of lead to decreased product levels, which can complicate
parallelization and automation [14,15]. However, it is well quantification and, thereby falsifies suggested clone rankings.
described in literature that screening P. pastoris libraries Thus, oxygen supply during screening is a crucial factor.
can be cumbersome due to elaborate handling procedures The available screening procedures mostly differ in the
[16]. The reason for the more elaborate screening is the following key parameters: the choice of cultivation system
frequently used alcohol oxidase (AOX1) promoter, which is (shake flask or MTP), the used carbon source (e.g., glucose
methanol dependent. It is tightly regulated and derived or glycerol) and the method of induction (media exchange,
from the methanol utilization pathway, thus, it is repressed multiple methanol additions or auto-induction) [16,18-20].
by glucose or glycerol. In the absence of repressing carbon- Furthermore, different culture media with or without complex
sources, gene expression can be induced by the addition of components [21] are used at different pH values using
methanol [17]. Due to this physiological peculiarity, different buffers [22]. It is known that the use of complex
common screening protocols recommend a two-stage media is not ideal in an industrial set-up. The raw materials
cultivation procedure [11]. Cells are firstly grown on in complex media vary from batch to batch, which may
repressing carbon-sources (e.g., glucose), harvested, and lead to low reproducibility and affects product quality and
then resuspended in a methanol containing medium without quantity [23]. Moreover, complex media are not suitable
any other carbon source [11,18]. This established media for strains that utilize auxotrophic selection markers [16].
change protocol bears the risk of cross contamination, loss Product recovery may be hampered if complex components
of cell material in individual cultures and, thereby, leads to are present in the medium. Thus, the next aim of this study
larger deviations in protein production between the tested was to elucidate strain behavior and protein production in
clones [16]. An alternative screening method that avoids defined mineral SYN6 medium [24]. All these aspects of
media exchange, was introduced by Weis et al. [16]. Here complex media components directly affect protein production
the P. pastoris cells are first cultured for 60 h in a glucose and should be considered during the screening process.
containing medium, where the extended cultivation time The selected cultivation system and more importantly
should ensure that glucose is completely depleted prior to the way it is operated, heavily impacts the oxygen supply
methanol induction. The induction itself is achieved by during the cultivation. The insufficient oxygen supply in
multiple additions of a medium/methanol mixture after small scale cultivation systems is often cited as the reason
60 h, 70 h, 82 h, and 106 h after inoculation, resulting in a for false screening results that are not reproducible in
total screening time of 130 h. The drawbacks of this stirred bioreactors [10,25,26]. To circumvent this problem,
method are the doubling of the final culture volume and the the maximum oxygen transfer capacity (OTRmax) needs to
long culturing time of 130 h. Besides, it is known that the be determined to ensure operating conditions that allow
disruption of a shaken cultivation changes the prevailing for non-limiting oxygen supply during screening. This
temperature. It can already lead to an oxygen limitation terminology (OTRmax) describes the maximum possible
during the sampling procedure [10]. As a result, the oxygen transfer rate (OTR) in the cultivation system under
interruption of the cultivation for medium or methanol the applied conditions. This means, we assume an oxygen
addition also might influence strain performance and, partial pressure in the liquid near to zero, resulting in a
thereby, strain selection. Lee et al. [19] suggest an auto- maximal driving concentration gradient. Thus, the term is
induction protocol that prevents the interruption of the used to highlight this special case and to differentiate
cultivation process, as methanol is added to the medium between OTRmax and OTR. The OTRmax is influenced by
right from the beginning of the cultivation. This procedure the shaking frequency, shaking diameter, shake flask size,
574 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

well-geometry, osmolality of the culture medium, and filling 30°C, cells were centrifuged and resuspended in 1 mL
volume. For shake flasks, the OTRmax can be estimated by BEDS solution, without DTT. Aliquots of 40 µL were made
the correlation of Meier et al. [27] and for MTPs via the and 500 ng of the desired DNA was added. The expression
correlation introduced by Lattermann et al. [28]. Therefore, vector was linearized prior to transformation, using the
the OTRmax is an important variable, which can be used to SacI restriction enzyme that cuts 208 bp downstream of the
apply suitable operating conditions that allow scalability of AOX promoter. Cells were incubated for 2 min on ice and
small scale cultivations to lab-scale fermenters. This has electroporated, using the GenePulserXcell™ (BioRad,
been successfully demonstrated for different microorganisms Hercules, CA, USA). The device was set to a voltage of
[29,30]. 1,500 V, a capacity of 25 μF, and a resistance of 200 Ω.
In this study, the standard screening method utilizing Electroporation was followed by a recovery step in YPD,
several methanol pulses in complex medium was compared supplemented with 1 M sorbitol for 2 h at 30°C. The
to a mineral auto-induction medium, using P. pastoris MutS recovered cells were plated on pre-warmed selection plates
strains, secreting green fluorescent protein (GFP) under the containing 1 M sorbitol and 200 µg/mL Zeocin.
control of the methanol-inducible AOX1 promoter. The
OTR of the cultures at shake flask and MTP scale were 2.2. Media
online monitored with a respective Respiration Activity All chemicals used for media preparation were of analytical
Monitoring System (RAMOS) to detect the oxygen grade and purchased from Carl Roth GmbH (Karlsruhe,
demand during the cultivation. The obtained OTR data is Germany), if not stated otherwise.
suitable to investigate the impact that the type and amount For the experiments, the microorganisms were either
of carbon source (glucose or glycerol) has on growth in grown in complex YPD medium or in mineral SYN6-MES
combination with the induction method of choice (pulse medium.
wise methanol addition or auto-induction). The resulting YPD medium is composed of 10 g/L yeast extract
auto-induction method was applied on a P. pastoris clone (granulated, CAS-No. 91079-40-2), 20 g/L peptone ex
bank and the screening results were compared to those casein (CAS-No. 8013-01-2) and 10 or 20 g/L glucose.
where a concentrated single methanol pulse was used. The yeast extract and peptone are dissolved in water and
autoclaved without glucose. Glucose is prepared as a 500 g/L
stock solution, autoclaved and added to the complex
2. Materials and Methods components to reach the desired final concentration.
SYN6-MES medium had the following composition
2.1. Strains [24]: Glycerol or glucose were prepared as 500 g/L stock
All GFP-producing strains are based on the P. pastoris host solutions and autoclaved separately. The basic SYN6
strain BSYBG11 (MutS), obtained from Bisy GmbH medium consists of 1.0 g/L KH2PO4, 7.66 g/L (NH4)2SO4,
(Hofstätten an der Raab, Austria). In this work, the 3.3 g/L KCl, 3.0 g/L MgSO4× 7H2O, 0.3 g/L NaCl, 27.3 g/L
BSYBG11 strain is referred to as “reference” (abbreviated 2-(N-Morpholino) ethanesulfonic acid, 4-Morpholinee-
as “Ref.” in the rest of the paper). It is used as a reference thanesulfonic acid (MES). All medium components were
control, as it does not contain a GFP expression cassette. dissolved and the pH was adjusted to 6.0 with 1 M NaOH.
For the GFP secreting P. pastoris BSYBG11 (MutS) strains, The basic medium solution is sterilized via autoclaving
the secretion signal mating factor α of Saccharomyces (121°C for 20 min). To obtain 1 L SYN6 medium, 940 mL
cerevisiae was cloned after the AOX promoter and was basic medium are supplemented with 10 mL of 100 g/L
fused to the 5’ end of the GFPmut 3 variant. The CaCl2 (autoclaved), 10 mL of a 100 × micro-elements
expression was under the control of the AOX1 promoter. stock solution, 10 mL of a 100 × vitamin stock solution,
The used GFP variant has been shown to decrease in signal 10 mL of a 100 × trace-elements solution and the residual
over time [31,32]. The expression cassette was randomly 20 mL are used to add a stock solution of the desired
integrated into the genome, following the protocol of Lin- carbon source. The stock solutions had the following
Cereghino et al. [33]. Briefly, P. pastoris cells were grown compositions: Micro-element stock solution: 6.65 g/L
overnight in YPD (yeast peptone dextrose) medium and EDTA (ethylenediamine tetraacetic acid disodiumsulfate),
reinoculated in the morning until an OD600 of 0.8 was 6.65 g/L (NH4)2Fe(SO4)2× 6H2O, 0.55 g/L CuSO4× 5H2O,
reached. Cells were then resuspended in 9 mL of BEDS 2 g/L ZnSO4× 7H2O and 2.65 g/L MnSO4× H2O. Vitamin
solution (10 mM bicine-NaOH, pH 8.3, 3% (v/v) ethylene stock solution: 0.04 g/L d-biotin and 13.35 g/L thiamine
glycol, 5% (v/v) dimethyl sulfoxide and 1 M sorbitol) with chloride. The d-biotin was dissolved in 10 mL of a (1:1)
1 mL 1 M dithiothreitol (DTT). After a 5 min incubation at mixture of 2-propanol and deionized water. Thiamin
Auto-induction Medium for P. pastoris MutS Srains 575

chloride was dissolved separately in 90 mL deionized 2.6. Offline sample analysis

water. Afterwards, the two solutions were mixed. Trace Glucose, glycerol, methanol and overflow metabolite
element stock solution: 0.065 g/L NiSO4× 6H2O, 0.065 g/L concentrations (ethanol) were determined by High Perfor-
CoCl2× 6H2O, 0.065 g/L boric acid, 0.065 g/L KI and mance Liquid Chromatography (HPLC). The HPLC device
0.065 g/L Na2MoO4× 2H2O. All stock solutions were filter (Ultimate3000; Dionex, Sunnyvale, CA, USA) was equipped
sterilized. with an organic acid resin: pre-column (40 × 8 mm) and an
organic acid resin column (250 × 8 mm), both from CS-
2.3. Pre-cultures Chromatographie Service GmbH (Langerwehe, Germany).
Pre-cultures were performed in 250 mL shake flasks at Additionally, a Rezex™ ROA-Organic-Acid H+ column
30°C with a shaking frequency of 300 rpm at a shaking (300 mm × 7.8 mm) from Phenomenex (Torrance, CA,
diameter of 50 mm for 10-14 h. The liquid culture volume USA) was used. Prior to the measurement, samples were
was 10 mL and 125 µL of a cryo-stock was added for centrifuged (10 min at 4,000 rpm), filter-sterilized (0.2 μm
inoculation. Medium and carbon source used for the pre- filter) and stored at 4°C. Sulphuric acid with a concentration
cultures were identical to the main culture medium. of 1 mM was used as elution agent. The flow rate was set
to 0.8 mL/min at a constant temperature of 40°C. A Shodex
2.4. RAMOS and offline shake flask cultivations RI-101 refractometer (Showa Denko Europe, Munich,
The OTR was monitored online at 30°C, using an in-house Germany) was used as detector. The software Dionex
built RAMOS device for shake flasks [34,35]. A commercial Chromeleon 6.8 (Thermo Fisher Scientific, Waltham, MA,
version is available from A. Kühner AG (Biersfelden, USA) was used for analysis.
Switzerland) or HiTecZang GmbH (Herzogenrath, Germany). The pH value was measured in cell free supernatant,
To use the RAMOS technology, special RAMOS shake using a HI2211 Basic pH/ Redox/°C Meter (Hanna
flasks were applied that allow for active aeration with Instruments, Vöhringen, Germany). The osmolality was
pressurized air. The bottom area of the shake flask that is measured with an Osmomat 3000 basic (Gonotec GmbH,
in contact with the culture broth, is identical in its geometry Berlin, Germany). Prior to the measurements, the device
to conventional shake flasks. Cultivations were performed was three-point calibrated, using calibration standards (500
in 250 mL shake flasks, in an orbital climo-shaker ISFX-1 and 850 mOsmol/kg) and deionized water.
from A. Kühner AG. Filling volume, shaking frequency,
temperature and number of replicates were varied and are 2.7. Determination of GFP fluorescence
indicated in the caption of the respective experiment. The If GFP was offline determined, a Multi-Detection Microplate
shaking diameter was constant at 50 mm. Reader (Synergy 4; BioTek, Winooski, VT, USA) was used.
For the measurement, samples were centrifuged (10 min,
2.5. BioLector cultivations 4,000 rpm) and 100 μL of cell-free culture supernatant
Two BioLector systems were used for cultivations. If were transferred to a black 96-well flat bottom plate (Type
dissolved oxygen tension (DOT) and pH were online No. 353219, Corning Incorporated Life Science, Tewksbury,
monitored, a commercially available BioLector system by MA, USA). From each biological sample, 2 technical
m2p-labs GmbH (Baesweiler, Germany) was used with replicates were measured. Medium, which was used for the
round, 48-well, deep-well micro titer plates containing corresponding cultivation, was used as a reference blank.
optodes (m2p-labs GmbH) in each individual well. The The excitation wavelength was set to 488 nm and the
MTP was sealed with a gas-permeable foil (Aeraseal Film; emission wavelength was 520 nm. GFP formation was also
Sigma-Aldrich, Burlington, MA, USA) to reduce evaporation measured online in both BioLector systems, with an
and prevent cross contamination. When the OTR was excitation wavelength set to 488 nm and the emission
online monitored within each individual well, an in house wavelength set to 520 nm.
built µRAMOS device, in combination with the BioLector
technology, was utilized [36,37]. For μRAMOS cultivations,
MTPs were sealed with a pierced polyolefin sealing foil 3. Results and Discussion
(HJ-Bioanalytik GmbH, Erkelenz, Germany).
All BioLector cultivations were conducted in round, 48- 3.1. Characterization of a standard complex media
well, deep-well microtiterplates (m2p-labs GmbH) with a screening
filling volume of 0.8 mL, a shaking frequency of 1,000 To gain deeper insight into this commonly used screening
rpm, a shaking diameter of 3 mm, at 30°C and a relative procedure, a GFP secreting P. pastoris strain was cultivated
humidity higher than 80%. The number of replicates is in YPD medium in 250 mL shake flasks. The OTR was
indicated in the caption of the respective experiment. online monitored using the RAMOS device [34]. The P.
576 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

Fig. 1. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec- Fig. 2. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec-
GFP) in shake flasks with YPD medium and step-wise methanol GFP) in shake flasks with YPD medium and early methanol
induction and online monitoring of the oxygen transfer rate induction and online-monitoring of the oxygen transfer rate
(OTR). Screening procedure adapted from Weis et al. [16]. The (OTR). Screening procedure is shortened as methanol is already
OTR (light blue line) is depicted for a cultivation with 10 g/L added after the end of the batch phase on glucose. The OTR (light
glucose, theoretical OTRmax 68.0 mmol/L/h according to Meier et blue line) is depicted for a cultivation with 10 g/L glucose,
al. [27] (A) or 20 g/L glucose, OTRmax 66.4 mmol/L/h according theoretical OTRmax 68.0 mmol/L/h according to Meier et al. [27]
to Meier et al. [27] (B) 0.5% v/v of methanol was added after (A) or 20 g/L glucose, OTRmax 66.4 mmol/L/h according to Meier
60 h, 70 h, 82 h, and 106 h (orange arrows). OTR duplicates et al. [27] (B) 0.5% v/v of methanol was added after 23 h, 33 h,
shown in Fig. S1. (C) and (D) depict corresponding offline 46 h, and 70 h (orange arrows). OTR duplicates shown in Fig. S2.
samples for glucose (pink triangles), methanol (orange diamonds), (C) and (D) depict corresponding offline samples for glucose
cell dry weight (brown circles), pH value (red crosses) and GFP (pink triangles), methanol (orange diamonds), cell dry weight
fluorescence (green stars). Offline samples N = 2, GFP fluorescence (brown circles), pH value (red crosses) and GFP fluorescence
N = 5. Orange box highlights a methanol concentration range of (green stars). Offline samples N = 2, GFP fluorescence N = 5.
0.5-2% (v/v). Open orange diamonds indicate the calculated Orange box highlights a methanol concentration range of 0.5-2%
methanol concentration after each pulse. Data are depicted for (v/v). Open orange diamonds indicate the calculated methanol
10 g/L glucose (C) and 20 g/L glucose (D). Cells with an initial concentration after each pulse. Data are depicted for 10 g/L
optical density (OD600nm) of 0.2 were cultured at 28°C in YPD glucose (C) and 20 g/L glucose (D). Cells with an initial optical
medium (pH = 6.0) with 10 g/L glucose (A and C) or 20 g/L density (OD600nm) of 0.2 were cultured at 28°C in YPD medium
glucose (B and D). Cultivation conditions: 250 mL unbaffled (pH = 6.0) with 10 g/L glucose (A and C) or 20 g/L glucose (B
shake flasks, 12 mL filling volume, 340 rpm shaking frequency, and D). Cultivation conditions: 250 mL unbaffled shake flasks,
50 mm shaking diameter. OTRmax: maximum oxygen transfer 12 mL filling volume, 340 rpm shaking frequency, 50 mm shaking
capacity, GFP: green fluorescent protein. diameter. OTRmax: maximum oxygen transfer capacity, GFP: green
fluorescent protein.

pastoris MutS strain was chosen, as it shows hardly any investigated using 10 or 20 g/L glucose (Fig. 1). Fig. 1A
growth on methanol, but methanol mainly functions as displays OTR and Fig. 1C shows the corresponding course
inducer for heterologous gene expression [38]. The maximum of CDW concentration using 10 g/L glucose. The screening
methanol uptake rate is not affected within the range of process starts with growth on glucose only and no
0.3% v/v to 3% v/v [39]. Therefore, we aimed to maintain methanol. This initial growth phase lasts from 0 to 11 h and
the methanol concentration within a range of 0.5% v/v to OTR and CDW increase exponentially, indicating unlimited
2% v/v to maintain inducing conditions. This concentration growth of the P. pastoris strain. The OTR reaches a
range is marked by an orange box in Figs. 1-4. GFP was maximum of 41 mmol/L/h before declining sharply due to
chosen as the model protein, as it can be easily detected the depletion of glucose. Depletion of glucose is validated
offline and, more importantly, online during BioLector by offline HPLC analysis (Fig. 1C). After glucose is
cultivations [40]. Parallel cultivations in conventional shake entirely consumed, breathing activity is still detectable
flasks were used for offline sampling to determine cell dry between 11 h and 25 h. During this cultivation period, the
weight (CDW), GFP fluorescence in the supernatant, pH CDW further increases from 7.8 g/L after 11 h to 10 g/L
value, glucose, and methanol concentrations over the time after 25 h, likely due to the metabolization of peptides and
course of the cultivation. As it was previously reported that amino acids present in the complex YPD medium.
glucose concentrations higher than 10 g/L negatively Moreover, no ethanol was formed or detected during this
impact production and cell viability [16], the protocol was period. This assumption is supported by the course of the
Auto-induction Medium for P. pastoris MutS Srains 577

Fig. 3. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec- Fig. 4. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec-
GFP) in shake flasks in SYN6 mineral medium. Induction with a GFP) in shake flasks with auto-indction using methanol (2% v/v)
concentrated (2% v/v) methanol pulse and online monitoring of in a mineral medium. The OTR (light blue line) is depicted for a
the oxygen transfer rate (OTR). The OTR (light blue line) is cultivation with 10 g/L glucose, theoretical OTRmax 49.0 mmol/L/h
depicted for a cultivation with 10 g/L glucose, theoretical OTRmax according to Meier et al. [27] (A). At the beginning (t = 0 h), 2 %
49.0 mmol/L/h according to Meier et al. [27] (A). When glucose (v/v) methanol was added. (B) depicts the corresponding offline
was depleted after 22 h, 2% (v/v) of methanol was added (orange samples for glucose (pink triangles), methanol (orange diamonds),
arrow). (B) depicts the corresponding offline samples for glucose cell dry weight (brown circles), pH value (red crosses) and GFP
(pink triangles), methanol (orange diamonds), cell dry weight fluorescence (green stars). Offline samples N = 2, GFP fluorescence
(brown circles), pH value (red crosses), and GFP fluorescence N = 3. Orange box highlights a methanol concentration range of
(green stars). Offline samples N = 2, GFP fluorescence N = 3. 0.5-2% (v/v). Glucose depletion after 12 h is estimated from the
Orange box highlights a methanol concentration range of 0.5-2% course of the OTR, as explained in Fig. 3. Cells with an initial
(v/v). Open orange diamonds indicate the calculated methanol optical density (OD600nm) of 0.2 were cultured at 30°C in mineral
concentration after the pulse. Glucose depletion is estimated from SYN6 medium with 200 mM MES buffer (pH = 6.0) and 10 g/L
the OTR batch peak and the time point is marked with an open glucose. Cultivation conditions: 250 mL unbaffled shake flasks,
triangle connected with a dashed line to the initially measured 12 mL filling volume, 300 rpm shaking frequency, 50 mm shaking
concentration. Cells with an initial optical density (OD600nm) of diameter. OTR: oxygen transfer rate, OTRmax: maximum oxygen
0.2 were cultured at 30°C in mineral SYN6 medium with 200 mM transfer capacity, GFP: green fluorescent protein.
MES buffer (pH = 6.0) and 10 g/L glucose. Cultivation conditions:
250 mL unbaffled shake flasks, 12 mL filling volume, 300 rpm
shaking frequency (reduced, compared to previous cultivations, as result of the strain’s MutS phenotype, which leads to slow
oxygen supply is not limited), 50 mm shaking diameter. OTRmax: methanol consumption, and lower oxygen demand [38].
maximum oxygen transfer capacity, GFP: green fluorescent protein.
Follow-up methanol additions of 60 µL were applied after
70 h, 82 h, and 106 h (indicated with orange arrows in
pH value. During the first 11 h of cultivation, the pH Fig. 1A). Over the period of the methanol pulses, a steadily
decreases to a value of 5 (Fig. 1C) due to the consumption decreasing OTR plateau emerges. The last pulse after
of glucose and ammonia [41,42]. When glucose is depleted, 106 h leads to a final OTR increase from 3.7 mmol/L/h to
the cells start to metabolize proteins and amino acids, which 7 mmol/L/h. It is followed by a slow decrease to 1.7 mmol/L/h,
leads to a pH increase, as ammonia is released. As a result, when the cultivation was terminated after 130 h. The
the final pH value for the 10 g/L cultivation (Fig. 1C) is 7.3. methanol concentration of the culture supernatant and in
Between 25 h and 60 h no metabolic activity is indicated the supplemented medium were measured via HPLC before
by the OTR signal and the offline data for CDW and GFP each addition. With this information, the concentration in
fluorescence also remain constant. After 60 h, GFP the culture broth after addition of the pulse was calculated.
expression is induced by the addition of 60 µL methanol, The calculated data points are illustrated as open diamond
resulting in a concentration of 0.5% v/v methanol in the symbols (Fig. 1C) while the filled diamond symbols
culture broth. The P. pastoris cells react almost immediately represent the measured concentration right before methanol
to the presence of methanol, indicated by the rise of the pulsing. The orange box highlights a methanol concentration
OTR to 13 mmol/L/h, where the signal plateaus. This is a range from 0.5-2% (v/v), as this is considered to be an
578 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

ideal concentration range to maintain inducing conditions. inducing conditions throughout the methanol pulsing phase
A decrease in methanol concentration between each pulse (60 h to 130 h). However, it also shows that the use of
can be clearly observed, which indicates methanol complex YPD medium leads to significantly changing pH
consumption. This observation is supported by the OTR values as no buffer is used. The pH values vary in the
data, showing that the oxygen is continuously consumed range from 4.8 to 7.3 and seemingly do not affect growth
during the methanol addition phase (60 h to 130 h). The or production. This finding is not surprising as P. pastoris
methanol concentrations never reaches zero and the constant is reported to be able to grow within a broad pH spectrum
presence of methanol leads to a continuous induction that between 3.3 to 7 [43]. Nevertheless, this phenomenon
results in a constant increase of the GFP fluorescence should be considered when screening clonal libraries, as
(Fig. 1C). metabolism and gene expression can be affected by pH
The OTR curve throughout the cultivation of the 20 g/L value variations [44]. In this case, GFP fluorescence is not
glucose experiment shows a similar trend to that observed affected within the observed pH value range. However, it is
using 10 g/L (Fig. 1B). This cultivation only differs in the reported that the GFP conformation changes, if the pH
amount of initial glucose. Replicates for the OTR drops below 4 or if it rises higher than 12, which would
measurements with and without methanol are included in affect the absorption spectrum [45]. The screening strategy
as supplementary data (Fig. S1 and S2). The GFP production shown above suggests methanol induction at a fixed time
was also induced via methanol addition after 60 h. During point after 60 h. On- and offline data (Fig. 1A and 1C)
growth on glucose, the OTR rises to a maximum of clearly show that growth on glucose already stops after
32 mmol/L/h after 12 h. Then, the OTR declines to 25 h (10 g/L glucose) or 30 h (20 g/L glucose). As a result,
21 mmol/L/h at 14 h and rises again to 50 mmol/L/h at the cells are in a starvation phase for 35 or 30 h, where they
17 h. The offline data shows that glucose was already show no breathing activity. Interestingly, using 20 g/L
depleted after 12 h (Fig. 1D), suggesting the second OTR glucose leads to ethanol formation (Fig. S3) and a slower
peak results from the consumption of previously formed increase in the OTR upon methanol addition (Fig. 1B)
ethanol. The formation of ethanol, acetate and arabitol compared to the 10 g/L glucose cultivation, where no
under aerobic high glucose conditions has already been ethanol was formed (Fig. 1A). The slower OTR increase
described [4,26]. It can be confirmed here via HPLC indicates a prolonged methanol adaption phase, which is
measurements as well as by the calculated respiratory supported by the offline measured methanol concentration
quotient (RQ) value corresponding to this experiment with (Fig. 1D). Even though ethanol is already consumed at the
20 g/L of glucose (Fig. S3). The applied operation conditions time point of induction, between 60 h and 70 h (Fig. S3),
ensured non-limiting oxygen supply as the theoretical almost no methanol is consumed and GFP fluorescence
maximum oxygen transfer capacity for 10 g/L glucose and does not increase. Only after the second methanol addition
for 20 g/L glucose were 68 mmol/L/h and 66 mmol/L/h, at 70 h, the course of the methanol concentration shows
respectively. These theoretical values were calculated methanol consumption and an increase in GFP fluorescence.
according to Meier et al. [25] and imply that the observed A possible explanation is that ethanol represses methanol-
ethanol formation occurs due to overflow metabolism on induced genes and, thereby, the AOX1 promoter [46,47].
excess glucose and not as a result of oxygen limitation. The observed differences between 10 g/L and 20 g/L
The course of the pH value in the first cultivation phase glucose are in accordance with Weis et al. [16]. This study
is also similar to the pH value of the 10 g/L glucose also described that the highest reporter protein activity was
experiment, but a slightly more acidic pH value of 4.5 is achieved using 10 g/L glucose. As insufficient oxygen
observed at the end of the batch phase after 14 h (Fig. 1D). supply can be excluded as a reason for the differences in
In the induction phase, after methanol addition at 60 h, the this study, it is likely that the ethanol formation has an
OTR shows a flat descent to 8 mmol/L/h until the next impact on the whole methanol metabolization pathway and
methanol pulse is added after 70 h. Methanol concentration its regulatory system [46,47]. Therefore, we conclude that
decreases only by 0.5 g/L until the second pulse is added. it is crucial to prevent ethanol formation in order to achieve
Moreover, no increase in GFP signal can be detected until full induction conditions and online monitoring systems
after the second pulse, when fluorescence constantly help determine the most suitable time of induction.
increases until the end of the cultivation. As during the observed starvation phase (lasting 35 or
The protocol tested here proposes a growth phase in 30 h), no GFP is produced, the overall screening procedure
methanol-free medium with glucose as carbon source for can be significantly shortened by starting the methanol
biomass production, before inducing protein production by additions once glucose is consumed. Therefore, the same
adding methanol. The on- and offline data (Fig. 1) shows screening procedure was repeated but methanol was added
that the tested screening protocol works and maintains directly after 23 h of cultivation (Fig. 2A), instead of 60 h
Auto-induction Medium for P. pastoris MutS Srains 579

(Fig. 1A). For the 10 g/L cultivation, the course of the OTR complex medium and is included as Fig. S4. The same
was similar to the one observed in Fig. 1A. Importantly, the experiment was performed, using SYN6 medium containing
final GFP fluorescence value was 20% higher than in the 200 mM MES buffer to prevent the pH drop that was
previous cultivation (Fig. 1A and 1C) with a 35 h starvation observed for the cultivations in complex medium (Figs. 1
phase. This indicates that a prolonged starvation phase has and 2). Fig. 3A displays the course of the online monitored
a negative effect on the strain performance. In contrast, the OTR, which exponentially increases to a maximum OTR
cultivation using 20 g/L glucose (Fig. 2B) shows no OTR of 30 mmol/L/h after 11 h of cultivation. Afterwards, the
increase after the first methanol pulse (23 h). Likewise, the OTR drops sharply, indicating that glucose is consumed.
methanol concentration does not decrease between the first This is in accordance with the HPLC data displayed in
(23 h) and second methanol pulses (33 h). The second Fig. 3B. In comparison to the pulsed cultivation carried out
pulse led to a slow OTR increase with a decrease in in complex medium (Figs. 1A and 2A), the maximum
methanol concentration, but only until the third pulse was OTR is at a lower level even though the same amount of
injected after 46 h. From there onwards, the OTR ranged initial glucose was used. This can be traced back to the fact
between 7 and 10 mmol/L/h until the cultivation was that in mineral medium the organisms need to synthesize
terminated at 96 h. The associated offline analysis of GFP all required amino acids and other essential precursors by
fluorescence shows no increase in fluorescence after the themselves, as no complex compounds are provided by the
first two pulses of methanol (Fig. 2D). An increase in medium. At 21 h, methanol was added as no more breathing
respiration activity and rise of GFP fluorescence can be activity was detected, and a steep OTR increase can be
detected only after the third methanol pulse (46 h). At the observed with a peak at 11 mmol/L/h. A decrease to
end of the cultivation, the observed GFP fluorescence was 7 mmol/L/h is then visible and the OTR slowly increases
half of that corresponding to the cultivation with a 30 h and plateaus at about 10 mmol/L/h. After approximately
starvation phase (Figs. 1D and 2D). 60 h of cultivation time, the OTR starts to decrease and
Comparing the results of the shortened screening protocol after 72 h no more oxygen is consumed, and the culture is
(Fig. 2) to those from the commonly used protocol, with an terminated. The offline analysis of methanol concentration
extended starvation phase prior to methanol induction over time (Fig. 3B) shows a nearly linear decrease after
(Fig. 1), it can be concluded that an earlier induction is addition of the methanol pulse at 21 h. The observed OTR
beneficial for cultivations using 10 g/L glucose. More course correlates to methanol consumption, since the drop
importantly, this shortens the overall procedure by 34 h. in the OTR signal (60 h) occurs at the same time as
This finding is not observed in the cultivations with methanol is depleted. As this study uses the P. pastoris
20 g/L glucose. The observation that GFP production is MutS phenotype, methanol is consumed at a very low rate,
delayed and does not start immediately upon methanol which explains why the OTR shows a plateau; the methanol
addition (Fig. 1B and 1D) is enhanced, when the P. pastoris present in the medium is metabolized at the maximum rate
cells do not experience a starvation phase (Fig. 2B and per cell. The CDW data indicates a steady increase of
2D). This finding is in accordance with literature, where it biomass throughout the entire cultivation. When glucose is
is described that additions of ethanol lead to a delay in consumed after 21 h, the CDW is approximately 6 g/L.
protein production, when gene expression is under the Methanol addition leads to an increase to a final CDW of
control of the AOX1 promoter [46,47]. As a conclusion, the 10 g/L at the end of cultivation (72 h). GFP fluorescence
screening procedure can only be shortened, if 10 g/L of intensity shows a steady increase over cultivation time
glucose is used in the cultivation or by preventing the (Fig. 3B). However, it is worth noting, that the GFP signal
formation of ethanol. The latter could be achieved by already increases prior to induction (Figs. 3B and 4B). A
applying fed-batch cultivation mode during screening, as possible explanation is basal GFP expression. The effect is
this would result in glucose limited conditions [4]. also observed in the BioLector system (Fig. 6), where the
GFP signal is measured online. Furthermore, the GFP
3.2. Comparing methanol addition and auto-induction fluorescence intensity detected in mineral medium was in
in defined mineral medium this case lower, compared to that in YPD complex medium
Based on the results observed in Fig. 1 and Fig. 2, 10 g/L (Figs. 1 and 2). This difference in productivity between
glucose were used, to prevent overflow metabolite formation. mineral and complex medium is often observed, and it is
In order to further simplify the screening procedure, a always strain and product specific. The observation is also
change was made from multiple methanol pulses towards in accordance with the data shown in Fig. S4. GFP
one concentrated methanol addition. Therefore, a concentrated production starts after induction and steadily increases,
methanol pulse was applied to achieve a concentration of leading to similar fluorescence levels as in Fig. 1A. The
2% v/v in the medium. The applicability was also tested in overall GFP production is also higher when compared to
580 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

the results obtained with SYN6 medium. A possible analysis is displayed in Fig. 4B. A similar progression for
explanation is that complex medium contains biosynthetic the OTR curve was observed during methanol consumption
precursors, e.g., peptides and amino acids, which can be in auto-induction medium compared to the cultivation were
directly channeled into anabolic pathways for protein methanol was added after 22 h (Fig. 3). However, it is worth
synthesis. This saves metabolic energy as these precursors noting that the first peak, where glucose is consumed, has
do not need to be newly synthesized by the cell [48]. On its maximum at 23 mmol/L/h, which is lower than the
the contrary, in mineral medium the organism needs to maximum 30 mmol/L/h of the initial methanol-free batch
generate all needed components by itself. Therefore, the phase for the cultivation shown in Fig. 3. The lowered
final product titer may be lower. A benefit of using mineral OTR maximum indicates that growth is negatively affected,
medium during screening is that defined media are if methanol and glucose are present at the same time. This
preferably used at larger scale for production, as complex is not surprising as glucose inhibits the methanol utilization
components often show lot to lot variations that can pathway [52]. Nevertheless, after 14 h, a similar total amount
negatively influence the process [23,49,50]. By using the of oxygen was consumed in both experiments (105 mmol/L
same medium for screening and production, the transferability O2 Fig. 3A and 95 mmol/L O2 Fig. 4A, respectively), and
is facilitated [16]. The transfer to defined SYN6 medium in the reached CDW was also highly comparable (5.6 g/L
combination with one concentrated methanol pulse lead to Fig. 3A and 5.8 g/L Fig. 4A, respectively). The methanol
successful GFP production. However, the cultivation still concentration data showed that for the auto-induction
needed to be interrupted to add methanol, once glucose procedure only 1.5% v/v was left in culture supernatant
was completely consumed. after 22 h (Fig. 4B), whereas 2% v/v methanol was detected
To omit methanol addition to the running cultivation, at the same time point for the single methanol injection
methanol was added at the beginning of the cultivation, to (Fig. 3B). The methanol concentration at the time point of
achieve auto-induction conditions (Fig. 4). Auto-induction induction is, therefore, lower in the auto-induction protocol
media are known for Escherichia coli cultivations [51]. as 0.5% v/v methanol has likely evaporated. Despite this
This approach was also briefly described for P. pastoris variation in methanol concentration, at the end of cultivation
screenings [19,20]. For the auto-induction method developed almost the same GFP intensity was measured for both
in this study, identical conditions as in Fig. 3 were applied. methods. One possible reason for this effect is that the cells
The only difference being that 2% v/v methanol was added in the auto-induction medium (Fig. 4) were able to adapt
to the medium at the beginning of the cultivation. The more quickly to methanol and started GFP production 2 h
OTR is displayed in Fig. 4A and the corresponding offline earlier (20 h) as in the pulsed cultivation (Fig. 3). The

Fig. 5. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec-GFP) in shake flasks using different carbon sources and methanol
induction methods. The OTR for 10 g/L glucose is depicted with auto-induction with 2% v/v methanol at the beginning of the cultivation
(dark blue line) and addition of one methanol pulse (2% v/v) (orange arrow) after 22 h (orange line), when glucose was depleted (A). It
is an overlay of the data depicted in Fig. 3A and Fig. 4A. The respiratory quotient (RQ) (light grey line) is depicted. Dotted black line
marks the average RQ of 1.1 during the batch phase on glucose (Additional equation 2) and the average RQ of 0.63 during the methanol
consumption phase (Additional equation 5). The OTR for 10 g/L glycerol is depicted with auto-induction of 2% v/v methanol at the
beginning of the cultivation (dark blue line) and addition of one methanol pulse (orange arrow) after 15 h (orange line), when glycerol
was depleted (B). The RQ (light grey line) is depicted. Dotted black line marks the average RQ of 0.77 (Additional equation 4) during
the batch phase on glycerol and the average RQ of 0.59 during the methanol consumption phase (Additional equation 5). Cells with an
initial optical density (OD600nm) of 0.2 were cultured at 30°C in mineral SYN6 medium with 200 mM MES buffer (pH = 6.0) and 10 g/L
glucose (A) or 10 g/L glycerol (B). Cultivation conditions: 250 mL unbaffled shake flasks, 10 mL filling volume, 350 rpm shaking
frequency, 50 mm shaking diameter.
Auto-induction Medium for P. pastoris MutS Srains 581

results suggest that adding methanol at the beginning of the terms of protein production, GFP was produced at almost
cultivation is a valid approach to achieve auto-induction in the same intensity.
P. pastoris. The only drawback observed is a negatively
affected growth behavior prior to methanol utilization, the 3.3. A glycerol/methanol mix is more suitable for auto-
effect is emphasized in Fig. 5A. Here, the OTR data from induction conditions
Fig. 3A and Fig. 4A are plotted in the same diagram Glucose as carbon source can lead to the formation of
together with the corresponding RQ value. However, in undesired overflow metabolites (Figs. 1, 2 and Fig. S3).
Furthermore, Weis et al. [16] reported, that glucose
concentrations higher than 10 g/L lead to reduced cell
viability and even cell death and the “Pichia Expression
Kit” (Invitrogen, Waltham, MA, USA) suggests glycerol
containing media for standard screening procedures [11].
The benefit of using glycerol is that ethanol formation is
not detectable in the OTR, even if concentrations up to
20 g/L glycerol are used (Fig. S5). Therefore, the auto-
induction protocol was tested using glycerol as carbon
source.
Cultivations were conducted in mineral SYN6 medium
with 10 g/L glycerol as carbon source (Fig. 5B) and were
compared to 10 g/L glucose cultivations (Fig. 5A). For both
carbon sources, the single methanol pulse method and the
auto-induction method were compared. The direct comparison
of OTR courses for the glucose cultivations shows the
already mentioned difference in the height of the OTR
batch peak, where the auto-induction method leads to a
lower OTR value (Fig. 5A). It is also visible that growth
rate during growth on glucose is reduced if methanol is
Fig. 6. Cultivation of Pichia pastoris (BSYBG11::pBSYAOXsec- already present in the medium, as the slope of the OTR
GFP) in microtiter plates with simultaneous online monitoring the signal differs. The methanol consumption phase shows a
oxygen transfer rate (OTR) and green fluorescent protein (GFP)
fluorescence. Cultivation was carried out with 10 g/L glucose (A) similar trend for both screening methods (glucose and
and (C) and 10 g/L glycerol (B) and (D) as carbon source. OTR glycerol) and the same OTR plateau at approximately
and GFP fluorescence were measured in parallel. (A) OTR (dark 10 mmol/L/h is reached. In comparison, the glycerol
grey line) without methanol addition (N = 2) and OTR (dark blue cultivations reach the same maximum OTR value during
line) with auto-induction of methanol in medium with 10 g/L
glucose (N = 3). For auto-induction, 2% (v/v) of methanol was the batch phase for methanol pulse induction and auto-
added at the beginning (t = 0 h) of the cultivation. (B) OTR (dark induction (Fig. 5B). The OTR course during the methanol
grey line) without methanol addition (N = 2) and OTR (light blue consumption phase, after glycerol depletion, is comparable
line) with auto-induction of methanol in medium with 10 g/L for both induction strategies (Fig. 5B). Besides the measured
glycerol (N = 2). For auto-induction, 2% (v/v) of methanol was
added at the beginning (t = 0 h) of the cultivation. (C) GFP OTR, the RQ is depicted. In Fig. 5A, the average RQ
fluorescence without methanol addition (grey symbols) and auto- during the batch phase on glucose is 1.1 and the average
induction of methanol (green symbols) in medium with 10 g/L RQ during the methanol consumption phase is 0.63. Both
glucose (only every 15th data point is displayed). For auto-
induction, 2% (v/v) of methanol was added at the beginning (t =
values are in accordance with the theoretically expected
0 h) of the cultivation. (D) GFP fluorescence without methanol value (Additional equations 2 and 5). The RQ values for
addition (grey symbols) and auto-induction of methanol (green the cultivations using glycerol (Fig. 5B) are 0.77 during the
symbols) in medium with 10 g/L glycerol (only every 15th data batch phase and 0.59 during the methanol consumption
point is displayed). For auto-induction, 2% (v/v) of methanol was
added at the beginning (t = 0 h) of the cultivation. Dashed vertical phase. These values also correspond to the theoretically
lines mark the end of the not induced growth phase. The dash- expected values [35] (Additional equations 4 and 5). Based
dotted line marks the end of the methanol consumption phase. on these observations, it can be concluded that glycerol is
Cells with an initial optical density (OD600nm) of 0.1 were cultured a more suitable carbon source for the auto-induction
at 30°C in mineral SYN6 medium with 200 mM MES buffer (pH
= 6.0). Cultivation conditions: 48-well round-well microtiter plate, screening method. The initial growth phase before induction
0.8 mL filling volume per well, 1,000 rpm shaking frequency, (Fig. 5B) shows almost identical, maximal OTR values,
3 mm shaking diameter. Cultivation was performed in at least suggesting the glycerol/methanol mix is less detrimental to
duplicates for each condition.
the cells than the glucose/methanol mix.
582 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

3.4. Applying an auto-induction protocol for high-

throughput strain screening
As the auto-induction protocol using mineral SYN6 medium
was shown to be a feasible alternative for protein production
at shake flask scale (Figs. 4 and 5), it was transferred to
MTP scale to evaluate its use for high-throughput screening.
This format also allows the online measurement of biomass
formation and GFP fluorescence when the BioLector
system is used. Additionally, the OTR at MTP scale was
measured with a µRAMOS device, allowing a more
thorough comparison to the results obtained at shake flask
scale (Fig. S6). Fig. 6 shows the comparison between Fig. 7. Screening for best-producing Pichia pastoris (BSYBG11::
pBSYAOXsec-GFP) strain in microtiter plates with online
glucose and glycerol cultivations using the auto-induction monitoring of the green fluorescent protein (GFP) fluorescence
protocol, a non-induced negative control was also included. and comparing methanol induction with a concentrated pulse and
The results for the measured OTR correlate with the data auto-induction. The online-measured GFP fluorescence is depicted
obtained in shake flasks (Fig. 5). for 7 different GFP producing strains (S1 to S7) and a reference
(Ref.) control. GFP production was induced by the addition of 2%
The online measured GFP signal shows, that GFP (v/v) methanol after 12 h (orange arrow) (A). The online measured
production is induced automatically once the primary GFP fluorescence is depicted for the same GFP producing strains
carbon source is depleted (Fig. 6). It is also observed that (S1 to S7) as in (A). GFP production was induced by the addition
higher GFP fluorescence was measured when glycerol was of 2% (v/v) methanol at the beginning of the cultivation (auto-
induction) (B). Cells with an initial optical density (OD600nm) of
used as carbon source (Fig. 6C and 6D). Once methanol is 0.8 were cultured at 30°C in SYN6 mineral medium with
completely consumed, GFP fluorescence also decreases. 200 mM MES buffer (pH = 6.0) and 10 g/L glycerol. Cultivation
The corresponding phases are marked in Fig. 6: The conditions: 48-well round-well microtiter plate, 0.8 mL filling
volume per well, 1,000 rpm shaking frequency, 3 mm shaking
dashed vertical lines mark the end of the initial growth diameter. Each cultivation was performed in at least duplicates.
phase, the cells are not induced. The dash-dotted lines Light shadow depicts the standard deviation.
mark the end of the methanol consumption phase.
The data shows that the auto-induction protocol can
successfully be transferred from shake-flask to MTP scale methanol induction phase on glycerol (42 h) compared to
and similar observations can be made at both scales glucose (48.5 h), as methanol is depleted earlier. Nevertheless,
(Fig. S6). Firstly, the obtained OTR values at MTP scale glycerol leads to higher GFP fluorescence, suggesting
emphasize that using glucose for auto-induction leads to a screening time could be further reduced if glycerol is used
delay in the biomass growth, compared to growth on for auto-induction screening instead of glucose.
glucose without the presence of methanol (Fig. 6A). In The newly developed auto-induction protocol at MTP
contrast, cultivations on glycerol and glycerol with 2% v/ scale was then used for screening a P. pastoris clone bank.
v methanol behave almost identical in the biomass growth Glycerol was selected as the carbon source due to the
phase (Fig. 6B). Furthermore, the online GFP measurements observed higher protein production in the previous
show that GFP expression is initiated as soon as the experiment (Fig. 6D). For the clone bank, the one methanol
P. pastoris cells start to metabolize methanol. This is pulse method was compared to the auto-induction method.
indicated by dashed vertical lines (Fig. 6C and 6D). When The performance of different GFP-secreting P. pastoris
comparing the two cultivations, it becomes apparent that strains was analyzed using the BioLector system and the
using glycerol leads to a higher OTR plateau during course of GFP intensity was online monitored (Fig. 7).
methanol consumption (15 mmol/L/h) compared to using Strain-specific increases of the GFP signal were observed
glucose (10 mmol/L/h). Although, glucose and glycerol after 20 h for both, pulse-mediated induction and auto-
were used in the same C-molar amounts, glycerol leads to induction. The clones S2 and S5 achieved the highest GFP
a higher biomass, as the degree of reduction of glycerol levels, while the lowest GFP level was detected in S1.
(4.67 per carbon) is higher and, thus, more energy-rich than Interestingly, the clones reached different GFP maxima and
glucose (4.0 per carbon; Table S1). The scattered light the time point of the GFP maxima varied over time. For
value, reached after growth on glycerol was completed, example, strain S1 reached its GFP maximum after 31 h
was also 15% higher than the biomass concentration reached compared to strain S2, which achieved its GFP maximum
on glucose (data not shown). This higher initial biomass after 55 h (Fig. 7A). After the maximum GFP fluorescence
prior to methanol consumption consequently leads to the is reached, the signal decreases, as it has been previously
described higher OTR plateau. This also leads to a shorter observed when using this GFP variant [31,32]. As discussed
Auto-induction Medium for P. pastoris MutS Srains 583

earlier, the drop in the GFP signal indicates methanol as carbon source in small-scale cultivation systems. For the
depletion (Fig. 5). Therefore, the length of the GFP first time, online monitoring techniques in shake flask and
production phase and the maximum GFP fluorescence MTP scale were applied in order to shed light into the
correlate with the duration of methanol induction. This metabolic activity of P. pastoris MutS during the screening
suggests that the tested P. pastoris clones not only show process. It is still suggested in literature [16,18] that a long
different GFP production behavior, but also differ in pause between initial growth on glucose and methanol
methanol consumption. It is likely that this effect is related induction is needed to prevent inhibition of the AOX
to gene copy number, integration locus or the orientation of promoter by glucose, glycerol, or the formed overflow
the expression cassette as observed in the study from metabolite, ethanol. Online monitoring technologies allow
Schwarzhans et al. [53]. However, this correlation would the detection of the end of the batch phase, overflow
need to be further investigated. formation and, therefore, the determination of a suitable
The auto-induction method resulted in a similar cluster induction time point that prevents a starvation phase. This
for strain performance compared to the pulsed cultivations. emphasizes the importance of online monitoring the
However, the observed GFP signals were higher for the cultivations already during the primary screening process.
methanol pulsed experiments, compared to auto-induction. We demonstrate that, if filling volume, shaking frequency
Moreover, the ranking between some strains varied slightly and the concentration of glucose are adequately selected,
(e.g., S2 to S5 and S3 to S6). Further analyses such as a auto-induction is an advantageous screening method. It
SDS-PAGE were not performed as no information regarding does not require multiple methanol additions to obtain a
protein functionality can be deduced. On the contrary, reliable primary screening result. The protocol can be
fluorescence detection only shows functional folded proteins, applied in shake flask and MTP format, and the overall
which was the desired outcome of these screening campaigns. screening time can be reduced from 132 h to 72 h of
Additional information for backscatter (biomass), DOT, cultivation using a mineral auto-induction medium with
and GFP signal are displayed in the supplementary data 10 g/L glucose. The protocol was also successfully applied
(Fig. S7). Overall, the data obtained using both methods for strain screening campaigns within a confidential
(auto-induction and one methanol pulse) lead to a similar industrial project. It was also shown that elevated glucose
ranking. However, the experimental effort was significantly concentrations lead to ethanol formation, even when the
reduced using the auto-induction method. This result oxygen supply is sufficient. The formation of ethanol
proves that the presented auto-induction screening method inhibits the AOX1 promoter [47] and in combination with
greatly simplifies primary screening. No interruptions of early methanol pulsing leads to poor screening results, as
the culture are necessary, as methanol additions during the gene expression is delayed [46]. These findings support the
cultivation are not needed. There is no delay between common practice of including a starvation phase, lasting up
initial growth phase and methanol induction, and the total to 3 days prior to induction with methanol [16,18]. The
cultivation time can also be reduced, still resulting in a starvation phase gives the cells enough time to consume
reliable clone ranking. Thus, this method is suitable for the ethanol produced. Therefore, ethanol is not present at
primary screening as it helps to reduce the number of the time point of induction and cannot act as a repressor.
strains that will be re-screened for protein production. The However, these findings also imply that the screening
high performing strains resulting from this auto-induction procedure can be shortened, if the applied operating
method can then be re-screened for reproducibility, and the conditions ensure that no ethanol is formed during the
best selected for cultivations in a 2 L stirred tank reactor to cultivation. This can be achieved by using a maximum
check for same performance in a bioreactor. glucose concentration of 10 g/L or by switching to glycerol
as carbon source. The use of glycerol as carbon source was
demonstrated in the here presented auto-induction medium
4. Conclusion and was successfully applied to a strain screening campaign
that led to a comparable rating of the tested clones regardless
Primary screening is a crucial step during bioprocess of the induction method.
development, as the selection of the best performing strain
has a major impact on the economic feasibility of the final
production process [10]. As P. pastoris is a well-established Acknowledgements
host organism, there are many different screening protocols
available that suggest different cultivation strategies. This The authors thank Dr. Monica Espinosa Gomez for critically
study investigated the effect of using complex or defined revising the manuscript.
mineral media and compared the use of glucose or glycerol
584 Biotechnology and Bioprocess Engineering 27: 572-585 (2022)

Authorsʼ Contributions enables precision genome engineering in the methylotrophic

yeast Pichia pastoris. J. Biotechnol. 235: 139-149.
7. Vogl, T., L. Sturmberger, T. Kickenweiz, R. Wasmayer, C.
DW designed the study, performed the experiments, Schmid, A. M. Hatzl, M. A. Gerstmann, J. Pitzer, M. Wagner, G.
analyzed the data and drafted the manuscript. RLM, LPM, G. Thallinger, M. Geier, and A. Glieder (2016) A toolbox of
and RH participated in the execution of the experiments. diverse promoters related to methanol utilization: functionally
verified parts for heterologous pathway expression in Pichia
AG performed the molecular cloning and provided the
pastoris. ACS Synth. Biol. 5: 172-186.
GFP secreting P. pastoris strains. LMB was also involved 8. Cregg, J. M., J. L. Cereghino, J. Shi, and D. R. Higgins (2000)
in the drafting of the manuscript. JB initiated and Recombinant protein expression in Pichia pastoris. Mol.
supervised the study, participated in data interpretation and Biotechnol. 16: 23-52.
9. Ahmad, M., M. Hirz, H. Pichler, and H. Schwab (2014) Protein
assisted in drafting the manuscript. All authors read and expression in Pichia pastoris: recent achievements and perspectives
approved the final manuscript. for heterologous protein production. Appl. Microbiol. Biotechnol.
98: 5301-5317.
10. Büchs, J. (2001) Introduction to advantages and problems of
shaken cultures. Biochem. Eng. J. 7: 91-98.
Ethical Statements 11. Invitrogen (2010) EasySelectTM Pichia Expression Kit. Manual
Part no. 25-0172. Invitrogen, Carlsbad, CA, USA.
The authors declare that they have no competing interests. 12. Klöckner, W. and J. Büchs (2012) Advances in shaking
Ethics statement and consent to participate are not applicable. technologies. Trends Biotechnol. 30: 307-314.
13. Lattermann, C. and J. Büchs (2015) Microscale and miniscale
fermentation and screening. Curr. Opin. Biotechnol. 35: 1-6.
14. Huber, R., D. Ritter, T. Hering, A. K. Hillmer, F. Kensy, C.
Data Availability Müller, L. Wang, and J. Büchs (2009) Robo-Lector - a novel
platform for automated high-throughput cultivations in microtiter
plates with high information content. Microb. Cell Fact. 8: 42.
The datasets generated and/or analysed during the current 15. Duetz, W. A. (2007) Microtiter plates as mini-bioreactors:
study are available from the corresponding author on miniaturization of fermentation methods. Trends Microbiol. 15:
reasonable request. 469-475.
16. Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A.
Glieder (2004) Reliable high-throughput screening with Pichia
pastoris by limiting yeast cell death phenomena. FEMS Yeast
Electronic Supplementary Material (ESM) Res. 5: 179-189.
17. Koutz, P., G. R. Davis, C. Stillman, K. Barringer, J. Cregg, and G.
Thill (1989) Structural comparison of the Pichia pastoris alcohol
The online version of this article (doi: 10.1007/s12257- oxidase genes. Yeast. 5: 167-177.
022-0006-z) contains supplementary material, which is 18. Boettner, M., B. Prinz, C. Holz, U. Stahl, and C. Lang (2002)
available to authorized users. High-throughput screening for expression of heterologous
proteins in the yeast Pichia pastoris. J. Biotechnol. 99: 51-62.
19. Lee, J. Y., H. Chen, A. Liu, B. M. Alba, and A. C. Lim (2017)
Auto-induction of Pichia pastoris AOX1 promoter for membrane
References protein expression. Protein Expr. Purif. 137: 7-12.
20. Kenzom, T., P. Srivastava, and S. Mishra (2015) Simplified high-
throughput screening of AOX1-expressed laccase enzyme in
1. Yamada, Y., M. Matsuda, K. Maeda, and K. Mikata (1995) The
Pichia pastoris. Anal. Biochem. 489: 59-61.
phylogenetic relationships of methanol-assimilating yeasts based
21. Matthews, C. B., A. Kuo, K. R. Love, and J. C. Love (2018)
on the partial sequences of 18S and 26S ribosomal RNAs: the
Development of a general defined medium for Pichia pastoris.
proposal of Komagataella gen. nov. (Saccharomycetaceae).
Biotechnol. Bioeng. 115: 103-113.
Biosci. Biotechnol. Biochem. 59: 439-444.
22. Holmes, W. J., R. A. Darby, M. D. Wilks, R. Smith, and R. M.
2. Macauley-Patrick, S., M. L. Fazenda, B. McNeil, and L. M.
Bill (2009) Developing a scalable model of recombinant protein
Harvey (2005) Heterologous protein production using the Pichia
yield from Pichia pastoris: the influence of culture conditions,
pastoris expression system. Yeast. 22: 249-270.
biomass and induction regime. Microb. Cell Fact. 8: 35.
3. Cregg, J. M., K. J. Barringer, A. Y. Hessler, and K. R. Madden
23. Diederichs, S., A. Korona, A. Staaden, W. Kroutil, K. Honda, H.
(1985) Pichia pastoris as a host system for transformations. Mol.
Ohtake, and J. Büchs (2014) Phenotyping the quality of complex
Cell. Biol. 5: 3376-3385.
medium components by simple online-monitored shake flask
4. Heyland, J., J. Fu, L. M. Blank, and A. Schmid (2010)
experiments. Microb. Cell Fact. 13: 149.
Quantitative physiology of Pichia pastoris during glucose-
24. Hellwig, S., C. Stöckmann, G. Gellissen, and J. Büchs (2005)
limited high-cell density fed-batch cultivation for recombinant
Comparative fermentation. pp. 287-318. In: G. Gellissen (ed.).
protein production. Biotechnol. Bioeng. 107: 357-368.
Production of Recombinant Proteins: Novel Microbial and
5. Looser, V., B. Bruhlmann, F. Bumbak, C. Stenger, M. Costa, A.
Eukaryotic Expression Systems. Wiley-VCH, Weinheim, Germany.
Camattari, D. Fotiadis, and K. Kovar (2015) Cultivation
25. Gupta, A. and G. Rao (2003) A study of oxygen transfer in shake
strategies to enhance productivity of Pichia pastoris: a review.
flasks using a non-invasive oxygen sensor. Biotechnol. Bioeng.
Biotechnol. Adv. 33: 1177-1193.
84: 351-358.
6. Weninger, A., A. M. Hatzl, C. Schmid, T. Vogl, and A. Glieder
26. Eck, A., M. Schmidt, S. Hamer, A. J. Ruff, J. Förster, U.
(2016) Combinatorial optimization of CRISPR/Cas9 expression
Schwaneberg, L. M. Blank, W. Wiechert, and M. Oldiges (2018)
Auto-induction Medium for P. pastoris MutS Srains 585

Improved microscale cultivation of Pichia pastoris for clonal 871-879.

screening. Fungal Biol. Biotechnol. 5: 8. 40. Su, W. W. (2005) Fluorescent proteins as tools to aid protein
27. Meier, K., W. Klöckner, B. Bonhage, E. Antonov, L. Regestein, production. Microb. Cell Fact. 4: 12.
and J. Büchs (2016) Correlation for the maximum oxygen 41. Kensy, F., E. Zang, C. Faulhammer, R. K. Tan, and J. Büchs
transfer capacity in shake flasks for a wide range of operating (2009) Validation of a high-throughput fermentation system
conditions and for different culture media. Biochem. Eng. J. 109: based on online monitoring of biomass and fluorescence in
228-235. continuously shaken microtiter plates. Microb. Cell Fact. 8: 31.
28. Lattermann, C., M. Funke, S. Hansen, S. Diederichs, and J. 42. Meissner, L., K. Kauffmann, T. Wengeler, H. Mitsunaga, E.
Büchs (2014) Cross-section perimeter is a suitable parameter to Fukusaki, and J. Büchs (2015) Influence of nitrogen source and
describe the effects of different baffle geometries in shaken pH value on undesired poly(γ-glutamic acid) formation of a
microtiter plates. J. Biol. Eng. 8: 18. protease producing Bacillus licheniformis strain. J. Ind. Microbiol.
29. Islam, R. S., D. Tisi, M. S. Levy, and G. J. Lye (2008) Scale-up of Biotechnol. 42: 1203-1215.
Escherichia coli growth and recombinant protein expression 43. Li, P., A. Anumanthan, X. G. Gao, K. Ilangovan, V. V. Suzara, N.
conditions from microwell to laboratory and pilot scale based on Düzgüneş, and V. Renugopalakrishnan (2007) Expression of
matched k(L)a. Biotechnol. Bioeng. 99: 1128-1139. recombinant proteins in Pichia pastoris. Appl. Biochem. Biotechnol.
30. Kensy, F., C. Engelbrecht, and J. Büchs (2009) Scale-up from 142: 105-124.
microtiter plate to laboratory fermenter: evaluation by online 44. Stöckmann, C., U. Maier, T. Anderlei, C. Knocke, G. Gellissen,
monitoring techniques of growth and protein expression in and J. Büchs (2003) The oxygen transfer rate as key parameter
Escherichia coli and Hansenula polymorpha fermentations. for the characterization of Hansenula polymorpha screening
Microb. Cell Fact. 8: 68. cultures. J. Ind. Microbiol. Biotechnol. 30: 613-622.
31. Cormack, B. P., R. H. Valdivia, and S. Falkow (1996) FACS- 45. Ward, W. W. and S. H. Bokman (1982) Reversible denaturation
optimized mutants of the green fluorescent protein (GFP). Gene. of Aequorea green-fluorescent protein: physical separation and
173: 33-38. characterization of the renatured protein. Biochemistry. 21: 4535-
32. Balleza, E., J. M. Kim, and P. Cluzel (2018) Systematic 4540.
characterization of maturation time of fluorescent proteins in 46. Inan, M. and M. M. Meagher (2001) The effect of ethanol and
living cells. Nat. Methods. 15: 47-51. acetate on protein expression in Pichia pastoris. J. Biosci.
33. Lin-Cereghino, J., W. W. Wong, S. Xiong, W. Giang, L. T. Bioeng. 92: 337-341.
Luong, J. Vu, S. D. Johnson, and G. P. Lin-Cereghino (2005) 47. Ohsawa, S., S. Nishida, M. Oku, Y. Sakai, and H. Yurimoto
Condensed protocol for competent cell preparation and (2018) Ethanol represses the expression of methanol-inducible
transformation of the methylotrophic yeast Pichia pastoris. genes via acetyl-CoA synthesis in the yeast Komagataella
Biotechniques. 38: 44-48. phaffii. Sci. Rep. 8: 18051.
34. Anderlei, T. and J. Büchs (2001) Device for sterile online 48. Hahn-Hägerdal, B., K. Karhumaa, C. U. Larsson, M. Gorwa-
measurement of the oxygen transfer rate in shaking flasks. Grauslund, J. Görgens, and W. H. van Zyl (2005) Role of
Biochem. Eng. J. 7: 157-162. cultivation media in the development of yeast strains for large
35. Anderlei, T., W. Zang, M. Papaspyrou, and J. Büchs (2004) scale industrial use. Microb. Cell Fact. 4: 31.
Online respiration activity measurement (OTR, CTR, RQ) in 49. Zhang, J., J. Reddy, B. Buckland, and R. Greasham (2003)
shake flasks. Biochem. Eng. J. 17: 187-194. Toward consistent and productive complex media for industrial
36. Flitsch, D., S. Krabbe, T. Ladner, M. Beckers, J. Schilling, S. fermentations: studies on yeast extract for a recombinant yeast
Mahr, U. Conrath, W. K. Schomburg, and J. Büchs (2016) fermentation process. Biotechnol. Bioeng. 82: 640-652.
Respiration activity monitoring system for any individual well of 50. Potvin, J., E. Fonchy, J. Conway, and C. P. Champagne (1997)
a 48-well microtiter plate. J. Biol. Eng. 10: 14. An automatic turbidimetric method to screen yeast extracts as
37. Ladner, T., M. Held, D. Flitsch, M. Beckers, and J. Büchs (2016) fermentation nutrient ingredients. J. Microbiol. Methods. 29:
Quasi-continuous parallel online scattered light, fluorescence and 153-160.
dissolved oxygen tension measurement combined with monitoring 51. Studier, F. W. (2005) Protein production by auto-induction in
of the oxygen transfer rate in each well of a shaken microtiter high density shaking cultures. Protein Expr. Purif. 41: 207-234.
plate. Microb. Cell Fact. 15: 206. 52. Hartner, F. S. and A. Glieder (2006) Regulation of methanol
38. Cereghino, J. L. and J. M. Cregg (2000) Heterologous protein utilisation pathway genes in yeasts. Microb. Cell Fact. 5: 39.
expression in the methylotrophic yeast Pichia pastoris. FEMS 53. Schwarzhans, J. P., D. Wibberg, A. Winkler, T. Luttermann, J.
Microbiol. Rev. 24: 45-66. Kalinowski, and K. Friehs (2016) Integration event induced
39. Khatri, N. K. and F. Hoffmann (2006) Impact of methanol changes in recombinant protein productivity in Pichia pastoris
concentration on secreted protein production in oxygen-limited discovered by whole genome sequencing and derived vector
cultures of recombinant Pichia pastoris. Biotechnol. Bioeng. 93: optimization. Microb. Cell Fact. 15: 84.

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