Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

Article abstract of DOI:10.1186/s12934-021-01577-4

Background: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. Results: A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high μ showed highest production rates, while methanol co-addition together with a decrease of μ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen. Conclusions: For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Full text of DOI:10.1186/s12934-021-01577-4

et al. Microb Cell Fact
https://doi.org/10.1186/s12934‑021‑01577‑4
(2021) 20:90
Garrigós‑Martínez
Microbial Cell Factories
Open Access
RESEARCH
Scalable production and application
of Pichia pastoris whole cell catalysts expressing
human cytochrome P450 2C9
Javier Garrigós‑Martínez1, Astrid Weninger2, José Luis Montesinos‑Seguí1, Christian Schmid2, Francisco Valero1,
Claudia Rinnofner3,4, Anton Glieder2,3* and Xavier Garcia‑Ortega1
Abstract
Background: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the
recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the
drug development process by simple, quick and scalable access of human drug metabolites is key for efcient and
targeted drug development in response to new and sometimes unexpected medical challenges and needs. However,
due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotrans‑
formations was still in its infancy.
Results: A scalable bioprocess for fne‑tuned co‑expression of hCYP2C9 and its essential complementary human
cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phafi) is presented. High‑throughput
screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with diferent
regulation patterns and a fuorimetric assay was used in order to fne‑tune hCYP2C9 and hCPR co‑expression, and to
identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst
production in bioreactors was carried out based on rational optimization criteria. Among the diferent alternatives
studied, a glycerol carbon‑limiting strategy at high µ showed highest production rates, while methanol co‑addition
together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity.
By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could
be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated
successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the
human enzyme catalyst for 3′hydroxyibuprofen.
Conclusions: For the frst time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was suc‑
cessfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative‑scale biotrans‑
formation of interest. The catalyst engineering procedure demonstrated the efciency of the employment of a set of
diferently regulated bidirectional promoters to identify transformants with most efective membrane‑bound hCYP/
hCPR co‑expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts
relying on co‑expressed enzymes such as other P450 catalysts or enzymes relying on co‑expressed enzymes for co‑
factor regeneration.
*Correspondence: a.glieder@tugraz.at
2 Institute of Molecular Biotechnology, Graz University of Technology,
Petersgasse 14, 8010 Graz, Austria
Full list of author information is available at the end of the article
© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
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is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
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Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 2 of 13
Keywords: Bidirectional promoters, Human CYP2C9, Ibuprofen, Pichia pastoris, Bioprocess optimisation, Whole cell
biocatalyst
associated hP450s seems preferable compared to solu-
ble expression of truncated P450s. Furthermore, to be
biologically active, CYPs require an electron transport
system which provides the electrons to the CYPs for oxy-
gen activation and substrate oxidation [15]. hCYPs rely
on the presence of a cytochrome P450 reductase (CPR),
which is needed for electron transfer from the co-factor
NAD(P)H. Te co-expression of hCPR often showed to
be more efective than taking advantage of the host’s own
CPR(s) [7].
Background
In order to avoid adverse drug reactions (ADRs) not only
the drug as such, but also the impact of its metabolites
on the organism are considered of great importance [1].
From the super-family of P450 enzymes from diferent
origins, Human cytochrome P450s (hCYPs) are a group
of heme-containing membrane-associated monooxy-
genases involved in the oxidation of monoterpenes,
saturated fatty acids and alkanes, vitamins, steroids and
eicosanoids as well as in the clearance of drugs and xeno-
biotics in humans [2, 3]. Among the human variants,
next to CYP3A4 and CYP2D6, CYP2C9 is one of the
most important drug oxidizing enzyme [4]. In particu-
lar, CYP2C9 oxidizes numerous drugs such as celecoxib,
diclofenac, etodolac, ibuprofen, indomethacin, lornoxi-
cam, mefenamic acid, suprofen, tenoxicam and, addition-
ally, also metabolizes endogenous compounds such as
arachidonic acid, linoleic acid, and non-drug xenobiotics
[4].
During the last years, to enable industrial biotransfor-
mations that require high conversion rates, easy han-
dling and low costs, the recombinant production of CYP
enzymes has been intensively investigated [6, 10, 12,
16–19]. Some relevant publications have reported several
cases for the recombinant expression of human P450s
in diferent cell factories [3, 20]. Specifcally, success-
ful expression of hCYP2C9 has been reported for insect
cells [21], Escherichia coli [22] and Schizosaccharomyces
pombe [23]. Tus, this enzyme was used for the prepara-
tion of milligram amounts of the 4′-hydroxy metabolite
of diclofenac using whole cells or isolated membranes
[22]. Te use of yeast expression systems as whole cell
biocatalysts was described by Neunzig et al., who demon-
strated the applicability of an ibuprofen conversion pro-
cess using fssion yeast for the co-expression of hCYP2C9
and CPR [24, 25], and efcient and selective 4′-hydroxy
diclofenac was synthesized on g-scale by the Bureik lab
[23]. In a comparative study Geier et al. highlighted the
methylotrophic yeast Pichia pastoris, recently re-classi-
fed as Komagataella phafi, as a preferable cell factory
for whole cell bioconversions using human CYP2D6 [7].
Due to those experiences and the broad availability and
establishment of the P. pastoris expression system in
thousands of labs, we were interested to study the relia-
bility, scalability and performance of P. pastoris whole cell
catalysts expressing hCYP2C9.
Drugs and drug-metabolite standards are required
for toxicological, biological and drug-to-drug interac-
tion studies. Teir synthesis is often challenging, since
selective hydroxylation of non-activated carbon atoms
is difcult to achieve by chemical synthesis. Tese stud-
ies are elucidated by the employment of tissue samples
(e.g. human liver homogenate) or microsomal prepara-
tions thereof [5–7]. Both are not scalable for preparative
synthesis on multi mg scale. Microbial P450 enzymes
provide an interesting alternative but frequently do not
produce the desired specifc products, or undesired mix-
tures which are diferent from the product spectrum
made by human P450s are obtained [6, 8–10]. Adaptation
by protein engineering is possible but in cases of unex-
pected arising needs for new drugs such as arising epi-
demic outbreaks, but such approach is usually too slow.
Terefore, based on their versatile and changing applica-
tions in drug metabolite synthesis, there is an increasing
interest and high demand for hCYPs in both pharmaceu-
tical and chemical industries, especially when applicable
on a preparative scale level [3, 11]. In this sense, biotrans-
formations performed with purifed enzymes are hardly
feasible on a large scale, since hCYPs usually present low
efciency and stability [11, 12]. To some extend this is
also related to the fact the hCYP’s are membrane asso-
ciated, so they can be considered as membrane proteins.
However, selective access of some substrates to the active
site is known to rely on the interface between enzyme
and the ER membrane [13, 14], expression of membrane
Pichia pastoris is currently considered as an efficient
alternative for recombinant protein production com-
bining the simplicity of bacterial expression systems
with some essential features of higher eukaryotic hosts
such as mammalian cells. Most of membrane pro-
teins (MPs) of interest such as CYPs often come from
eukaryotic organisms, thus bacterial expression sys-
tems often fail to express such genes to get biologically
active and corrected folded enzymes. This fact has
been described to be due to the lack capacity to per-
form post-translational modifications (e.g. disulphide

Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 3 of 13
bridge formation or glycosylation), the inefficient
secretory pathways needed to direct the protein to the
membrane, as well as to the differences in lipid bilayer
composition [26, 27] and availability of different intra-
cellular compartments. On the other hand, mamma-
lian expression systems are able to produce complex
proteins, and especially MPs [27]. However, their cell
growth is significantly slower and more expensive
compared to microbial cells. Thus, on the basis of its
high efficiency, wide availability and cost-effectiveness,
P. pastoris has become one of the most frequently used
host systems for the production recombinant proteins,
including membrane-anchored and soluble proteins—
either intra or extracellular [27]. Usually, most pub-
lished studies focused on the recombinant expression
of MPs address issues related to protein degradation,
ER folding problems and post-translational modifica-
tions in order to increase final product titers, since it
may have a relevant impact on its production [28, 29].
However, to the date, there has not been reported any
study that assess the impact of operational strategies
in the recombinant production of MPs targeted to be
anchored in the cell membrane. So, as a highly relevant
factor to design efficient scalable processes, this work
aimed to determine the interrelation between the spe-
cific production rate (qp) and the specific growth rate
(µ) of the culture for active hP450 catalyst production
and biotransformations in bioreactors. This relation-
ship is called production kinetics, and can be consid-
ered the key to design optimal bioprocess strategies for
recombinant protein production (RPP) [30–36].
Results and discussion
Strain generation
Expression systems based on engineered bidirectional
promoters ofer a simple and quick solution to enable
multi-gene co-expression in which the expression of
each gene can be optimally tuned towards to achieve the
desired objective [37]. To fully exploit the potential of
this bidirectional expression system, a test set of 7 alter-
native promoters with diverse regulation patterns was
used in diferent co-orientations to drive the expression
of the full-length genes: CYP2C9 (AL359672) including
its hydrophobic N-terminal sequence and redox partner
hCPR (accession AAH34277.1). In contrast to self-suf-
cient soluble bacterial P450s, here the CPR is covalently
linked to the heme domain of the P450 catalyst, thus dif-
ferent co-expression levels and regulatory profles were
evaluated in order to achieve the best performance of the
whole cell biocatalyst requiring two separate functionally
expressed peptide chains, anchored to the membrane in
optimal orientation and proximity to its redox partner.
Tis study included the strong methanol inducible pro-
moters PDAS1 and PDAS2 (and their shortened variants
P
DAS1-552 and PDAS2-699), as well as the promoters PPDC and
PDF, two derepressed promoters in which the recombi-
P
nant expression is activated upon glucose/glycerol deple-
tion, and additionally it is further inducible in presence
of methanol. In earlier studies using GFP as a model pro-
tein, PDAS1 presented a strong methanol-inducible per-
formance, reaching at least half of the expression level
of the commonly used AOX1 promoter, while PDAS2 even
outperformed PAOX1 expression [38]. On the other hand,
the expression of the PPDC promoter (native catalase gene
promoter) is repressed in the presence of glucose or glyc-
erol and derepressed upon the depletion of these carbon
sources. Its expression can further be induced with meth-
anol and remarkably also with oleic acid [39]. Besides the
The aim of this work was to demonstrate a model
case for the production of highly active and selective
CYP2C9/CPR whole cell biocatalysts using the sim-
ple, widely available and efficient expression system
P. pastoris. It takes into consideration the essential
scalability required for catalyst manufacturing by the
design and implementation of a reliable and optimized
cultivation process for whole cell catalyst production.
Therefore, in a complementary approach, the cata-
lyst expression bioprocess was optimized by evaluat-
ing different co-expression ratios of hCYP and hCPR
using a bidirectional and functionally balanced expres-
sion system and also further combined with a ration-
ally designed bioprocess at bench-top bioreactor scale
taking into consideration the relevant impact of pH,
specific growth rate and the use of methanol as co-
substrate on whole cell biocatalyst production. The
efficiency of such produced whole cell catalysts was
demonstrated by preparative scale biotransformation
(0.5 L) of ibuprofen to produce the main hCYP2C9
ibuprofen metabolite 3-hydroxyibuprofen.
PPDC, an orthologous promoter, i.e. PPDF, which exhibits
a similar regulation profle, was employed to regulate
gene transcription. Tese promoters, which allow difer-
ent levels of induction, enable to some extend to uncou-
ple recombinant protein expression and cell growth for P.
pastoris and therefore become interesting tools for bio-
process optimization.
Earlier studies reported the efciency of CYP2D6 in P.
pastoris whole cells, which was detectable by an spectros-
copy method based on carbon monoxide (CO) diference
[7]. However, highest CYP levels determined by CO-
spectroscopy could not be correlated to highest conver-
sions levels when using whole cells. Working with whole
cells, their conversion efciencies are infuenced by elec-
tron and substrate supply [7], and it also afected by the
complex interactions of multiple parameters (e.g. CYP/
CPR ratio, membrane permeability, NADH production/

Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 4 of 13
regeneration or expression level of ROS degrading
enzymes between among others) [40]. Tus, this work
is focused on the achievement of active whole cells with
optimized CYP/CPR ratios rather than a maximization
of P450 as determined by carbon monoxide spectroscopy
following a previously described bidirectional expression
strategy [37].
For the identifcation of the catalyst with the highest
activity, two alternative whole cell bioconversions assays
suitable for High Troughput Screening (HTS) were used
[41]. One based on diclofenac conversion, and the other
one based on the fuorescence of the product 7-hydroxy-
4-(trifuoromethyl)-2H-chromen-2-one (HFC). Both
analysis methods were used for comparing the best
clone’s performance of each construct, as shown in Fig. 1.
Te qualitative behaviour of both screening methods was
rather similar, construct 2C9-PDC/PDF-CPR showed
the best conversion (53%) in the diclofenac-based assay
(2 mM). Te same construct with inverted promoter ori-
entation 2C9-PDF/PDC-CPR, also presented good sub-
strate conversion reaching 39%, while all other constructs
showed only 20% conversion of diclofenac, or even lower.
Similar results were obtained using the alternative fuo-
rometric MCF-based assay, demonstrating the applicabil-
ity of this simple assay for the identifcation of cells with
highest P450 activity in simple 96-deep well plate assays
with low cell densities cultures. As for the diclofenac
screening, the expression system built on PPDF/PPDC com-
bination provided the best results, reaching a maximum
value of 28 RFU per OD600. However, due to the low
sample numbers the activity of samples obtained from
bench-top fermenters, was directly determined by HPLC
for the drug target substrate diclofenac.
Fig. 1 a Diclofenac and 7‑methoxy‑4‑(trifuoromethyl)‑2H‑chro
men‑2‑one (MCF) oxidation reactions catalysed by P. pastoris CYP2C9/
CPR whole cell biocatalyst. b Screening of producer clones based
on bidirectional expression system using diferent promoter variants
towards to identify the most active CYP2C9/CPR whole cells
Te applied screenings identifed transformants with a
PPDF promoter for CYP expression and PPDC for the CPR
(clones 2C9-PDC/PDF-CPR) as most promising for the
further bioprocess development in bench-top fermenters.
obtained from bench-top fermenters displayed similar
diclofenac oxidation activities than cells from shake fask,
a thorough study of hCYP2C9/hCPR whole cell produc-
tion was carried out including chemostat cultivation and
bioprocess optimization in fed-batch cultures.
Production of P. pastoris CYP2C9/CPR whole cell biocatalyst
in bioreactor cultures
For the very frst time, CYP2C9/CPR expressing P. pas-
toris cells were cultivated successfully in bioreactor.
Previous unpublished studies in our lab prior to this cul-
tivation optimization demonstrated unexpected difcul-
ties in the reproduction of biocatalyst activities observed
in shake fasks after cultivation in methanol induced 5 L
bioreactor cultures. Tus, it was considered interesting
to compare hCYP2C9/hCPR expressing cells cultivated
in either shake fask and bioreactor regarding their ability
to convert diclofenac. As presented in Additional fle 1:
Fig. S1, the conversion rates obtained per mg of biomass
dry cell weight (DCW) for catalysts generated in this
study were rather similar. After confrmation that cells
Among the physical–chemical parameters that could
afect the bioprocess performance, pH was identifed to
have signifcant impact. Te control and adjustment of
pH can hardly be controlled or adapted in 96 well plate
cultures, with high bufer concentrations as the only way
to keep at least a desired pH constant. Previous experi-
ments in micro scale deep well plate cultures showed
best results with a starting pH of 8, which most likely
drop to lower values during cultivation. Tus, the efect
of diferent pH values (pH 5–7) on the production of
CYP2C9/CPR whole cells in chemostat cultivations at

Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 5 of 13
3 g L−1 (FB5). Te last strategy was selected since it was
previously reported to be the best condition for Rhizopus
oryzae lipase production under the control of PAOX1 pro-
moter [42].
intermediate D=0.10 h−1 was assessed systematically.
Working in steady state conditions for a constant specifc
growth rate (µ), provides a robust and reliable cultiva-
tion system that allows studying the efect of a specifc
operational parameter while keeping the others constant.
Activity tests showed that the best condition was pH=5,
obtaining a specifc enzyme activity up to threefold
higher than in the other conditions tested (Fig. 2). Con-
sequently, further fed-batch (FB) cultivations were always
carried out at pH=5.
In designed co-substrate cultivations using the MutS
host strain phenotype, it was necessary to maintain a
low µ—0.05 h−1, otherwise methanol utilisation (MUT)
genes might be repressed [43, 44]. Consequently, metha-
nol would not be consumed and its impact as promoter
inducer might not be properly assessed.
Concerning biomass generation, the identifed most
active transformant (named P. pastoris 2C9-PDC/PDF-
CPR) was able to grow as expected in all three methanol-
free FB cultures (Fig. 3). Although some growth efect
might have been expected for such membrane protein
overexpression strain, the recombinant expression did
not have any signifcant limiting efect on the cell growth.
Most of the cultures were grown up to reach 100 g L−1
of DCW. Nevertheless, the fermentation performed at
0.15 h−1 (FB1) had to be stopped at a biomass concen-
tration around 80 g L−1. At this point, the temperature
control could no longer keep constant the tempera-
ture at 25 °C due to the high amount of heat generated
at high specifc growth rate. Anyway, numerous samples
were taken throughout the process, which allowed the
determination of the production-related macrokinetic
parameters needed for its comparison with the rest of FB
cultures.
Biomass growth characterization in fed‑batch cultures
Since the producer strain identifed by screening for best
performing transformants was based on the bidirectional
expression construct 2C9-PDC/PDF-CPR, the regula-
tion pattern of the used promoters allowed to implement
either methanol-induced and/or methanol-free bio-
process strategies or consecutive induction schemes by
derepression followed by induction. Terefore, as the
current trend of P. pastoris bioprocesses is to avoid the
use of toxic and fammable methanol due to its opera-
tional associated drawbacks, a primary set of methanol-
free FB cultures with glycerol as sole carbon source was
performed. In these cultures, the efect of the specifc
growth rate (µ) on biocatalyst production was evalu-
ated by performing glycerol-limiting cultures at diferent
nominal µ (FBs 1–3). Furthermore, the potential boosting
efect of methanol on biocatalyst production was addi-
tionally assessed by growing the culture at µ=0.05 h−1
on glycerol-limiting conditions and adding methanol
as co-substrate either by pulses as the simplest strategy
(FB4), or keeping a constant methanol concentration of
Due to the low biomass-to-methanol yield, YX/MetOH
,
and the slow specifc methanol uptake rate, qMetOH, of the
P. pastoris phenotype (MutS strains), the use of metha-
nol as a co-substrate did not modify the growth curves
Fig. 2 Efect of culture pH for the production of P. pastoris CYP2C9/
CPR whole cells biocatalyst. Chemostat cultivations were carried out,
setting the dilution rate to 0.10 h−1. Error bars represent the activity
assay standard deviation determined by triplicates
Fig. 3 a Biomass time‑profle of the fed‑batch cultures
performed. Experiment labels: FB1: μ =0.05 h−1; FB2: μ =0.10 h−1
FB3: μ =0.15 h−1; FB4: μ =0.05 h−1 +MetOH pulses; FB5:
μ =0.05 h−1 +constant MetOH=3 g L−1
;

Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 6 of 13
substantially when comparing with methanol-free pro-
cesses. Moreover, CYP2C9/CPR production did not alter
signifcantly other relevant cell physiology rates, as indi-
cated by the specifc glycerol uptake rate (qGly) and bio-
mass-to-substrate yield (YX/Gly) (Table 1). Te obtained
values were similar to those usually reported as standard
for P. pastoris strains producing diferent recombinant
proteins [45–47].
Production of CYP2C9/CPR in P. pastoris whole cells
in fed‑batch cultures
Regarding whole cell biocatalyst activity, the production-
time profles were analysed for the fve alternative FB
strategies. Te obtained results (Fig. 4), demonstrated
signifcant diferences in terms of biocatalyst activity
between the methanol-free fed-batches cultures (FB 1–3)
and those induced with methanol (FB 4–5). In methanol-
free processes, a saturation trend was observed in late
stage of the profles at low and mid µ—0.05–1; 0.1 h−1,
whereas at the highest µ tested—0.15 h−1—always a
growth-coupled production profle was observed. On
the contrary, the methanol induced processes present
an exponential trend up to 3.28 AU L−1, thus reaching
biocatalyst activity relevantly higher than methanol-free
fermentations. Specifcally, when further inducing the
system with methanol a twofold increase in biocatalyst
activity could be observed.
Fig. 4 Biocatalyst activity time‑profles obtained in fed‑batch
cultures 1–5. Experiment labels: FB1: µ =0.05 h−1; FB2: µ =0.10 h−1
FB3: µ =0.15 h−1; FB4: µ =0.05 h−1 +MetOH pulses; FB5:
µ =0.05 h−1 +constant MetOH=3 g L−1
;
expressing a heterologous protein regulated by difer-
ent promoters. In several previous studies was reported
that cell growth and protein production were coupled
[33, 45, 48, 49]. As depicted in Fig. 5, the qp in this study
was clearly correlated with µ within the experimental
range tested. Results pointed out a slight increase of qp
when increasing the specifc growth rate from 0.05 h−1
(FB1) to 0.10 h−1 (FB2). However, a bigger increase was
found when growing cultures at 0.15 h−1, reaching up to
a 2.5-fold qp increase respect to the worst condition. In
contrast, the µ efect on YP/X values might be considered
negligible as yield values are rather similar for all three
fed-batches, around 11.5 AU gX−1 (FB 1–3).
As presented in Fig. 4, a signifcant but low amount of
active biocatalyst was obtained consistently already at the
end of the batch phase in all fve cultures. Tis fact is an
efect of the derepressed expression in non-limiting glyc-
erol conditions of the promoters used, PPDF/PPDC. How-
ever, later, at glycerol-limiting conditions the expression
was boosted as expected. Te similar starting point at the
end of the batches carried out demonstrated also the high
reproducibility between the diferent bioreactor runs.
It has been widely described that the specifc pro-
duction rate (qp) is importantly afected by µ when
With respect to the efect of methanol utilization on
biocatalyst activity production, bioreactor cultivations
with the same µ (FB1, FB4 and FB5) but either adding or
not adding methanol were compared. In all the cultures,
the use of this co-substrate further induced the protein
expression, signifcantly increasing the production rates.
Table 1 Main process parameters obtained in the diferent fed‑batch cultures performed for the production of active hCYP2C9/hCPR
P. pastoris whole cell biocatalysts
Run
YX/S
gX gS
qGly
qMetOH
Biocatalyst
activity
qp
YP/X
AU gX
YP/MetOH
−1
−1
gGly gX−1 h−1
gMetOH gX−1 h−1
AU gX−1 h−1
AU
−1
AU L−1
gMetOH
FB1
FB2
FB3
FB4
FB5
0.47
0.51
0.46
0.55
0.53
0.102
0.159
0.272
0.084
0.087
–
1.78
1.52
1.17
3.28
2.68
0.67
0.78
1.71
1.15
1.10
13.4
10.4
12.9
21.9
21.1
–
–
–
–
–
0.029
0.023
39.7
47.8
µ: specifc groth rate, YX/S: Biomass to substrate yield; qGly: specifc glycerol uptake rate; qMetOH: specifc methanol uptake rate; qP: specifc producrtion rate; YP/X: Product
to biomassa yield; YP/MetOH: Product to methanol yield;
FB1: 0.05 h−1; FB2: 0.10 h−1; FB3: 0.15 h−1; FB4: 0.05 h−1 +MetOH pulses; FB5: 0.05 h−1 +MetOH constant 3 g L−1

Garrigós‑Martínez et al. Microb Cell Fact
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(YP/X), the process should be prolonged in time, growing
the cells not only at a low µ, but also adding methanol as
co-substrate, preferably through pulses. In this way, up to
3.28 AU L−1 could be achieved, this representing a three-
fold increase respect to the worst condition. On the con-
trary, if qp is selected as the parameter to be optimized,
the best operational strategy would be a carbon-limit-
ing fed-batch operation using glycerol as a sole carbon
source at high µ. Specifcally, setting the µ at the highest
value tested—0.15 h−1 (FB3); a threefold increase could
be achieved respect to worst conditions.
However, from an application point of view, in order
to select the optimal bioprocess strategy to produce an
active biocatalyst like the presented in this work, the
whole workfow including downstream steps should also
be taken into consideration during the optimization pro-
cess. Since this bioprocess approach uses whole cells,
downstream processing can be considered rather simple
and not costly. Terefore, the need to reach high bio-
catalyst activities, which is essential to be able to aford
high cost separations, should not be considered as prior-
ity. Tus, taking into consideration the diferent criteria
discussed previously, the methanol-free carbon-limiting
strategy based on glycerol at highest µ should be con-
sidered the best alternative that allows maximization of
the product generation rates (qp). Despite lower biocata-
lyst activity levels reached, avoiding the use of methanol
would allow to avoid the important methanol-associ-
ated operational drawbacks. Tese relevant drawbacks,
including high oxygen demand, heat production, and
additional costs related to methanol storage, transporta-
tion and handling need to be considered for further scal-
ing steps of the bioprocess [38].
Fig. 5 Product‑related parameters obtained in fed‑batch cultures:
specifc product generation rate (qp) and product‑to‑biomass
yield (YP/X). Experiment labels: FB1: µ =0.05 h−1; FB2: µ =0.10 h−1
FB3: µ =0.15 h−1; FB4: µ =0.05 h−1 +MetOH pulses; FB5:
µ =0.05 h−1 +constant MetOH=3 g L−1
;
More specifcally, it led to qp improvements of up to 1.72-
fold, and up to 1.63-fold of YP/X. On the one hand, this
can be due to the efect that recombinant gene expres-
sion is highly induced in presence of methanol with both
promoters used, PPDF and PPDC. However, the observed
efects might also be attributed to other diferences in
carbon source dependent systems-level organization,
such as for example co-factor regeneration, membrane
permeability, or the expression levels of other proteins
[40]. As a summary, a comparison of the main process
parameters is presented in Table 1. When comparing
both methanol-based culture strategies implemented,
strikingly, controlling the methanol concentration in
the culture broth (FB5) only provided a slight increase
of about 20% in terms of active biocatalyst produced
per gram of methanol fed (YP/MetOH) compared to the
methanol-pulses based strategy (FB4). Moreover, other
product-related parameters—qp, YP/X—are rather similar
when controlling the methanol at a constant concentra-
tion. Consequently, since the results obtained are similar,
the methanol pulses-based process can be considered as
preferable due to its technical simplicity comparing with
a cultivation strategy based on a closed-loop methanol
control.
Preparative scale demonstration
Biomass produced by controlled cultivation in biore-
actors was applied to evaluate its activity for prepara-
tive biotransformation of ibuprofen to its respective
CYP2C9 metabolites. During Phase I metabolism in the
human liver the drug ibuprofen is mainly converted into
1-hydroxyibuprofen, 2-hydroxyibuprofen, and 3-hydrox-
yibuprofen [24]. CYP2C8 and CYP2C9 are known as the
main enzymes responsible for its stereoselective hydrox-
ylation [50]. However, as reported by Neunzig et al.,
CYP2C9 is the only known CYP able to produce relevant
amounts of 3-OH-ibuprofen [24]. To evaluate the feasi-
bility of a preparative scale production using P. pastoris,
CYP2C9 expressing cells made by reliable cultivation in
bioreactors were applied in a whole cell biotransforma-
tion approach using a reaction volume of 500 mL and
an initial substrate concentration of 2 mM. Within 16 h
a rather linear increase in conversion to 25% of total
product was observed (Fig. 6), which further increased
In order to maximize the bioprocess efciency, dif-
ferent optimization criteria based on diverse per-
formance indexes can be considered; among them,
biocatalyst activity, productivity and yield are the most
often selected. In P. pastoris fermentation processes,
the culture is often limited by the maximum amount of
biomass that can be reached, about 100 g L−1 in DCW.
Ten, the optimization method should aim to maximise
the production before reaching this limiting cell concen-
tration. To maximise the product activity (P) and yield

Garrigós‑Martínez et al. Microb Cell Fact
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Page 8 of 13
confrmed the potential of recombinant CYP2C9 cata-
lysed biotransformations using P. pastoris whole cells as
biocatalyst. In terms of space–time yield and specifc pro-
duction rate, values obtained with the P. pastoris whole
cell biocatalyst were signifcantly higher—up to 55% and
21%, respectively—than the reported in the literature, in
which the same proteins were expressed in a fssion yeast
system [24]. Moreover, the ratios between the two prod-
ucts (M2/M1) were in average around 5, and therefore,
even higher than previous published values [24], con-
frming the high selectivity of this biotransformation for
the 3-OH-metabolite. Te main reaction parameters are
presented in Table 2.
Despite the promising results obtained in the proof-
of-concept ibuprofen biotransformation, the biocatalytic
reaction could be further improved by addressing the
impact of some important parameters such as dissolved
oxygen availability, efciency of NADPH regeneration
system and/or heme group generation.
Conclusions
Recombinant production of hCYPs has been intensively
investigated and it is expected to continue attracting high
attention by the pharmaceutical and chemical industry.
Especially, a fast track from catalyst design to biocatalyst
manufacturing and scalable biotransformation is essential
to react on urgent and sometimes unexpectedly arising
needs in drug development. In this work, a whole scala-
ble bioprocess development for fne-tuned co-expression
of CYP2C9 and CPR in the broadly available and efcient
expression host P. pastoris has been successfully attained.
To achieve this objective, an innovative bidirectional
expression system using a small representative bidirec-
tional promoter library with diferent regulation patterns
was successfully implemented to achieve an optimized
fne-tuned co-expression clone for the hCYP29 and its
natural redox partner hCPR. Furthermore, for the frst
time, functionally active P. pastoris CYP2C9 whole cell
biocatalysts were obtained from reliable and optimized
bioreactor cultures, which fnally were successfully tested
for a biotransformation of interest based on the oxidation
of ibuprofen. Te biocatalyst used in the preparative test
could convert more than 40% of the added substrate in
a simple batch biotransformation and, more importantly,
Fig. 6 a Ibuprofen oxidation reactions catalysed by human CYPs.
b Preparative scale ibuprofen oxidation by P. pastoris CYP2C9/CPR
whole cells biocatalyst. Substrate concentration: 2 mM, reaction
volume: 500 mL
up to 40% within 86 h of reaction. As expected from
small scale results (data not shown), metabolite 2 (35%),
which was identifed as 3-OH-ibuprofen by NMR spec-
troscopy (Additional fle 2), was mainly produced, while
metabolite 1 was only found to an extent of 5% (2-OH-
ibuprofen). No relevant amounts of metabolite 3 could be
detected (1-OH-ibuprofen), which was observed as a typ-
ical product in previous experiments employing a fungal
bifunctional P450 homologue of the bacterial CYP102
[6].
A total product concentration of 0.8 mM was obtained
starting with 2 mM of substrate ibuprofen with 0.70 mM
of 3-OH-ibu and 0.1 mM of 2-OH-ibu, respectively
as products and no 1-OH-ibuprofen. Tese results
Table 2 Main parameters obtained for the preparative scale conversion of ibuprofen
Reaction time (h)
Average substrate conversion (%)
Product formed
(mM)
Space–time yield Specifc prod. rate
Yield (µmol
product g
(µmol product L−1 (µmol product g
d−1
M1
)
whole cells−1 d−1
)
whole cells−1
)
M1
M2
M2
M1
M2
M1
M2
86
41.7
0.14
0.69
39
194
1.22
6.06
4.39
21.7

Garrigós‑Martínez et al. Microb Cell Fact
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it demonstrated high selectivity for the product hydroxy- recombination. Due to the Pichia clonal variability, High
lated at position three.
Troughput Screening (HTS) were used for the further
Moreover, a deep study of using CYP2C9/CPR express- identifcation of the catalyst with the highest activity.
ing P. pastoris whole cells focused on the production
kinetics has been carried at bench top bioreactor scale.
Whole cell cultivation
It has been demonstrated that the µ has a positive efect
High‑throughput small scale cultures
on qp, demonstrating that the production is coupled with
Microscale 96 deep well cultivation was performed based
the cell growth. Terefore, selecting a high µ in the bio-
on Weis et al. [41]. Single colonies were transferred to
process is advantageous to maximize qp on methanol-
one well containing 250 µL of BMD1% (bufered mini-
free strategies. However, if the objective is to maximise
mal dextrose 1%) and cultivated for 60 h at 320 rpm and
the YP/X and, therefore, the biocatalyst activity, a bio-
28 °C. Subsequently, 250 µL BMM2% (bufered minimal
process using mixed substrates based on the co-feeding
methanol 1%) were added to induce gene expression.
of glycerol and methanol should be recommended. In
8, 24 and 32 h after the frst induction, 50 µL BMM10
these strategies, µ is decreased to the lowest value tested
(bufered minimal methanol 5%) were added. After 48 h
(0.05 h−1) to promote the methanol consumption.
of methanol induction, cells were harvested by centrifu-
Altogether, it is expected that this work will become
gation at 500 rpm. Cultivation in shake fask (Tomson
a model, not only for the targeted fast development of
2 L ultra-yield fasks) was performed accordingly starting
new human CYP catalysts, but also as applicable reli-
with 200 mL.
able bioprocesses development for respective whole cell
biocatalyst production and the respective catalysed bio-
Chemostat cultures
transformations employing recombinant human CYPs
Chemostat cultures were performed based on the meth-
odology previously described by Garcia-Ortega et al.
[48], but using glycerol as a substrate instead of glu-
cose. In this case, the diferent cultures conditions com-
pared were performed at a constant specifc growth
rate (µ) of 0.10 h−1, as intermediate value considering
µmax ≈0.20 h−1. All the operating parameters were kept
constant as reported in the reference along the cultiva-
tions except the pH, which was controlled at diferent
values of 5, 6 and 7.
expressed by P. pastoris. In a similar fashion, other bio-
catalysts relying on optimized co-expression rations can
be developed. Tis progress will support fast and efcient
drug development pipelines, which become especially
challenging and important in case of pandemic diseases,
where new drug candidates need to be advanced to the
next phase within very short timelines.
Materials and methods
Strain generation
Escherichia coli/P. pastoris shuttle vectors were based
on plasmid pPpT4 [51] and adapted as entry vectors for Fed‑batch cultures
quick and simple cloning of bidirectional promoters by Main details regarding medium composition, opera-
bisy (bisy GmbH, Hofstaetten, Austria). Synthetic DNA tional parameters and process strategies are described
sequences were for these human open reading frames in previous works [45]. All the fed-batch cultures were
were obtained from IDT (Integrated DNA Technologies based on a carbon-limited operational strategy using a
Inc., Coralville, Iowa, USA). By using Gibson assem- pre-programmed exponential feeding profle of glycerol
bly [52], a test set of 7 bidirectional promoters obtained feeding solutions, which allows keeping a constant spe-
from bisy GmbH (PDAS1/DAS2, PDAS2/DAS1, PPDC/PDF, PPDF/ cifc growth rate (µ) throughout the process, reaching a
PDC, PDAS1-D6/DAS2-D8, PDAS2-D8/DAS1-D6, and PDAS2-699/DAS1- pseudo-stationary state. Upon co-substrate processes,
552) and described previously by Vogl et al. [30] to drive two strategies were compared, one based on periodic
expression of full length hCYP2C9 (AL359672) includ- methanol pulse additions and another in which the meth-
ing its hydrophobic N-terminal sequence and hCPR anol concentration was kept constant at 3 g L−1 by means
(accession AAH34277.1) were generated using the men- of a closed control loop. For this last case, the methanol
tioned cloning plasmids. Tese cloning plasmids were concentration was determined by a Methanol Sensor Sys-
transformed into the P. pastoris strain BSYBG11 MutS tem (Raven Biotech Inc., Vancouver, Canada), and con-
(Δaox1, MutS), which presents a slow methanol utilisa- trolled by performing a predictive-PI control strategy.
tion phenotype, which was obtained from Bisy GmbH. Furthermore, of-line methanol determinations by HPLC
Generation of competent P. pastoris cells and transfor- were used to validate the control system. Further details
mation were performed according to Lin-Cereghino et al. about the methanol control system are described else-
[53]. In this strain generation the target expression cas- where [54, 55].
sette is integrated into the Pichia genome by homologous

Garrigós‑Martínez et al. Microb Cell Fact
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Page 10 of 13
Analytical methods
Biomass determination
diclofenac to reach a 2 mM substrate concentration and
reactions were carried at 28 °C and 200 rpm. After 20 h
of incubation, 50 µL were taken and mixed with 450 µL
of MetOH/acetonitrile (1:1), to stop the reaction and to
extract both the substrate (diclofenac) and the product
(4′OH-diclofenac). Both substrate and product concen-
trations were determined by HPLC–UV. Before its analy-
sis, samples were centrifuged at 12000×g for 2 min and
supernatants were fltered (0.45 µm).
Biomass concentration was measured by DCW using a
protocol described elsewhere [56]. RSD was estimated to
be under 3%. Biomass concentration was also measured
by optical density at 600 nm using a DR3900 spectropho-
tometer (Hach Lange, Colorado, USA). RSD was esti-
mated to be below 3%.
Te product-related results of the fermentations are
presented in terms of activity units (AU). One AU is
defned as the amount of enzyme which catalyses the
production of 1 µmol of 4-hydroxy-diclofenac per hour
under the assay conditions. Tus, this parameter pro-
vides an enzyme titer value in terms of (AU L−1).
Carbon source and by‑products quantifcation
Te amounts of both carbon sources (glycerol, methanol)
and potential fermentation by products were analysed by
HPLC. Te chromatography procedure and the column
specifcations were described elsewhere [57]. RSD was
estimated to be lower than 1%.
Whole cell bioconversions
High throughput screening (HTS)
HPLC–UV/MS
HPLC/UV analysis were performed on a HPLC Dionex
Ultimate 3000 (Dionex Corporation, Sunnyvale, CA,
USA) series using a CORTECSTM C18+2.7 μm
4.6×150 mm column (Waters), at a temperature of 40 °C
and a detector wavelength of 275 nm. Te mobile phase
was composed of Trifuoroacetic acid 0.1% (v/v) in water
(Eluent A) and Trifuoroacetic acid 0.095% (v/v) in Ace-
tonitrile 80% (Eluent B). Gradient-elution was performed
at 0.7 mL min−1 as follows: 0.00–0.50 min: 95% A, 5% B;
0.50–13 min: 55% A, 45% B; 13.00–13.50 min: 20% A,
80% B; 13.5–15.00 min: 0% A, 100% B; 15.00–15.50 min:
0% A, 100% B; 15.50–20.00 min: 95% A, 5% B. Retention
times were 12.04 for diclofenac and 8.22 4′-hydroxy-
diclofenac. For calculations, peak areas of the UV chro-
matograms recorded at 239 nm were used.
Whole cell screenings using diclofenac as substrate were
carried out in 96-well microtiter plates. After the har-
vest, cells were washed twice with 200 µL of assay bufer
(100 mM KPi, pH 7.4) and then the biomass concentra-
tion was determined by optical density determination
(OD600). Cells were frozen at −20 °C for several days to
obtain a better membrane permeability. Subsequently,
4 µL of a 100 mM diclofenac stock solution in methanol
(MetOH) were added to each well and conversions were
performed for 20 h at 28 °C and 320 rpm. Reactions were
stopped by addition of 200 µL of MetOH/acetonitrile
(1:1) and vigorously mixed. After centrifugation (10 min,
3200×g) 200 µL of supernatant were transferred into a
fresh 96 well microtiter plate (polypropylene, V-shaped),
sealed and analysed by HPLC–MS.
HPLC–MS was performed on a HPLC 1200 series
(Agilent technologies, Santa Clara, CA, USA) equipped
with an MSD SL detector with an electron spray ioniza-
tion (ESI) unit as described previously [58]. A Kinetex®
2.6 μm C18 100 Å, LC Column 50×4.6 mm column
(Phenomenex) was used. Te mobile phase was com-
posed of water (acidifed with 0.1% formic acid) and
acetonitrile (ACN). Gradient-elution was performed at
1.3 mL/min as follows: 0.00–1.00 min: 10% ACN; 1.00–
3.50 min: 10–100% ACN; 3.50–4.00 min: 10% ACN. To
allow a fow rate of 1.3 mL min−1, a splitter (2:1 ratio)
was implemented in front of the ESI unit. 4′-hydroxy-
diclofenac (m/z 312) eluted after 2.2 min and diclofenac
(m/z 296) after 2.4 min. For analysis of ibuprofen and
its metabolites a stepwise gradient was used at 25 °C as
described previously [6]: 0.00–1.00 min: 20% ACN; 1.00–
3.00 min: 20–100% ACN; 3.00–5.00 min: 100% ACN;
5.00–6.00 min: 20% ACN re-equilibration. Metabolites
were detected at 239 nm (VWD) and MS spectra were
used to support data evaluation.
Fluorometric whole cell screenings based on the
7-methoxy-4-(trifluoromethyl)-2H-chromen-2-one
(MFC) assay were performed using 95 μL of the cell sus-
pension in assay solution using black microtiter plates.
Te reaction was started by the addition of 5 μL of 1 mM
MFC to reach a fnal concentration 50 μM. Fluorescent
metabolite formation was quantifed measuring the fuo-
rescence of the product 7-hydroxy-4-(trifuoromethyl)-
2H-chromen-2-one (HFC). Te fuorescence signal of the
product formed (ex 410 nm/em 538 nm) was monitored
every minute for a period of 1 h using a SynergyMX plate
reader (BioTek Instruments Inc, USA). During the reac-
tion the microtiter plates were kept at 30 °C.
Bioconversion reactor
For bioreactor samples, the enzymatic assays were per-
formed in 50 mL falcon tubes. In this case, the washed
cells were diluted to OD600 of 5 as a standard OD600
for all reactions. Ten, 2 mL of cells were mixed with

Garrigós‑Martínez et al. Microb Cell Fact
(2021) 20:90
Page 11 of 13
Standortagentur Tirol, Government of Lower Austria and Business Agency
Vienna through the Austrian FFG‑COMET—Funding Program.
Preparative scale conversion of ibuprofen
Preparative scale conversions were performed as
described previously [6, 58]. A total reaction volume of
500 mL including the whole cell catalysts correspond-
ing to OD600 = 150 was used for oxidation of 206 mg
ibuprofen dissolved in DMSO. Conversion was per-
formed in a bafed 2 L shake fask over a period of 86 h.
Samples of 200 µL were withdrawn at diferent times
and analysed as described above.
Authors’ contributions
AW and CS constructed the producer strains and performed the HTS. JGM
performed all the cultures in bench‑scale bioreactors. CR performed the pre‑
parative scale demonstration. JLM, FV, AG and XGO, conceived and supervised
the study. JGM, CR, JLM, FV, AG and XGO drafted the manuscript. All authors
read and approved the fnal manuscript.
Funding
This work received funding from the EU project ROBOX (Grant Agreement
No. 635734) under EU’s H2020 Programme Research and Innovation actions
H2020‑LEIT BIO‑2014‑1.
Nuclear magnetic resonance spectroscopy (NMR)
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
For recording NMR spectra, a Bruker Avance III
300 MHz FT NMR spectrometer with autosampler was
used (300.36 MHz-H-NMR and 75.5 MHz-C-NMR).
Te residual protonated solvent signal serve as inter-
nal standard for interpretation of the chemical shifts
δ (H-,C-NMR). To facilitate the interpretation, the
C-spectra were proton decoupled to gain better identi-
fcation of the peaks.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Te chemical shift δ is indicated in ppm (parts per
million) and the coupling constant J in Hz (Hertz). For
the signal multiplicities the following abbreviations
were most commonly used: s (singlet), bs (broad sin-
glet), d (doublet), t (triplet), q (quadruplet), m (multi-
plet). Quaternary carbons are labelled as Cq.
Competing interests
The authors of TU Graz and bisy declare that they have commercial interests
in the use of bidirectional promoters for protein coexpression. Bisy GmbH
owns and commercializes tools for recombinant bidirectional coexpression of
genes.
Author details
1Department of Chemical, Biological and Environmental Engineering, School
of Engineering, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdan‑
yola del Vallès), Spain. 2Institute of Molecular Biotechnology, Graz University
of Technology, Petersgasse 14, 8010 Graz, Austria. 3Bisy GmbH, Wuenschen‑
dorf 292, 8200 Hofstaetten/Raab, Austria. 4Austrian Centre of Industrial
Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12934‑021‑01577‑4.
Additional fle 1: Figure S1. Whole cell hydroxylation of diclofenac by P.
pastoris CYP2C9/CPR whole cells cultivated respectively in bioreactor and
in shake fask (UYF=ultra‑yield fask). The wild type strain BSYBG11 was
used as a negative control.
Received: 9 September 2020 Accepted: 7 April 2021
Additional fle 2. Nuclear Magnetic Resonance Spectroscopy (NMR)
analyses. Figure S2A. Product 1, 1H NMR (300 MHz, DMSO): δ 12.16 (bs,
1H); 7.15 (s, 4H); 3.96 (bs, 1H); 3.62 (q, J =7.0 Hz, 1H); 2.61 (s, 2H); 1.31
(d, J =14.4 Hz, 3H); 1.05 (s, 6H) ppm. Figure S2B. Product 1, 13C NMR
(75.5 MHz, DMSO): δ 175.5; 138.5; 137.4; 130.4; 126.6; 69.3; 49.0; 44.3; 29.2;
18.5 ppm. Figure S2C. Product 2, 1H NMR (300 MHz, DMSO): δ 12.25 (s,
1H); 7.24–7.07 (m, 4H); 4.51 (s, 1H); 3.62 (dd, J =13.8; 6.8 Hz; 1H); 3.23 (dd,
J =15.1; 8.3 Hz, 2H); 2.68 (dd, J =13.2; 5.7 Hz, 1H); 2.25 (dd, J =13.1; 8.3 Hz,
1H); 1.76 (dd, J =12.9; 6.5 Hz, 1H); 1.34 (d, J =7.0 Hz, 3H); 0.78 (d, J =6.6 Hz,
3H) ppm. Figure S2D. Product 2, 13C NMR (75.5 MHz, DMSO): δ 175.5;
139.4, 138.5; 129.1; 127.2; 65.6; 44.3; 38.7; 37.6; 18.6; 16.5 ppm.
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