Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

Article abstract of DOI:10.3762/bjoc.11.190

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ～50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knockout strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO₂ represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

Full text of DOI:10.3762/bjoc.11.190

Engineering Pichia pastoris for improved NADH regeneration:
A novel chassis strain for whole-cell catalysis
Martina Geier1, Christoph Brandner1, Gernot A. Strohmeier1,2, Mélanie Hall3,
Franz S. Hartner4,5 and Anton Glieder*5
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 1741–1748.
1Austrian Centre of Industrial Biotechnology (ACIB GmbH),
doi:10.3762/bjoc.11.190
Petersgasse 14, Graz, 8010, Austria, 2Institute of Organic Chemistry,
Graz University of Technology, Stremayrgasse 9, Graz, 8010, Austria,
Received: 03 June 2015
3
Department of Chemistry, University of Graz, Heinrichstrasse 28,
Accepted: 26 August 2015
Published: 25 September 2015
Graz, 8010, Austria, 4Sandoz GmbH, Biochemiestrasse 10, 6250,
Kundl, Austria and 5Institute of Molecular Biotechnology, Graz
University of Technology, Petersgasse 14, Graz, 8010, Austria
This article is part of the Thematic Series "Sustainable catalysis".
Guest Editor: N. Turner
Email:
Anton Glieder* - a.glieder@tugraz.at
©
2015 Geier et al; licensee Beilstein-Institut.
*
Corresponding author
License and terms: see end of document.
Keywords:
bioreduction; cofactor regeneration; dihydroxyacetone synthase;
methanol utilization pathway; whole-cell biotransformation
Abstract
Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor,
methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel
and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH
regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration
via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol
dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching
~
50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-
out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the
cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the
intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for
NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regen-
eration pathway expression and source of energy.
Introduction
Bioreductions represent a sustainable strategy to obtain enan- oxidoreductases, which mainly depend on nicotinamide cofac-
tiopure molecules which serve as chiral building blocks for fine tors as electron donors and acceptors. As these cofactors are
chemicals and drugs [1,2]. Such reactions are catalyzed by required in stoichiometric amounts to drive the reaction to
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completion and the simple addition of these compounds is not Here, we present a novel, engineered platform strain for the use
acceptable from an economical point of view, efficient in situ in whole-cell bioreductions and NADH-dependent biosynthetic
regeneration of the consumed cofactor is required. During the pathways based on the methylotrophic yeast Pichia pastoris
recent years, a broad range of cofactor regeneration systems (Komagataella phaffi). P. pastoris is well known as an excel-
based on enzymatic, chemical, photochemical or electrochem- lent host for recombinant protein production [8,9]. More
ical processes have been developed [3].
recently it also attracted attention as a suitable whole-cell cata-
lyst [10-15]. The target of the presented engineering approach is
In living (growing) cells, NAD(P)H can be recycled via the the methanol utilization (MUT) pathway, which is schemati-
cellular metabolism of a co-substrate such as glucose. Alter- cally depicted in Figure 1. This pathway enables methylo-
natively, cofactor regeneration enzymes such as D-glucose-6- trophic yeasts to use methanol as sole carbon source [16]. In the
phosphate dehydrogenase, formaldehyde dehydrogenase and initial step, methanol is oxidized by alcohol oxidase (AOX) to
formate reductase can be co-expressed ensuring cofactor supply formaldehyde, which is further metabolized either in the assimi-
also in non-growing, resting cells. In addition to the cofactor latory or in the dissimilatory pathway. In the latter one,
supply, the application of whole cells circumvents time- formaldehyde spontaneously reacts with glutathione to
consuming enzyme isolation and purification steps and S-hydroxymethylglutathione which is oxidized in a first step by
improves enzyme stabilities during a process [4]. Nowadays, the glutathione- and NAD+-dependent enzyme formaldehyde
efforts are made to further increase the cofactor availability dehydrogenase (FLD) to S-formylglutathione. S-Formylgluta-
within cells and to allow the use of alternative and cheaper thione hydrolase (FGH) then hydrolyses this compound to
co-substrates by strain engineering [1,5-7].
formate and glutathione. In a second NAD+-dependent step,
Figure 1: Simplified schematic representation of the methanol utilization pathway in Pichia pastoris. The main pathways and the respective enzymes
are shown. AOX: alcohol oxidase; FLD: glutathione-dependent formaldehyde dehydrogenase; FGH: S-formylglutathione hydrolase; FDH: formate
dehydrogenase; CAT: catalase; DAS: dihydroxyacetone synthase; DAK: dihydroxyacetone kinase; DHA: dihydroxyacetone; GAP: D-glyceraldehyde-
3-phosphate; DHAP: dihydroxyacetone phosphate; FBP: D-fructose 1,6-bisphosphate; F6P: D-fructose 6-phosphate; Xu5P: D-xylulose 5-phosphate.
By deleting dihydroxyacetone synthase the dissimilatory pathway leading to the formation of NADH is strengthened.
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formate is oxidized to CO2 by formate dehydrogenase (FDH). strains were generated to investigate their impact on cofactor
Thus, 2 equivalents of the cofactor NADH are generated via the regeneration: the single knock-outs Δdas1 and Δdas2 as well as
dissimilatory pathway and full oxidation of the single carbon the double knock-out Δdas1 Δdas2.
molecule methanol. Alternatively, in a simplified model
formaldehyde is condensed with D-xylulose-5-phosphate and The targeted gene disruption was accomplished with knock-out
subsequently converted into dihydroxyacetone and D-glycer- cassettes as described earlier [19,20]. The cassettes were
aldehyde-3-phosphate by dihydroxyacetone synthase (DAS), harboring a ZeocinTM resistance marker for the selection of
thereby contributing to biomass production on methanol.
integrative transformants and the FLP recombinase system [21]
to enable marker recycling. Thereby, marker-free knock-out
Modifying the dissimilatory part of the MUT pathway has strains were obtained which only have one 34 bp FLP recombi-
already been shown to improve the substrate conversion by nation target sequence (FRT) left in the targeted locus. The
NADH-dependent enzymes in P. pastoris [7]. Over-expression cassettes were designed such that the complete coding sequence
of the formaldehyde dehydrogenase, which was identified as the including the start and stop codons of the corresponding DAS
main bottleneck for an efficient cofactor recycling via the gene was deleted as schematically depicted in Figure 2. In the
dissimilatory pathway, significantly improved the production case of Δdas1 Δdas2, the coding sequences of both genes were
rates of NADH-dependent whole-cell biotransformations. In deleted in one step, thereby also removing HOB3, coding for a
contrast, the present study focuses on redirecting the flux in the hypothetical guanosine nucleotide exchange factor.
MUT pathway by disrupting the assimilation pathway. Thus,
the co-substrate methanol should be exclusively redirected to Characterization of knock-out strains
cofactor regeneration in theory, increasing the available NADH In a first step, the generated das knock-out strains were charac-
concentration in whole-cell biotransformations, while cells terized by determining their specific growth rate on different
should not be able to grow any more if methanol is the sole carbon sources. As expected, the growth of the single and
carbon source.
double knock-out strains on D-glucose and glycerol was not
significantly impaired, showing growth rates in the same order
of magnitude as the wild type strain (Table 1). A distinct pheno-
type was observed when methanol was used as the sole carbon
Results and Discussion
Construction of knock-out strains
The assimilatory pathway was disrupted by the knock-out of source for growth. A slight, but residual growth was observed
dihydroxyacetone synthase. In contrast to other methylotrophic for the double knock-out strain, although both dihydroxyace-
yeasts, P. pastoris has two genes encoding two isoforms of this tone synthase genes, that are linking methanol to biomass pro-
enzyme (DAS1 and DAS2) [17,18]. Therefore, three knock-out duction, were deleted. In the course of 38 h, the optical density
Table 1: Specific growth rates of P. pastoris CBS7435 and generated das knock-out strains on different carbon sources. Values represent mean
values ± standard deviations of the growth rate during the exponential growth phase determined in biological triplicates.
Strain
Growth rate [h−1]
D-Glucose
Glycerol
Methanol
P. pastoris CBS7435
0.25 ± 0.03
0.23 ± 0.06
0.23 ± 0.03
0.26 ± 0.03
0.19 ± 0.05
0.21 ± 0.04
0.20 ± 0.02
0.20 ± 0.04
0.15 ± 0.03
0.12 ± 0.03
P. pastoris CBS7435 Δdas1
P. pastoris CBS7435 Δdas2
P. pastoris CBS7435 Δdas1 Δdas2
0.09 ± 0.01
0.01 ± 4 × 10−3
Figure 2: Representation of the genomic region coding for dihydroxyacetone synthases. The DAS1 and DAS2 coding sequences are located in close
proximity on chromosome 3 in opposite direction. The two genes are separated by a short sequence encoding a hypothetical guanosine nucleotide
exchange factor (HOB3). The target sites for the respective das knock-out cassettes are indicated schematically.
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NADH-dependent biotransformations
of the P. pastoris Δdas1 Δdas2 cultures on methanol doubled.
This remaining carbon flux into cellular metabolism might also performed in knock-out strains
enable the continuation of protein expression and thus biocata- To evaluate the NADH regeneration potential of the generated
lyst production, while still maintaining a strong methanol flux knock-out strains, they were employed as hosts in whole-cell
towards oxidation to CO2 and NADH regeneration.
bioreductions based on the 2,3-butanediol dehydrogenase
from S. cerevisiae YAL060W (BDH1). This enzyme was
Deleting only one isoform of the dihydroxyacetone synthase did shown to specifically use NADH as cofactor, displaying high
not have such a severe impact on the methanol depending turnover numbers in the reduction of racemic acetoin
growth of P. pastoris. The growth rates were reduced by ~20% (kcat = 98,000 min−1) [22].
and by ~40% for the Δdas1 and Δdas2 single knock-out strains,
respectively.
In an initial screening step, BDH1 transformants in the respec-
tive wild type and knock-out backgrounds were evaluated by
The behavior of the das knock-out strains in heterologous performing acetoin conversions in the 96 well deep-well format.
protein production was tested by expressing green fluorescent After biomass production on a D-glucose containing medium,
protein (GFP) under the control of the AOX1 promoter (PAOX1), methanol was fed as the sole carbon source to induce the pro-
which is the most prominent, methanol inducible promoter for duction of the BDH1 enzyme and to induce the dissimilatory
P. pastoris.
pathway. After 12 hours of induction acetoin was added to the
growing cells as substrate in addition to the co-substrate
A possible negative effect on PAOX1 driven protein expression methanol. The substrate conversions into the two products
might arise from changes in the energy metabolism due to the (2R,3R)-butane-2,3-diol and meso-butane-2,3-diol obtained
interrupted input into the pentose phosphate cycle. In addition, a after 16 h reaction time are shown in Figure 4A. Performing the
possible accumulation of methanol oxidation products due to BDH1-catalyzed acetoin reduction with the wild type strain P.
the lack of the two key metabolizing enzymes might be unfa- pastoris CBS7435 resulted in an average conversion of ~5%
vorable for recombinant protein production. However, as shown (40 individual clones tested). Employing the single knock-out
in Figure 3, the PAOX1 driven expression of GFP was not nega- strains Δdas1 and Δdas2 in the same reaction did not yield
tively affected by deleting the DAS gene(s). In fact, the obtained substantially higher conversions. The average conversions were
expression levels in the generated single knock-out strains were in the same order of magnitude as for the wild type strain (~6%
increased up to ~30% in comparison to the wild type P. pastoris and ~7% for the Δdas1 and Δdas2 strain, respectively), indi-
strain. These findings are of importance for the use of the cating that the NADH regeneration was not significantly impro-
knock-out strains in whole-cell processes, as impairments in ved in these strains. In contrast to the single knock-out strains,
recombinant protein production, i.e., the production of the the acetoin conversion was 5.5-fold higher when using the
actual biocatalyst, would reduce its applicability.
double knock-out strain in comparison to the wild type (average
conversion ~29%), indicating a more efficient cofactor supply.
The higher conversions with the double-knock out strain were
even obtained with fewer cells per reaction as it grew much
slower on methanol than the wild type and the single knock-out
strains. These first findings confirm the assumption that the
NADH formation via methanol oxidation is increased when the
assimilatory pathway is disrupted, if there is a NADH require-
ment in the cells due to, e.g., a biocatalytic reaction. The latter
is only efficiently realized when both genes coding for the dihy-
droxyacetone synthase proteins are deleted.
The time course of acetoin conversions with representing BDH1
transformants in the wild type and Δdas1 Δdas2 background
were investigated under optimized conditions in shake flasks.
To avoid differences in the amount of cells employed in the
biotransformation and to obtain higher product yields, cells
corresponding to 3000 OD600 units were harvested 12 h after
the first methanol induction and resuspended in 50 mL of
buffered minimal medium before the conversions were started
Figure 3: Relative expression levels of the green fluorescent protein
(GFP) in the das knock-out strains. GFP expression obtained in the
wild type P. pastoris strain indicated as relative fluorescence units per
OD600 unit was set as 100%. GFP fluorescence was measured after
7
±
0 h of methanol induction. The shown values represent mean values
standard deviations of 40 individual transformants.
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Figure 4: BDH1-based whole-cell conversions of rac-acetoin. (A) Conversions of 25 mM rac-acetoin in the different knock-out strains were performed
in 96 well deep-well plates for 16 h. The shown conversions represent mean values ± standard deviations derived from reactions using whole cells
from 40 individual BDH1 transformants. (B) Time-resolved biotransformations of 50 mM substrate were conducted in the wild type (grey squares) and
in the Δdas1 Δdas2 strain background (white squares). Background activity by endogenous acetoin reducing enzymes of P. pastoris was observed
(grey triangles: P. pastoris CSB7435 wild type, white triangles: P. pastoris Δdas1 Δdas2).
by the addition of substrate. During the first two hours of the out strain P. pastoris CBS7435 Δdas1Δdas2 displayed better
whole-cell biotransformation, the conversions obtained with performance in dehydrogenase based whole-cell biotransforma-
each of the strains were in the same order of magnitude (see tions without the need of an external electron source. Higher
Figure 4B). After longer reaction times, hardly any residual conversions were achieved in comparison to reactions carried
BDH1 activity was observed with the wild type strain (final out with the wild type Pichia strain due to an extended NADH
conversion of ~50%), while the reaction reached 100% conver- supply by the oxidation of methanol.
sion after 6 h in the engineered strain. These findings clearly
indicate that the NADH supply for the oxidoreductase can be Methanol, which is a cheap carbon source, fulfills several roles
attained over a prolonged reaction time in the P. pastoris Δdas1 in the presented biotransformations: It acts as an inducer of the
Δdas2 strain, which directly translates into increased yields in PAOX1 regulated production of the redox enzyme as well as of
the whole-cell reactions.
the endogenous NADH regeneration system at the same time.
The cofactor regeneration itself is based on methanol as co-sub-
It has to be noted, that an additional acetoin reduction by strate: the oxidation of methanol is irreversible and represents a
endogenous Pichia dehydrogenases was also observed for the strong thermodynamic driving force while only CO2 is
wild type and the double knock-out strain without the BDH1 produced as side product. Furthermore, methanol can serve as
expression cassette, reaching ~35% and ~8% substrate conver- solvent for the substrate and methylotrophic yeasts are already
sion, respectively. As for the recombinant strains, two products naturally adapted to elevated concentrations of methanol.
were detected, namely (2R,3R)-butane-2,3-diol and meso-
butane-2,3-diol.
The redesigned Pichia strain, thus, represents a valuable host
for whole-cell applications where NADH regeneration is an
issue. No additional over-expression of cofactor regenerating
Conclusion
Efficient cofactor recycling is not only required for an optimal enzymes such as formate dehydrogenase, which only yields one
enzyme performance, but is also crucial to render the resulting molecule of NADH per carbon atom, is required. Due to its
process economical and sustainable. For this purpose, biotrans- simplicity it might further boost the use of recombinant whole-
formations based on whole cells represent an elegant strategy as cell catalysts in chemical and pharmaceutical industry.
the cell metabolism can be exploited for cofactor supply. We
Experimental
have generated a novel platform strain for whole-cell catalysis
based on the methylotrophic yeast P. pastoris by engineering its General
methanol utilization pathway. By deleting the pathway for Unless stated otherwise, all chemicals were obtained from
methanol assimilation, we forced flux through the dissimilatory Sigma-Aldrich (Steinheim, Germany) or Carl-Roth (Karlsruhe,
pathway in which theoretically two molecules of NADH can be Germany) with the highest purity available. ZeocinTM was
formed per molecule of methanol. The resulting double knock- obtained from InvivoGen (San Diego, CA, USA). Phusion®
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High Fidelity Polymerase for DNA amplification and further strains were determined by measuring the optical density
DNA modifying enzymes were purchased from Thermo Fisher (OD600) of cultures in biological triplicate during the exponen-
Scientific Inc. (Waltham, MA, USA). E. coli Top10 (Invitrogen, tial growth phase.
Carlsbad, USA) was used for all cloning steps and plasmid
propagation. The P. pastoris strain CBS7435 as well as the GFP expression in knock-out strains
plasmid pPp_T4_S were obtained from the Pichia pool of TU The plasmid pPp_T4_S_GFP was linearized with SmiI and used
Graz [20].
to transform the wild-type as well as the das knock-out P.
pastoris strains. Single colonies were transferred to 96 well
deep-well plates for standard cultivation and protein production
Generation of das knock-out strains
The generation of knock-out strains was based on linear exci- as described previously [24]. For comparison of the expression
sion cassettes harboring a FLP recombinase system for levels, the GFP fluorescence (488 nm excitation, 507 nm emis-
inducible marker recycling. A schematic representation of such sion) and the optical density (OD600) of the cell suspension
a knock-out cassette is provided in Supporting Information were measured with a Synergy MX Microplate Reader.
File 1, Figure S1. Knock-out cassettes were essentially
constructed as described in [20]. Locus specific integration Cloning of model enzyme
sequences, i.e., DNA fragments of approximately 1.5 kb To evaluate the cofactor regeneration of the generated knock-
directly located up- and downstream of the corresponding out strains, the 2,3-butanediol dehydrogenase of S. cerevisiae
coding sequences, were amplified from genomic DNA. The YAL060W (BDH1) [22] was employed as NADH-dependent
corresponding primers were designed based on the genome model enzyme. The corresponding gene was ligated with the P.
sequence of P. pastoris CBS7435 and are summarized in pastoris expression vector pPp_T4_S_after digestion with
Supporting Information File 1, Table S1. The resulting knock- EcoRI/NotI. The thus resulting construct, pPp_T4_S_BDH1,
out cassettes were employed for the transformation of P. was linearized with SmiI for the subsequent transformation of
pastoris CBS7435 wild type cells according to the condensed the Pichia wild-type as well as das knock-out strains.
protocol by Lin-Cereghino et al. [23]. Transformants were
selected on YPD agar plates containing 100 mg/L ZeocinTM. Screening of BDH1 transformants
The correct integration of the knock-out cassette was confirmed Strains harboring the expression cassette for BDH1 were grown
by PCR using genomic DNA as template and primer pairs for 60 h in 250 µL of BMD medium in 96 well deep-well
binding within the cassette and either up- or downstream of the plates. Protein expression was started by the addition of 250 µL
targeted locus (see Supporting Information File 1, Table S2). In BMM2 (1% (v/v) in methanol) approximately 12 h prior to the
a next step, the marker was recycled by inducing the FLP start of the bioreduction reaction. The BDH1-mediated biore-
recombinase. Therefore, a single colony from a correct knock- duction was started by adding BMM10 (5% (v/v) methanol)
out strain was used to inoculate 50 mL of YPD media (250 mL containing 250 mM rac-acetoin. After a reaction time of 16 h,
baffled shake flask) and grown for 24 h at 28 °C. After 24 h of 300 µL of the reaction supernatant were extracted twice with
growth, methanol was added to a final concentration of 0.5% to 400 µL ethyl acetate containing 50 mM n-butanol as internal
start recombinant protein production, which was maintained by standard for the determination of acetoin conversions. The
daily methanol addition for 96 h. An aliquot of these cultures combined organic phases were dried over Na2SO4 and
was plated on YPD agar plates to obtain single colonies. These subjected to GC-FID analyses as described below.
were subsequently streaked out on YPD agar plates supple-
mented with 100 mg/L ZeocinTM to identify clones that had Kinetic studies of biotransformations
excised the cassette and, thus, were ZeocinTM-sensitive. The Biotransformations in shake flasks were conducted with strains
marker recycling was further validated by PCR using primers harboring one copy of the BDH1 expression cassette as deter-
that bind in the genomic region directly up- and downstream of mined by quantitative real-time PCR [25]. The corresponding
the deleted locus (see Supporting Information File 1, Table S3) strains were grown for 60 h in 200 mL BMD medium (2 L
and Sanger sequencing of the resulting PCR product.
baffled shake flasks) and protein expression was started by the
addition of 20 mL BMM10 approximately 12 h prior to the start
of the bioreduction reaction. For BDH1 catalyzed acetoin
Growth rate studies
Liquid Pichia cultures were grown in buffered minimal medium conversion, cells corresponding to 3000 OD600 units were
containing 200 mM KPi (pH 6.0), 13.4 g/L yeast nitrogen base harvested by centrifugation (3000g, 10 min, rt) and resus-
and 0.4 mg/L biotin supplemented either with 2% (w/v) pended in 50 mL of buffered minimal medium. The reaction
D-glucose (BMD), 1% (w/v) glycerol (BMG) or 0.5% (v/v) was started by adding 10 mL of the substrate solution (300 mM
methanol (BMM). Growth rates of the generated das knock-out rac-acetoin, 50% (v/v) methanol, 200 mM KPi, pH 6.0).
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Schematic representation of knock-out cassette architecture
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The research leading to these results has received funding from
the Innovative Medicines Initiative Joint Undertaking project
CHEM21 under grant agreement n°115360, resources of which
are composed of financial contribution from the European
Union’s Seventh Framework Programme (FP7/2007-2013) and
EFPIA companies’ in kind contribution. In addition, the work
has been supported by the Federal Ministry of Science,
Research and Economy (BMWFW), the Federal Ministry of
Traffic, Innovation and Technology (bmvit), the Styrian
Business Promotion Agency SFG, the Standortagentur Tirol,
the Government of Lower Austria and ZIT - Technology
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