Metabolic Engineering xxx (xxxx) xxx–xxx

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Metabolic Engineering
journal homepage: www.elsevier.com/locate/meteng

Review

Metabolic engineering of Pichia pastoris

David A. Peñaa,b, Brigitte Gassera,b,c, Jürgen Zanghellinia,b, Matthias G. Steigera,b,
⁎
Diethard Mattanovicha,b,
a
Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria
b
Austrian Centre of Industrial Biotechnology, Vienna, Austria
c
CD-Laboratory for Growth-Decoupled Protein Production in Yeast, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria

A R T I C LE I N FO A B S T R A C T

Keywords: Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn.
Pichia pastoris Komagataella spp.) has been increasingly employed as a platform to produce metabolites of varying origin. We
Komagataella summarize here the impressive methodological developments of the last years to model and analyze the me-
Metabolic engineering tabolism of P. pastoris, and to engineer its genome and metabolic pathways. Efficient methods to insert, modify
Synthetic biology
or delete genes via homologous recombination and CRISPR/Cas9, supported by modular cloning techniques,
Systems biology
Bioeconomy
have been reported. An outstanding early example of metabolic engineering in P. pastoris was the humanization
of protein glycosylation. More recently the cell metabolism was engineered also to enhance the productivity of
heterologous proteins. The last few years have seen an increased number of metabolic pathway design and
engineering in P. pastoris, mainly towards the production of complex (secondary) metabolites. In this review, we
discuss the potential role of P. pastoris as a platform for metabolic engineering, its strengths, and major re-
quirements for future developments of chassis strains based on synthetic biology principles.

1. Introduction Komagataella strains employed in biotechnological applications.

The yeast commonly known as Pichia pastoris has first been de-
scribed as Zygosaccharomyces pastori by the French mycologist and cy-
tologist Alexandre Guilliermond (Guilliermond, 1920). In the 1950s,
Herman Phaff isolated several related strains from oak trees in Cali-
fornia, and renamed the species as Pichia pastoris (Phaff et al., 1956).
Such as only few other yeasts, it shows a distinct ability to grow on
methanol as the sole carbon and energy source, which is based on
several highly overexpressed genes encoding the enzymes of the me-
thanol assimilation and dissimilation pathways (Wegner and Harder,
1987). In the 1980s the feature to highly express genes such as AOX1
(encoding for methanol oxidase, the first step of methanol utilization)
only when methanol is present, was utilized to develop a strong and
methanol inducible expression system (Cregg et al., 1985). In 1995 P.
pastoris was re-classified into the newly established genus Komagataella
(Yamada et al., 1995), which was later split into several species
(Kurtzman, 2005), so that former P. pastoris strains now split up into the
two species K. pastoris and K. phaffii. Today six species of the genus
Komagataella are described (Naumov et al., 2013). Thus, the widely
accepted name Pichia pastoris stands for at least two different species.
To avoid confusion and to include all strains used in biotechnology we
use the established name P. pastoris here as a synonym for all
At a time when the genome of Saccharomyces cerevisiae was se-
quenced (Goffeau et al., 1996), laying foundations for metabolic en-
gineering in this yeast (Nielsen, 1998), nearly nothing was known of
genomics, metabolism (except for methanol metabolism) and cell
biology of P. pastoris. It took until 2009 when two genome sequences
were published (De Schutter et al., 2009; Mattanovich et al., 2009),
followed by further (re)-sequencing and re-annotation of several
strains: both the K. pastoris type strain CBS704/DSMZ70382 and K.
phaffii CBS7435 and its commercial variant GS115 (Kuberl et al., 2011;
Love et al., 2016; Sturmberger et al., 2016; Valli et al., 2016). Thor-
oughly annotated genome sequences are available at www.
pichiagenome.org and have formed the basis for detailed systems
biology analyses of P. pastoris since then (Zahrl et al., 2017).
P. pastoris is a methylotrophic yeast, capable to oxidize methanol for
energy production, and to assimilate it as sole carbon source for growth
and product formation. After oxidation of methanol to formaldehyde,
assimilation begins with the reaction of dihydroxyacetone synthase, a
specialized transketolase, transferring formaldehyde to xylulose-5-
phosphate (Xu5P), resulting in glyceraldehyde-3-phosphate and dihy-
droxyacetone. Xu5P is then regenerated via a cyclic pathway (called the
Xu5P cycle) involving reactions of the pentose phosphate pathway.
Russmayer et al. (2015a) showed recently that the entire Xu5P cycle is

⁎
Corresponding author at: Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
E-mail address: diethard.mattanovich@boku.ac.at (D. Mattanovich).

https://doi.org/10.1016/j.ymben.2018.04.017
Received 22 February 2018; Received in revised form 16 April 2018; Accepted 23 April 2018
1096-7176/ © 2018 Published by Elsevier Inc. on behalf of International Metabolic Engineering Society.

Please cite this article as: Peña Navarro, D.A., Metabolic Engineering (2018), https://doi.org/10.1016/j.ymben.2018.04.017
D.A. Peña et al.
localized in peroxisomes, utilizing a set of specialized enzymes encoded
by duplicated genes that acquired transcriptional regulation by me-
thanol. This finding sheds light on the potential benefit of compart-
mentalization of an entire specialized pathway and may be utilized for
metabolic pathway engineering in the future.
Besides methanol, glucose and glycerol are frequently used as
carbon sources. Glycerol is utilized quite fast (qSmax ca. 0.37 h−1 in
mineral media) with a high biomass yield making it a useful substrate to
accumulate biomass, recombinant proteins and potentially metabolites.
The presence of four putative H+/glycerol symporters encoded in the
genome is an explanation for the efficient utilization of glycerol
(Mattanovich et al., 2009).
Glucose uptake is limited in P. pastoris compared to S. cerevisiae,
with a nearly tenfold difference in maximum specific glucose uptake
rates (qSmax ≈ 0.35 h−1 and 2.88 h−1, respectively). Under fully
aerobic conditions the glycolytic flux does not exceed the respiratory
capacity, so that nearly no fermentative by-products are formed
(Hagman et al., 2014). P. pastoris is therefore classified as a canonical
Crabtree-negative yeast. This limitation of glucose uptake in compar-
ison to S. cerevisiae has been ascribed to the low number of hexose
transporter genes and a potentially more rigid regulatory network of
carbon metabolism. The lower glycolytic flux comes with a higher
fraction of pentose phosphate pathway (PPP) flux with a split ratio of
40–50% PPP flux (Baumann et al., 2010; Nocon et al., 2016). This
provides much more NADPH regeneration per carbon compared to S.
cerevisiae, which has a split ratio in glucose surplus conditions in the
range of 4–17% (Gombert et al., 2001; Maaheimo et al., 2001;
Velagapudi et al., 2007). Apart from these fundamental differences in
glucose utilization it was shown recently that P. pastoris reduced its
maintenance demand threefold under extreme calorie restriction
(Rebnegger et al., 2016) different to S. cerevisiae where no change was
observed (Vos et al., 2016).
This review is intended as a comprehensive summary of the devel-
opment and current state of the art of metabolic engineering in P.
pastoris and the underlying specific techniques to analyze and engineer
its metabolism. For comprehensive reviews on cell engineering meth-
odology and process design, the reader is referred to reviews by
Schwarzhans et al. (2017a) and Yang and Zhang (2018). The first one
focusses on metabolic and cell engineering, systems biology and an
extensive discussion of clonal variability. The latter addresses most
specifically bioreactor cultivation design as a tool for optimization of
protein production. More specific reviews on P. pastoris engineering
focused on glycoengineering (Laukens et al., 2015), mathematical
modeling (Theron et al., 2018), systems biotechnology (Zahrl et al.,
2017) and CRISPR-Cas technologies in different yeasts (Raschmanová
et al., 2018).
Based on conclusions from past achievements, novel developments
will be discussed here in the light of recent synthetic biology progress,
drawing conclusions for the upcoming potential of this yeast for
modern metabolic engineering.
2. Methods of metabolic engineering of P. pastoris
Until recently, specific methods to engineer and analyze the meta-
bolism of P. pastoris have been quite underdeveloped, allowing only
single solutions to specific questions rather than targeted construction
of chassis platforms for advanced metabolic engineering. Since the
publication of the first genome sequences in 2009 this development has
gained great momentum. This section summarizes the current state of
the art of methods to quantify intracellular metabolites and metabolic
fluxes, to model and predict metabolic states, and to engineer cellular
metabolism of P. pastoris for the production of new biomolecules or the
enhanced productivity and quality of heterologous proteins. Based on
the impressive methodological developments, enabling advanced
Design-Build-Test cycles for metabolic engineering, P. pastoris holds a
position at the forefront of synthetic biology.
2
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2.1. Quantification of cellular metabolism
2.1.1. Metabolomics
The set of metabolites synthesized and taken up by an organism
constitute its metabolome (Fiehn, 2002). “The metabolome can be de-
fined as the complete complement of all small molecule (< 1500 Da)
metabolites found in a specific cell, organ or organism” (Wishart,
2007). This definition by Wishart includes all small metabolites but
excludes polymers like DNA, RNA, proteins, polymeric carbohydrates
and others. Importantly, also metabolites detected and not necessarily
synthesized by the organism are included in this definition. Metabo-
lomics is the technology used to measure and describe qualitatively and
quantitatively the metabolic state of a cell. It directly provides in-
formation about alterations in the cellular metabolism and can reveal
potential targets for metabolic engineering. However, compared to
other omics technologies like transcriptomics or genomics, metabo-
lomics has the challenge that the chemical diversity of metabolites is
substantially larger. For genomics, transcriptomics and to a lesser ex-
tent for proteomics, the analytes (DNA, RNA, proteins) are chemically
homogenous, which facilitates the establishment of a general metho-
dology to quantify these entities.
For P. pastoris, a first benchmark study providing quantitative me-
tabolite data was published 2012 by Carnicer et al. (2012a). In this
work more than 30 metabolites including amino acids, organic acids
and some sugar phosphates were quantified after controlled cultivation
in a chemostat, using an optimized quenching protocol. Importantly, an
interspecies comparison between S. cerevisiae and P. pastoris was per-
formed, highlighting some differences between the two species. While
the metabolite concentrations of upper glycolysis and the tricarboxylic
acid (TCA) cycle were comparable, the concentrations of metabolites of
the lower part of glycolysis (2-phosphoglycerate/3-phosphoglycerate
and phosphoenolpyruvate pools) were lower in P. pastoris. Interestingly,
also the pool of trehalose-6-phosphate was significantly larger in S.
cerevisiae compared to P. pastoris (Carnicer et al., 2012a).
An important part in metabolomics is dealing with sample pre-
paration techniques due to two main reasons: first, as already men-
tioned, chemically heterogeneous compounds need to be analyzed, and
second, the cellular metabolism needs to be arrested rapidly to measure
metabolites with a high turnover rate. In order to stop cellular meta-
bolism, cells are quenched and consecutively washed to remove ex-
tracellular metabolites. During this procedure it is important that cells
are not retained for a long period of time in the pure quenching solution
as this leads to substantial leakage of metabolites to the quenching
solution. For P. pastoris cells, a rapid filtration and washing step on a
filter is best suited to remove extracellular metabolites and reduce
unwanted leakage effects (Russmayer et al., 2015b). The initial
quenching procedure established by (Carnicer et al., 2012a) for P.
pastoris using 60% (v/v) methanol was further optimized by buffering
the quenching solution to reduce the leakage of metabolites during the
quenching process. In order to provide a framework for further buffer
optimizations the parameters influencing the leakage of a certain me-
tabolite were investigated. Hereby, it was found that size and charge-
related properties of a metabolite play a major role in controlling me-
tabolite loss, which are: molecular weight, the van-der-Waals volume,
the charge-weighted positive surface area, charge-weighted negative
surface area and the total polar surface area. Depending on the main
research question and the metabolites of interest, these influence fac-
tors can help to decide which buffer systems are best suited. The buffer
system that has shown so far the best properties towards reducing
leakage losses is Tris, buffered (0.125 M) to a pH of 8.2, with a NaCl
concentration of 0.055 M and a methanol concentration of 60% (v/v)
(Mattanovich et al., 2017). To gain metabolites out of the quenched
biomass for their analysis, the extraction method of choice employs
boiling ethanol (Carnicer et al., 2012a). However, in another early
study a direct comparison of boiling ethanol and repeated freeze-thaw
with methanol plus sonication showed that also the freeze-thaw
D.A. Peña et al.
extraction method can be as efficient as the boiling ethanol procedure
(Tredwell et al., 2011).
In order to accurately quantify the absolute concentrations of me-
tabolites, internal standards are used which are either labeled with 13C
or 15N. 13C labeled standards are preferred for the obvious reason that
carbon is present in most cellular metabolites. As for many metabolites
such labeled standards are commercially not available, it is common
practice to utilize a uniformly 13C- labeled cell extract (Lu et al., 2017).
The preparation of such a standard for P. pastoris and its application
during the quenching and extraction procedure was described by
Neubauer et al. (2012).
Metabolomic datasets can be used for different research applica-
tions. One aspect is to analyze the metabolite concentrations in strains
producing recombinant proteins compared to control strains (Jorda
et al., 2014). For example, analysis on protein producing strains sup-
plemented with all amino acids showed that costly amino acids were
preferentially taken up (Heyland et al., 2011), suggesting a regulatory
logic applied under the limited energy and redox availability caused by
recombinant protein production. Another research question was to
characterize the metabolite levels on different carbon sources, espe-
cially on methanol for P. pastoris, compared to glucose or glycerol
conditions. Connected to this aspect, a quantitative analysis of in-
tracellular free metabolites has been used to study the metabolic impact
of assimilation of methanol as a carbon source (Russmayer et al.,
2015a). Under methanol assimilation conditions, also the special me-
tabolite sedoheptulose-1,7-bisphosphate was detected, which is re-
quired for the xylulose-monophosphate cycle enabling methanol as-
similation.
In many metabolomic studies, the main focus is on metabolites of
the central carbon and energy metabolism, which are typically com-
prising the metabolite classes of amino acids, organic acids, sugar
phosphates and alcohols. However, a study investigating the physiolo-
gical impacts of hypoxia also focused on the lipid metabolism and on
the so called lipidome. Changes in lipid metabolites including fatty
acids, phospholipids and sphingolipids were quantified under normoxic
and hypoxic conditions showing significant differences (Adelantado
et al., 2017).
Most studies conducted with P. pastoris measuring intracellular
metabolites levels were targeted approaches. In contrast, Tredwell et al.
(2017) followed an untargeted strategy using NMR to evaluate the
impact of recombinant protein production on metabolomic profiles.
Thereby, the untargeted NMR metabolic profiling strategy was com-
bined with a transcript analysis of unfolded protein response (UPR)
relevant gene transcripts (HAC1, KAR2, PDI1). Correlations between
these UPR markers and certain metabolite signals (isoleucine, aspartate,
arabitol) were detected assuming that such data can be used as in-
dicators to detect UPR stress. The benefit of replacing transcript ana-
lysis of one or a few genes with a metabolomics method remains
however to be proven.
2.1.2. Metabolic flux analysis
13
C metabolic flux analysis is a powerful tool to experimentally
determine the flux distribution in a cell (Sauer, 2006; Zamboni et al.,
2009). The methodology is used on a frequent basis to measure the
metabolic flux distributions in P. pastoris, and Table 1 gives an overview
about 13C metabolic flux experiments carried out with this organism.
In order to calculate fluxes, it was first necessary to verify which
reactions occur in the central carbon metabolism of P. pastoris. As
mentioned earlier, genome data for P. pastoris was not available until
2009. Therefore, the first labeling experiments were also used to
identify the biochemical pathways and to investigate if they differ from
S. cerevisiae. It was found that the proteinogenic amino acids in P.
pastoris are in principle synthesized as in S. cerevisiae and that the re-
solved flux ratios of P. pastoris are more similar compared to S. cerevisiae
than to Scheffersomyces (Pichia) stipitis (Solà et al., 2004). However,
later it was found that the leucine pathway has a different
3
Metabolic Engineering xxx (xxxx) xxx–xxx
compartmentalization compared to S. cerevisiae. This information is
especially important for flux analysis experiments as the leucine pre-
cursor alpha-isopropylmalate is synthesized from alpha-ketoisovalerate
consuming cytosolic acetyl-CoA instead of mitochondrial acetyl-CoA
(Förster et al., 2014). Current experiments show that also other amino
acid biosynthesis pathway enzymes have a different compartmentali-
zation in P. pastoris compared to S. cerevisiae. In particular, biosynthetic
enzymes of the arginine and lysine pathways were found in the per-
oxisome or cytosol rather than in mitochondria (own unpublished
data). In a recent publication comparing S. cerevisiae and the methy-
lotrophic yeast Hansenula (Ogataea) polymorpha it was highlighted that
it is important to carefully evaluate compartmentalization constraints
in modeling approaches and not to extrapolate assumptions regarding
compartmentalization from other organisms without further evidence
(Lehnen et al., 2017).
In order to obtain absolute flux values, it is necessary to determine
the biomass composition of the cell, besides other important parameters
such as metabolite uptake and secretion rates. In 2009, the macro-
molecular composition of P. pastoris cells under different oxygenation
conditions was investigated and compared to a strain producing a re-
combinant protein. It was found that the amino acid composition of P.
pastoris is significantly different from the amino acid composition de-
termined for the yeast S. cerevisiae. Furthermore, it was found that
different oxygenation levels have an impact on the general biomass
composition and need to be taken into account (Carnicer et al., 2009).
The determined biomass datasets were integrated into a stoichiometric
model together with flux ratios of a METAFoR analysis yielding abso-
lute fluxes (Baumann et al., 2010). Biomass composition differs mark-
edly in cells grown on different carbon sources as well, which should be
taken into consideration for metabolic flux balancing (Tomàs-Gamisans
et al., 2018).
Different analytic techniques can be used to analyze labeling pat-
terns of 13C labeling experiments. Due to the high abundance of pro-
teinogenic amino acids, NMR (Solà et al., 2004) as well as GC-MS based
techniques (Heyland et al., 2011) were used to determine the labeling
profile in P. pastoris cultures. Later also techniques were established to
directly measure the labeling pattern of free intracellular metabolites
based on GC-MS (Mairinger et al., 2015; Mairinger et al., 2018) and LC-
MS (Jorda et al., 2013).
13
C flux measurements were often used to determine the flux
changes between a protein producing strain compared to a control
strain (Table 1). In several studies, the flux profile in protein producing
P. pastoris was shown to change towards an up-regulation of the PPP
pathway. Consistently, the engineering of the PPP pathway by over-
expression of the first two enzymatic steps (encoded by ZWF1 and
SOL3) further increased recombinant protein production as discussed in
Section 3.2 (Nocon et al., 2016).
2.2. Metabolic modeling
Various (ad hoc) modeling efforts, very recently reviewed by Theron
et al. (2018), have been undertaken to understand and optimize espe-
cially protein production in P. pastoris. Here we outline recent devel-
opments in the modeling and genome-wide analysis of P. pastoris me-
tabolism. Constraint-based analysis is a key approach in the systems
analysis of metabolism and has proven extremely useful in providing
mechanistic insights into metabolism. Together with (genome-scale)
metabolic models, constraint-based analysis enables the prediction of
intracellular steady-state flux distributions and the study of the geno-
type-phenotype relations in silico (Lewis et al., 2012).
For P. pastoris several genome-scale metabolic models are currently
available (Caspeta et al., 2012; Chung et al., 2010; Saitua et al., 2017;
Sohn et al., 2010; Tomas-Gamisans et al., 2016; Tomàs-Gamisans et al.,
2018; Ye et al., 2017). The latest two genome-scale metabolic models
represent two independent consensus reconstructions that integrated
and updated the knowledge of the previous reconstructions. While
 D.A. Peña et al.

Table 1
13
Main studies applying C metabolic flux analysis to P. pastoris.
13
Research question related to C labeling experiment Labeled substrate Measured metabolites Analytical procedure Ref.

Verification of amino acid biosynthesis pathways and flux ratio analysis after growth 10% (w/w) of [U-13C6] glucose or [U-13C3] glycerol Proteinogenic amino acids NMR (2D [13C,1H]- (Solà et al., 2004)
on either glucose and glycerol COSY)
Analysis and regulation of the methanol metabolism in the presence of a second carbon different mixtures of glycerol and methanol, always 10% (w/w) Proteinogenic amino acids NMR (2D [13C,1H]- (Solà et al., 2007)
source of [U-13C3] glycerol and [U-13C1] methanol COSY)
Flux changes between a protein producing strain and a control strain in fed-batch 10% (n/n) [U-13C6] glucose Proteinogenic amino acids GC-MS (Heyland et al., 2010)
experiments
13
Amino acid supplementation to unburden cellular metabolism during protein 10% (n/n) [U-13C6] C- glucose Proteinogenic amino acids GC-MS (Heyland et al., 2011)
production
The metabolic flux distribution during growth on a mixed feed of glucose and 12% (w/w) [U-13C6] glucose and [U-13C1] methanol Proteinogenic amino acids NMR (1H-13C-HSQC) (Jordà et al., 2012)
methanol while producing recombinant protein

4
13 13
Mapping of metabolic fluxes of glycolysis, pentose phosphate and methanol 80% [1– C1] glucose and 20% [U- C6] glucose; 100% Intracellular metabolites GC-MS, LC-MS (Jorda et al., 2013)
assimilation pathways [U-13C1] methanol
Comparison of fluxes between recombinant protein producing strain (Rol) and control 80% [1–13C1] glucose and 20% [U-13C6] glucose; 100% Intracellular metabolites GC-MS, LC-MS (Jorda et al., 2014)
strain in chemostat [U-13C1] methanol
Comparison of fluxes between high expression recombinant protein producing strain 80% [U-13C] glucose, 20% [1–13C] glucose Intracellular free amino GC-MS (Nie et al., 2014)
(beta-galactosidase) and low expression control strain acids
Comparison of fluxes between recombinant protein producing strain (hSOD) and 17% [U-13C6] glucose; Proteinogenic amino acids GC-MS (Nocon et al., 2014)
control strain
13 13
Flux distribution in chemostat cultivations of P. pastoris comparing glucose to a mixed 20% [U- C6] glucose; 20% [U- C1] methanol and 20% Proteinogenic amino acids GC-MS (Russmayer et al., 2015a)
substrate of methanol/glycerol [U-13C3] glycerol;
Measurement of split ratios between glycolysis and pentose phosphate pathway (PPP) 100% [1,6–13C; 2,3,4,5–12C] glucose; Intracellular metabolites GC-MS (Nocon et al., 2016)
analyzing strains engineered in the PPP
Differences in fluxes comparing medium with and without glutamate 80% [U-13C] glucose, 20% [1–13C] glucose Intracellular free amino GC-MS (P. Liu et al., 2016)
acids
13
High anabolic use of the TCA cycle on glucose and comparison of flux profile to S. 10% (w/w) U- C labeled Proteinogenic amino acids NMR (Zhang et al., 2017)
cerevisiae and P. stipitis
Metabolic Engineering xxx (xxxx) xxx–xxx
D.A. Peña et al.
Tomàs-Gamisans et al. (2018) focused on extending the phenotypic
capabilities by providing accurately measured biomass compositions
during growth on glucose, glycerol and methanol as sole carbon source,
Ye et al. (2017) put their focus on increasing the coverage of their
model, which currently contains 1243 annotated genes, 2407 reactions,
and 1740 metabolites. In comparison, the latest version of the con-
sensus genome-scale reconstruction of S. cerevisiae, YEAST v7.0 (Aung
et al., 2013) contains 910 genes, 3498 reactions and 2384 metabolites.
These recent model expansions led to an improved predictability of P.
pastoris growth capabilities over a wide range of different carbon and
nitrogen sources in industrially relevant conditions. Overall, current
genome-scale metabolic models of P. pastoris allow one to robustly
predict cellular growth rates. In fact, these models were already used to
guide rational metabolic engineering projects, e.g., to improve re-
combinant protein production (Nocon et al., 2014; see Section 3.2 for
details), or to support culture media optimization (Matthews et al.,
2018). To improve the predictive quality of these models not only with
respect to growth but also with respect to recombinant protein yield,
Irani et al. (2016) attempted to reconstruct the native as well as the
humanized N-glycosylation pathways in P. pastoris. Although including
N-glycosylation into the analysis reduced the predicted protein yield
(compared to a purely metabolic analysis), the model typically over-
estimated achievable protein yields by orders of magnitude. Thus, fu-
ture development will need to take other processes (e.g., protein folding
and secretion efficiencies) into account in order to realistically model
protein production.
Constraint-based modeling provides means to analyze the metabolic
steady-state behavior determined by the stoichiometric constraints of
the biochemical reaction network, while regulatory constraints are
often not considered. In the case of P. pastoris the situation is ag-
gravated by the fact that knowledge on metabolic regulation specific to
P. pastoris is all but known. Computational methods that aim to com-
pensate these deficiencies by the integration of multi-omics data sets
have so far proven unsuccessful (Machado and Herrgård, 2014).
Moreover, recent findings by (Hackett et al., 2016) questioned the
importance of transcriptional regulation on changes in nutrient condi-
tions at least in S. cerevisiae. They report that many changes in flux
levels are due to changes in the metabolome (by Michaelis-Menten-like
kinetics) rather than changes in the enzyme levels. These observations
motivate the increased interest in the development of large-scale kinetic
models of metabolism (Vasilakou et al., 2016).
A (quasi-)dynamic analysis of P. pastoris was recently presented by
Saitua et al. (2017). It coupled the uptake and secretion dynamics of
key metabolites with the steady-state behavior of metabolism in a well-
established framework known as dynamic flux-balance analysis
(Höffner et al., 2013; Sánchez et al., 2014). By doing so, the authors
could analyze the dynamic rearrangements in the intracellular flux
distributions. Moreover, they derived optimized feeding strategies and
beneficial single gene deletion strategies that led to a predicted increase
in production of recombinant human serum albumin. However, the
experimental viability of the predictions and the computational scal-
ability of the method to predict more complex intervention strategies
remain to be further investigated.
Next to dynamic constraints, (global) resource allocation constraints
have received much attention in recent years (Berkhout et al., 2013;
Goelzer and Fromion, 2011; Molenaar et al., 2009; O'Brien and Palsson,
2015). In essence, these methods try to take enzyme levels into account
and assign a “price” on the expression of each protein/enzyme. As total
resources are limited, cells have to carefully adjust their resource al-
location, which – in turn – allows one to calculate optimal enzyme
distributions and corresponding flux distributions. Although im-
pressively accurate results were achieved in Bacillus subtilis (Goelzer
et al., 2015), Escherichia coli (O'Brien et al., 2013) and S. cerevisiae
(Sánchez et al., 2017) resource allocation has not been studied in P.
pastoris yet as these methods tend to be quite data intensive. They re-
quire i.a. estimations of catalytic rates, detailed knowledge of protein
5
Metabolic Engineering xxx (xxxx) xxx–xxx
synthesis steps, and/or absolute protein abundances – data that is not
readily available for non-classical model organisms.
2.3. Tools for metabolic engineering and synthetic biology of P. pastoris
2.3.1. Genomic integration: prerequisite for genetic engineering
Applying metabolic and cell engineering strategies requires ad-
vanced tools to perform genetic modifications and to introduce syn-
thetic pathways into the host (Wagner and Alper, 2016). Since the early
days of P. pastoris research, gene overexpression has mostly been per-
formed by stably introducing the gene expression cassette into the
genome, ideally into a targeted locus via a single crossover knock-in
strategy based on homologous recombination (HR). Although HR effi-
ciencies are lower in comparison to S. cerevisiae, the number of trans-
formants obtained is usually sufficient to select strains for protein
production. Using the standard electroporation-based transformation
protocol described in Gasser et al. (2013), adapted from Cregg and
Russell (1998), or the high-efficiency protocol applying pretreatment
with lithium acetate prior to electroporation (Wu and Letchworth,
2004), transformation efficiencies of 103–104 colonies per µg linearized
DNA are typically obtained, with around 85% of the obtained trans-
formants containing the expression cassette in single or multiple copies.
Methods to increase multi-copy transformants which are often asso-
ciated with higher gene expression and product levels have been re-
cently reviewed by Piva et al. (2017).
Also the generation of gene knockouts was described early on, e.g.
for protease-deficient mutants (Gleeson et al., 1998) or AOX1/2-defi-
cient strains (Cregg et al., 1989), however, targeting efficiencies have
often been a bottleneck for this purpose. While in principle also the
knock-in approach can be used to disrupt gene functions (Vervecken
et al., 2004) in most cases generation of knock-outs relies on gene re-
placement by double crossover (Da Silva and Srikrishnan, 2012). De-
pending on the genomic locus to be disrupted and the length of
homologous flanking regions, gene replacement efficiencies can range
from < 0.1–80% (Nett and Gerngross, 2003; Schwarzhans et al., 2016;
Vogl et al., 2018a), and often a high number of false positive trans-
formants was observed. For obvious reasons genes are easily dis-
ruptable when their deletion has no negative impact on strain fitness
while the disruption of genes with a negative impact on growth leads to
much lower frequencies of positive clones. In the latter case positive
clones face a negative selection pressure during cultivation so that
clones with incorrect integration into random loci may dominate. As
long as sufficiently long homologous arms (approximately 1000 bps on
each side) are provided, the probability of obtaining correctly in-
tegrated clones is still rather high, however, if short or no flanking
regions are presented non-homologous end joining (NHEJ) is prevailing
(Näätsaari et al., 2012; own unpublished data). Deletion of the NHEJ
machinery allowed to use significantly shorter homologous flanking
regions (down to 250 bps on each side). However, the downside of this
approach is that a specific genetic background bearing the Δku70 mu-
tation needs to be used, which shows 10–30% reduced growth rates,
reduced transformation efficiencies and decreased stress tolerance due
to impaired DNA damage repair (Näätsaari et al., 2012; Weninger et al.,
2018).
In contrast, the split-marker approach (Heiss et al., 2013) and
genome editing by CRISPR/Cas9-mediated homology directed repair
HDR (Gassler et al., In press; Weninger et al., 2018, 2016) improve or
trigger HR, independent of the strain background. The latter has also
the advantage of marker-free genetic manipulations. Alternatively, re-
combinase-based marker recycling strategies such as Cre/loxP (Marx
et al., 2008; Pan et al., 2011) and FLP/FRT (Cregg and Madden, 1989;
Näätsaari et al., 2012; Perez-Pinera et al., 2016) are available for P.
pastoris. While episomal plasmids are usually not in use for the ex-
pression of recombinant genes due to their segregational instability in
non-selective conditions (Hong et al., 2006; Liachko and Dunham,
2014; Sreekrishna et al., 1987), they proved to be an excellent choice
D.A. Peña et al.
for genome engineering approaches such as the expression of Cas9 or
Cre recombinase. Efficient curing of the episomal plasmid can be
achieved during one round of plating, meaning that the final production
strain is free of the DNA modifying enzyme.
2.3.2. Genome editing with CRISPR/Cas9
In addition to gene replacements and targeted integration, CRISPR/
Cas9 can be used to create indel mutations via the NHEJ DNA repair
mechanism at targeted genomic loci if no homology target (also called
donor DNA) is provided. Frequencies are highly dependent on the
genomic locus and the used guide RNAs, and range from 90% to 100%
for easily disruptable genomic loci such as AOX1, DAS1/2 or the gly-
cerol kinase encoding gene GUT1, but can be as low as < 5% for dif-
ficult loci and/or specific sgRNAs. It has been shown that in P. pastoris
CRISPR/Cas9 preferentially creates deletions of one single bp (and to a
lesser extent 2 or 3 bps) upstream of the Cas9 cleavage position, in-
dicating a difference to S. cerevisiae where insertions and deletions of
different lengths (DiCarlo et al., 2013) were observed.
Independently, both Weninger et al. (2016) and Gassler et al. (In
press) found that Cas9 exerts some toxicity to the host cells. Corre-
spondingly, the use of weaker promoters for Cas9 expression (such as
PPFK300 or PLAT1) was shown to enhance transformation efficiency and
growth rate (Prielhofer et al., 2017). Furthermore, both groups strongly
recommend testing at least 2 or 3 different guide RNAs per locus. Also,
the capacity for multiplexing (targeting two or more loci simulta-
neously) has been demonstrated, with slightly reduced targeting effi-
ciencies (70% compared to approximately 90%) compared to single
sgRNA expression (Weninger et al., 2016).
CRISPR/Cas9-mediated HDR allows the efficient and selection
marker-free integration of DNA fragments into the genome with the
possibility to replace, disrupt or tag a specific genomic locus (Singh
et al., 2017). Thereby researches take advantage of the fact that the
double-strand break generated by Cas9 recruits the HR machinery and
thus increases HR efficiency at the targeted locus. Weninger et al.
(2018) suggested the use of a ku70 background strain to achieve high
efficiency of marker-free HDR. However, this reduces transformation
efficiencies drastically and is not suited to generate indel mutations.
The Cas9/sgRNA episomal plasmid described by Gassler et al. (In press)
(part of the CRISPi kit available at Addgene #1000000136) and linear
donor DNA fragments with flanking regions of approximately 1000 bp
each enabled targeting efficiencies above 50% on a routine basis, si-
milar to indel generation, in a wild type strain. Please note that genes
essential for growth can still not be deleted by this method, however, in
such a case Cas9-mediated indels (as described above) or dCas9-medi-
ated CRISPR interference (Lian et al., 2018; Qi et al., 2013) might be
suitable to diminish gene function.
For genes especially hard to knock-out such as och1 (Krainer et al.,
2013; Nett and Gerngross, 2003; Vervecken et al., 2004), where also
CRISPR/Cas9 only yielded 50% mutation efficiency (Weninger et al.,
2016), also more time and work intensive indirect methods might be
considered. Examples are the simultaneous expression of the gene to be
knocked out from an episomal plasmid (Chen et al., 2013) or as part of
a loxP-flanked disruption cassette (Shibui and Hara, 2017), followed by
curing of it. Another promising approach to increase HR efficiency was
the addition of hydroxyurea during cell transformation that arrests the
cells in the S/G2 phase of the cell cycle, where HR is prevalent to NHEJ
(Tsakraklides et al., 2015).
2.3.3. Novel modular cloning strategies and libraries of synthetic parts for
P. pastoris
For rapid optimization of single expression cassettes and whole
synthetic pathways, efficient and high throughput modular cloning
strategies are essential. To this end, seamless modular cloning strategies
such as Gibson assembly, Golden Gate assembly (GGA), or Restriction
site free cloning (RSFC) have been adapted and established for use in P.
pastoris. Initially GGA and RSFC were used to design or optimize single
6
Metabolic Engineering xxx (xxxx) xxx–xxx
expression units by assembly of standardized and commonly used ge-
netic parts such as promoters, secretion signals, tags and transcription
terminators (Obst et al., 2017 – MoClo Pichia Toolkit, available at
Addgene #1000000108; Schreiber et al., 2017; Vogl et al., 2015). Ad-
ditionally, a library of nuclear localization signals was reported
(Weninger et al., 2015).
The GGA toolbox was then extended to allow simultaneous ex-
pression of up to eight genes from one vector and the repertoire of
synthetic parts encompasses 20 promoters, 10 transcription termina-
tors, 5 integration loci and 4 resistance markers (Prielhofer et al., 2017)
– GoldenPiCS kit, available at Addgene #1000000133. The GoldenPiCS
kit is fully compatible with the GoldenMOCS system, which was de-
signed to perform metabolic engineering tasks in different cellular hosts
with the same cloning platform (Sarkari et al., 2017). Gibson assembly
has also been used for the same purpose, with a set of 49 promoters and
20 terminators characterized (Vogl et al., 2016). Both studies found
that it is crucial to avoid repetitive sequences (such as the same pro-
moter or terminator) in vector or pathway design in order to prevent
loop out during the transformation step, which resulted in only partial
integration of the desired pathway.
Multiple enzyme pathways can be expressed using combinatorial
assembly of gene cassettes (by Gibson assembly or the GoldenPiCS
toolkit) or using single cassettes with polycistronic expression con-
structs based on 2 A peptide (Geier et al., 2015; Siripong et al., 2018),
thus enabling efficient testing of complex heterologous pathways and
fine-tuning of enzyme expression. Due to the combinatorial nature of
multigene pathway assembly, a high number of variants is possible,
thus screening for the best combination is likely becoming the limiting
factor. To explore the possible design space, advanced high throughput
screening methods are needed.
For efficient genetic engineering and pathway expression, tunable
and differently strong promoters are a prerequisite to drive gene ex-
pression. Apart from overexpression of heterologous genes, exchange of
native promoters with other promoters is a way to overcome feedback
inhibition, optimize enzyme abundance and increase the metabolic flux
towards a product of interest (Liu et al., 2015; Marx et al., 2008). Ef-
ficient and regulatable P. pastoris promoters are often derived from
carbon-source utilization pathways such as the methanol utilization
pathway (MUT) or the rhamnose-utilization pathway (reviewed by Vogl
and Glieder, 2013). Natively, these promoters are induced by the
availability of the respective carbon source and shut off on glucose or
glycerol surplus. Although the AOX1 promoter is still the most com-
monly used control element for protein production applications, a wide
variety of other MUT promoters have been described and tested re-
cently (Gasser et al., 2015; Prielhofer et al., 2017; Vogl et al., 2016).
Together with systematically engineered promoter variants (Hartner
et al., 2008; Yang et al., 2018), and synthetic promoters (Vogl et al.,
2014) a large toolbox of synthetic parts is available, which allows
methanol-induced or in some cases also de-repressed gene expression. If
methanol-free expression systems are intended, strong constitutive
promoters such as PGAP or PTEF1 are frequently used (Vogl and Glieder,
2013). Alternatively, the PG promoter series which is induced in glu-
cose-limiting conditions as often encountered during production pro-
cesses (Prielhofer et al., 2013) or the thiamine-regulatable PTHI11 pro-
moter (Landes et al., 2016) provide for methanol-independent
regulated promoters. Vitamin or amino acid regulated promoters such
as PSER1, PMET3, and PTHR1 also allow for repression and induction of
pathways independent of the carbon source and can be used to speci-
fically down-regulate otherwise essential gene functions (Delic et al.,
2013). Additionally, less strong constitutive or regulatable promoters,
as provided e.g. in the GoldenPiCS toolbox (Prielhofer et al., 2017) can
be of interest for metabolic engineering applications and to balance
multigene expression in synthetic pathways. Altogether, more than 100
promoters have been described for use in P. pastoris.
Recently, a non-orthogonal combination of promoter engineering
(by duplication or deletion of transcription factor binding sites) and
D.A. Peña et al.
overexpression of the respective transcription factors (TF) has been
reported for PGAP (Ata et al., 2017). A similar approach was followed up
for PAOX1, where single overexpression of two out of three different TFs
involved in regulation of the MUT pathway (Mxr1, Mit1) converted P.
pastoris to methanol-free PAOX1 expression (Vogl et al., 2018b). Con-
currently, it was reported that knockout of three repressing transcrip-
tion factors (Δmig1Δmig2Δnrg1) in combination with Mit1-over-
expression renders PAOX1 methanol-independent (Wang et al., 2017). As
an example of a truly orthogonal system, a β-estradiol-inducible circuit
consisting of an estradiol-inducible zinc-finger transcription factor and
corresponding artificial binding sites was shown to be operational in P.
pastoris (Perez-Pinera et al., 2016).
Less effort was so far put into transcription terminators (TT), which
have also been implicated with mRNA stability. Recent high-
throughput screening in S. cerevisiae emphasized that TTs are important
regulatory elements which should be considered for the fine-tuning of
gene expression and metabolic pathways as terminator selection can
influence the flux through a metabolic pathway (Curran et al., 2013;
Wei et al., 2017; Yamanishi et al., 2013). Apart from the commonly
used AOX1-TT and the S. cerevisiae derived CYC1-TT, two recent studies
provided more insight into this topic in P. pastoris: while Vogl et al.
(2016) focused on TTs of MUT genes, Prielhofer et al. (2017) assessed
the efficiency of TTs of strongly expressed P. pastoris genes including
many ribosomal protein genes. All tested P. pastoris TTs showed rather
similar reporter levels, slightly exceeding ScCYC1-TT by maximum
50%. The so far tested P. pastoris terminators had a size of 174–507 bps
(Prielhofer et al., 2017; Vogl et al., 2016), whereas significantly shorter
(35–75 bps long) and even synthetic terminator sequences have been
identified for S. cerevisiae (Curran et al., 2015). As TTs seem to be
functional across yeast species (Wagner and Alper, 2016), it will be of
interest to test and implement such short TTs also into the synthetic P.
pastoris toolbox.
As stated above, the traditional method is to integrate the expres-
sion cassette(s) into the genome of P. pastoris. Only a few integration
loci have been comparatively analyzed so far (3´-AOX1, 5´-AOX1,
AOX1, 5´-ENO1, 5´-RGI2, 5´-GAP/TDH3, GUT1, rDNA locus), which all
seemed to allow gene expression from a single expression cassette at
similar strength (Prielhofer et al., 2017; Vogl et al., 2018a).
A recent approach involving in vivo self-ligation cloning of short
overlapping DNA sequences and episomal vectors (Camattari et al.,
2016) is a promising step towards in vivo single step pathway con-
struction similar as described for S. cerevisiae (Shao and Zhao, 2009),
however, efficient assembly of larger multi-gene pathways in P. pastoris
will need further optimization. Episomal vectors (using e.g. the native
P. pastoris ARS, the K. lactis Pan-ARS or the P. pastoris mitochondrial
ARS sequences) are also a useful possibility for screening experiments
e.g. for the assessment of activity of enzyme variants, however, they are
not suitable for protein production processes so far, as they require a
constant positive selection pressure due to their segregational in-
stability (Camattari et al., 2016; Liachko and Dunham, 2014;
Schwarzhans et al., 2017b).
Apart from precise genetic engineering of industrial strains, adap-
tive laboratory evolution (ALE) is a valuable tool to engineer their
genomes, especially when the desired traits are more complex and ge-
netically not well defined. Evolution and subsequent reverse en-
gineering proved to be efficient for increasing stress tolerance and
product levels in many host organisms (Dragosits and Mattanovich,
2013). So far, the application of laboratory evolution has been rather
limited in P. pastoris, which might be due to the lack of easily selectable
traits connected to protein production. In this respect, ALE has been
used to select for enhanced growth on methanol yielding some clones
that also produced more heterologous protein (Moser et al., 2017).
Mating as another approach to engineer genomes without exact
knowledge of the involved genetic traits was not fully accessible for P.
pastoris until recently. Mating type switching and the rapid haploidi-
zation after mating prevented the efficient applications of methods like
7
Metabolic Engineering xxx (xxxx) xxx–xxx
quantitative trait loci mapping (Swinnen et al., 2012). The recent de-
velopment of mutant P. pastoris strains with defined stable mating types
(Heistinger et al., 2018) further enhances the accessibility of the genetic
repertoire of this yeast.
3. Metabolic engineering applications
3.1. Engineering of protein glycosylation
Many secretory proteins are glycosylated, and it is no surprise that
glycosylation of recombinant therapeutic proteins is a major quality
issue for biopharmaceutical production. After polypeptide translocation
into the ER, glycans are either linked to asparagine residues (N-glycans)
or to serine or threonine residues (O-glycans). While the uniform core
structure of N-glycans consists of two N-acetyl glucosamine and three
mannose residues forming a branch, there is a species dependent wide
variety of sugar structures attached to the core. N-glycans of yeast
proteins, as of most fungi, mainly consist of mannose residues adjacent
to the two N-acetyl-glucosamine (GlcNAc) residues, while human pro-
teins contain so-called complex N-glycans consisting of GlcNAc, ga-
lactose and sialic acid adjacent to the core mannose residues. Concerns
that mannose-rich glycans of yeast made proteins may be immunogenic
(Jacobs and Callewaert, 2009) as well as the positive impact of complex
glycan pattern on efficacy and stability of therapeutic proteins (Fukuda
et al., 1989) prompted to engineer N-glycosylation patterns in P. pas-
toris. In fungi the first committed step towards high-mannose glycan
formation is an α-1,6 mannosylation by Och1, so that the deletion or
inactivation of this enzyme was the first engineering step towards hu-
manization of N-glycans in P. pastoris. After removal of terminal man-
nose residues by mannosidases, the complex glycan structure is built up
by the consecutive activity of GlcNAc transferases, galactosyl trans-
ferase and sialyl transferase. These steps required complex metabolic
engineering to provide both the sugar transferases and the precursors
(activated sugars) at the right cellular localization, thus ensuring the
correct consecutive additions of sugars to the glycans. Targeting of
mannosidases and sugar transferases to consecutive locations in the
secretory pathway was achieved with gene fusion libraries of fungal
membrane protein leader sequences with mannosidases and trans-
ferases of animal and fungal origin, followed by screening for correct
glycan patterns (Choi et al., 2003; Hamilton et al., 2003, 2006). The
base strain lacking Och1 activity expressed 3 recombinant genes:
Kluyveromyces lactis uridine 5′-diphosphate (UDP)-GlcNAc transporter,
the mouse mannosidase MnsIA catalytic domain fused to the N-terminal
localization peptide of the ER protein Sec12 from S. cerevisiae, and
human GlcNAc transferase GnTI fused to the leader sequence from the
S. cerevisiae Golgi protein Mnn9 (Choi et al., 2003). Additional over-
expression of Drosophila melanogaster mannosidase II (ManII) and rat
GlcNAc transferase GnTII, both fused to the N-terminal localization
signal of the S. cerevisiae Golgi protein Mnn2 lead to the predominant
formation of GlcNAc2Man3GlcNAc2 N-glycans (Hamilton et al., 2003).
Addition of galactose was achieved by overexpression of Schizo-
saccharomyces pombe galactose epimerase, Drosophila melanogaster UDP-
Gal transporter, and human β-1,4 galactosyl transferase (Li et al.,
2006). Several further steps needed to be engineered for terminal sia-
lylation (Hamilton et al., 2006). After screening more than 120 per-
mutations of genes involved in sialic acid synthesis, transport and
transfer, five enzymes were selected: human UDP-N-acetylglucosamine-
2-epimerase/N-acetylmannosamine kinase (GNE), human N-acet-
ylneuraminate-9-phosphate synthase (SPS), human CMP–sialic acid
synthase (CSS), mouse CMP–sialic acid transporter (CST), and a library
of chimeric sialyltransferases linked to yeast type-II transmembrane
localization peptides (ST). After some further optimization 90% sialy-
lated N-glycans (Sia2Gal2GlcNAc2Man3GlcNAc2) were achieved.
Similarly, (Jacobs et al., 2009) described the conversion of a wild
type P. pastoris strain to a strain producing Gal2GlcNAc2Man3GlcNAc2
N-glycans by the consecutive transformation and screening with five so-
D.A. Peña et al.
called GlycoSwitch vectors, encoding ManI (Trichoderma reesei man-
nosidase), GnTI, GalT, ManII, and GnTII, respectively.
It should be noted that any modification of N-glycosylation also
affects all native yeast proteins and thus potentially strain fitness. This
aspect was more recently discussed for OCH1 deletion strains (Krainer
et al., 2013) which should also affect further engineered strains.
However, it was shown that glycoengineered P. pastoris was able to
produce recombinant human erythropoietin (Hamilton et al., 2006) and
IgG in the lab and pilot scale (Ye et al., 2011). Recently, a strategy to
obtain simplified homogeneous glycosylation profiles in recombinant
proteins was implemented in P. pastoris. The approach, termed Glyco-
delete, is based on the expression of the fungal endoglycosidase endoT,
which specifically cleaves N-glycan structures leaving behind a single
N-GlcNAc attached to the protein (Claes et al., 2016)
Yeast O-glycosylation differs from mammalian-type O-glycosylation
as well. In P. pastoris, O-glycans consist of short linear α-1,2-linked
mannose chains with potential β-1,2-linked or phosphorylated mannose
at the outer ends. After deletion of the genes responsible for β-1,2-
mannosylation and α-1,2-phosphomannosylation, a T. reesei mannosi-
dase gene was integrated into P. pastoris, leading to single mannose O-
glycans (Hamilton et al., 2013). Following the same engineering
strategy for the addition of GlcNAc, galactose and sialic acid as de-
scribed above led to the formation of complex sialylated O-glycans in P.
pastoris (Hamilton et al., 2013).
3.2. Metabolic engineering to enhance protein production
Recombinant protein production is a metabolically costly process. It
diverts several cellular machineries from their evolutionary goal of cell
growth and maintenance, drains precursors from the central carbon
metabolism, consumes redox and energy cofactors, and can create en-
ergetic inefficiencies in the metabolism (Kafri et al., 2016; Klein et al.,
2015; Wu et al., 2016). Given that sufficient gene copies of a protein of
interest are expressed under a strong promoter, the synthesis of high
quality protein requires high cellular capacities for translation, folding,
posttranslational modifications and localization of the protein (Fig. 1).
Therefore, any of these molecular machineries and the metabolic re-
sources they consume can become a bottleneck for recombinant protein
production. Recycling of stalled cellular machineries and misfolded or
low quality protein greatly reduces cell energetic efficiency and activate
8
Metabolic Engineering xxx (xxxx) xxx–xxx
stress responses (Geiler-Samerotte et al., 2011). Additionally, high
protein production reduces cell fitness by diverting resources from the
synthesis of macromolecules needed to sustain an optimal growth rate.
Depending on the extent of growth rate reduction, this could lead to
low productivities, especially if the product is growth-coupled
(Rebnegger et al., 2014).
Metabolic engineering strategies have been applied to address the
constraints of protein production described above, by improving the
supply of precursors and increasing the cellular redox and energy effi-
ciency. A study by Nocon et al. (2014) demonstrated the use of genome-
scale metabolic modeling to predict gene knock-out and overexpression
targets in the central carbon metabolism that would increase protein
production. Many of those in-silico targets were successfully validated
experimentally in P. pastoris. These included the deletion of genes di-
verting carbon towards fermentative pathways, the overexpression of
malate dehydrogenase, which could increase the supply of mitochon-
drial NADH, and the overexpression of the PPP. Further combinatorial
overexpression of the enzymes in the oxidative part of the PPP resulted
in improved titers of protein (Nocon et al., 2016). This could be ex-
plained by an increased supply of NADPH and precursors (e.g. ery-
throse-4-phosphate or ribose-5-phosphate) or a reduced oxidative
stress. Exploring the central role of NADH for protein production in P.
pastoris, a recent study introduced a NADH oxidase from Lactococcus
lactis (noxE) to balance the excess of NADH generated from methanol
dissimilation (Jayachandran et al., 2017). The increased thermo-
dynamic efficiency of the pathway yielded a higher methanol uptake, a
corresponding upregulation of several enzymes in the methanol meta-
bolism, and a 37% higher titer of Lipase B from Candida antarctica
(CalB). However, NADH consumption by the heterologous oxidase re-
duced the supply of ATP from oxidative phosphorylation, resulting in a
lower adenylate energy charge (AEC). Upon expression of the adenylate
kinase gene ADK1 from S. cerevisiae the AEC was restored above the
wild type level, resulting in a 1.5-fold higher titer of secreted CalB
protein.
A general metabolic response to high recombinant protein produc-
tion is the depletion of intracellular amino acids concentrations
(Carnicer et al., 2012b; P. Liu et al., 2016). Thus, the supply of amino
acids can be engineered to improve protein production in P. pastoris.
For example, overexpression of GCN4, encoding a general transcrip-
tional activator of amino acid biosynthesis, was shown to double the
Fig. 1. Metabolic constraints of recombinant protein pro-
duction. (A) Genome-scale metabolic modeling related to
protein production is yet to include constraints beyond the
metabolic limitations. (B) Recombinant protein produc-
tion drains energy, precursors and redox potential from
the central carbon metabolism across all production steps.
D.A. Peña et al.
secretion of a glucose oxidase (Gu et al., 2015) or a fungal carbox-
ylesterase (Gasser et al., 2014). Furthermore, direct overexpression of
metabolic enzymes in the anabolism of serine, isoleucine, alanine and
aromatic amino acids resulted in improvement of 1.2–1.8 fold in pro-
tein accumulation and several fold increases in most of the amino acids
pools (Gasser et al., 2014). Thus, there seems to be a potential to op-
timize the synthesis pathways of amino acids by tuning enzyme abun-
dance or their kinetics (Bar-Even et al., 2011).
The metabolic burden of protein production can be alleviated by
supplying key amino acids, vitamins and fatty acids to the cell meta-
bolism (Heyland et al., 2011; P. Liu et al., 2016; Matthews et al., 2018).
This underlines the tight energetic and redox constraints imposed by
high recombinant protein production. Hence, particularly expensive
macromolecules building the cellular machinery for protein synthesis
and processing could be engineered to free up scarce metabolic re-
sources. For example, modifying the lipid composition of P. pastoris
cells by altering culture conditions has been shown to improve protein
secretion (Adelantado et al., 2017; Baumann et al., 2011). These
changes, which were more pronounced in sphingolipid and sterol
composition, were obtained by applying hypoxic conditions or an in-
hibitor of ergosterol synthesis. In the future, direct metabolic en-
gineering of lipid synthesis and degradation pathways could be ex-
plored to fine tune lipid composition for improve protein secretion in P.
pastoris.
Besides direct metabolic limitations of protein synthesis, which are
already amenable to metabolic modeling and analysis, folding and se-
cretion have been shown to be potential major bottlenecks of re-
combinant protein production (Gasser et al., 2008). As in other hosts,
many of these limitations have been addressed by cell engineering
(Zahrl et al., 2017), however actual productivities still lag behind those
predicted by metabolic models. Integration of constraints downstream
of direct metabolic costs of protein synthesis into metabolic models, as
suggested by Feizi et al. (2013), will therefore be necessary to improve
the predictions from genome-scale models (Fig. 1), and to enable sys-
tems engineering of the synthesis and secretion of proteins.
3.3. Metabolic engineering for the production of metabolites
Given the proven history of P. pastoris as an industrial cell factory
for protein production, there has been an increasing interest in ex-
ploring its potential to produce primary and secondary metabolites
(Fig. 2). Many of the published studies harnessed the distinct phy-
siology and genetic makeup of P. pastoris to introduce heterologous
enzymes and optimize native metabolic pathways. In 2006, He et al.
(2006) described the overproduction of S-adenosyl-L-methionine
(SAM), probably the first example of metabolic engineering in P. pas-
toris. SAM is a component of the sulfur metabolism acting as a methyl
group donor in several biochemical reactions. It has potential applica-
tions in the treatment of many medical conditions including Alzhei-
mer's disease, osteoarthritis and liver disease. To engineer P. pastoris for
SAM production, conversion of L-methionine to SAM was first improved
by overexpression of the native methionine adenosyltransferase gene
SAM2. Then, the gene encoding the enzyme cystathionine-β-synthase
was deleted, resulting in higher supply of the precursor homocysteine.
The combined strategies resulted in a 45-fold increase in the production
of SAM and a titer of 4.4 g/L, when methionine was supplied in the
culture. In another study, the natural D-arabitol accumulation of P.
pastoris as a redox sink (Baumann et al., 2010) and a protective re-
sponse to high osmotic conditions (Dragosits et al., 2010) was exploited
to evolve a strain with improved D-arabitol production (Cheng et al.,
2014). The resulting strain was used as platform to express hetero-
logous D-arabitol and xylitol dehydrogenases, enabling direct synthesis
of 0.29 g/L-h of the sweetener xylitol. Similarly, the metabolic pathway
of L-valine was harnessed to produce isobutanol and isobutyl acetate
from glucose (Siripong et al., 2018), in one of the few examples of bulk
chemical production in P. pastoris. In the study, a strain was first
9
Metabolic Engineering xxx (xxxx) xxx–xxx
Fig. 2. Primary and secondary metabolites produced in P. pastoris. Specific
pathways towards products already made in P. pastoris, and to their precursors
are highlighted in yellow and orange, respectively. Common metabolic nodes at
key precursors (pathways highlighted in red) indicate targets for future en-
gineering of chassis strains as platforms for the development of multiple pro-
duction strains for different metabolic products.
constructed by overproduction of three native enzymes diverting pyr-
uvate into the intermediate 2-ketoisovalerate. Then, the alcohol dehy-
drogenase gene ADH7 from S. cerevisiae and a 2-keto acid decarboxylase
gene from Lactobacillus lactis were expressed to convert 2-ketoisovale-
rate into isobutanol. Shake flask cultivations of the engineered strain
accumulated up to 2.2 g/L isobutanol, proving the functionality of the
pathway. Furthermore, isobutyl acetate was produced upon expression
of the alcohol O-acyltransferase gene ATF1 from S. cerevisiae. Also re-
cently, expression of a lactate dehydrogenase gene from Bos taurus
enabled the construction of a strain converting 63% of glycerol into
lactic acid, which was secreted by a newly characterized native trans-
porter (de Lima et al., 2016).
P. pastoris has also a distinct capacity to produce functional cyto-
chrome P450 enzymes (Geier et al., 2012). This has encouraged efforts
to engineer it as host for the synthesis of plant secondary metabolites. A
remarkable example is the production of the valuable fragrance com-
pound (+)-nootkatone from glucose (Wriessnegger et al., 2014). By
expressing the membrane associated premnaspirodiene oxygenase gene
from Hyoscyamus muticus and a cytochrome P450 reductase from Ara-
bidopsis thaliana, trans-nootkatol could be efficiently synthesized from
supplemented (+)-valencene. Next, an endogenous alcohol dehy-
drogenase was identified that efficiently converted trans-nootkatol to
(+)-nootkatone. In order to eliminate the need for (+)-valencene
supplementation, valencene synthase from Callitropsis nootkatensis was
further introduced, which in combination with a truncated hydroxy-
methylglutaryl-CoA reductase, enabled the direct synthesis of 0.2 g/L
(+)-nootkatone. The potential of P. pastoris to produce pharmaceuti-
cally relevant triterpenoides was also demonstrated with the synthesis
of dammarenediol-II from glucose (Liu et al., 2015). Dammarenediol-II
is a platform molecule that can be converted into various bioactive
ginsenosides by glycosylation and oxidation with cytochrome P450-
dependent monooxygenases. Simple expression of a dammarenediol-II
synthase from Panax ginseng (PaDDS) in P. pastoris resulted in only
0.030 mg dammarenediol-II/g dry cell weight (DCW). Hence, in order
to boost the supply of the intermediate 2,3-oxidosqualene, ERG1 en-
coding for the rate-limiting enzyme squalene epoxidase was over-
expressed, and the competing consumption of 2,3-oxidosqualene for the
synthesis of ergosterol was reduced by downregulation of the native
lanosterol synthase gene ERG7. Finally, the resulting strain reached a
D.A. Peña et al.
25-fold higher accumulation of dammarenediol-II. A subsequent study
explored the use of a protein scaffold to co-localize the squalene
epoxidase and PaDDS, which naturally localizes to subcellular lipid
particles (Zhao et al., 2016). The strategy was shown to double the yield
obtained in a strain without the scaffold, possibly due to the channeling
of 2,3-oxidosqualene towards dammarenediol-II. However, the ob-
tained yield was still almost 10-fold lower than in the approach of
pathway optimization performed by Liu et al. (2015).
In 2013, Gao and coworkers described the synthesis of 6-methyl-
salicylic acid (6MSA), the first polyketide to be produced in P. pastoris
(Gao et al., 2013). Polyketides form a large family of bioactive and
structurally complex compounds produced as secondary metabolites by
diverse organism including bacteria, fungi and plants. Production of
6MSA in P. pastoris required the functional expression of the 6MSA
polyketide synthase gene from Aspergillus terreus as well as the phos-
phopantetheinyl transferase gene from Aspergillus nidulans (npgA). The
latter is needed in yeast to activate the acyl carrier protein (ACP) do-
main of the heterologous polyketide synthase, enabling the transfer of
malonyl units to the growing acyl chain. The heterologous pathway,
consuming cytosolic acetyl-CoA and malonyl-CoA, showed to be func-
tional in P. pastoris, enabled a titer of 2.2 g/L 6MSA. In a subsequent
study, the complex mycotoxin citrinin was produced by expressing six
genes from Monascus purpureus under the control of the methanol-in-
duced AOX1 promoter (Xue et al., 2017). Citrinin was detected in a
concentration 0.6 mg/L after 24 h of methanol induction, with further
cultivation reducing the titer because of the low pH (2.4) in the culture.
Interestingly, correct identification of an intron in the heterologous
polyketide synthase gene from M. purpureus enabled an efficient release
of the ketoaldehyde from the ACP domain, which had previously hin-
dered citrinin production (Gao et al., 2013). Indeed, in-silico prediction
of introns is still a challenge for the functional expression of natural
product pathway genes in yeasts (Billingsley et al., 2016). In a recent
work, the synthesis of two further polyketides, lovastatin and mon-
acolin J, was reported (Liu et al., 2018). Lovastatin and derivative
compounds of monacolin J are widely used for the treatment of car-
diovascular diseases because of their cholesterol-lowering activity. For
production in P. pastoris, a biosynthetic pathway for dihydromonacolin-
L (DML) was first constructed by expression of the genes encoding for
lovastatin nonaketide synthase, enoyl reductase and thioesterase from
A. terreus. Further oxidation of DML by heterologous cytochrome P450
enzymes from A. terreus resulted in monacolin-L and monacolin-J,
which could be converted to lovastatin by heterologous expression of a
lovastatin diketide synthase gene from the same host. After observing
accumulation of intermediate metabolites, the synthesis pathway was
split to express the upstream DML synthesis module and the remaining
pathway in two separate strains to be co-cultured, thereby reducing the
individual metabolic burden. After optimizing the relative strain
abundance, the resulting co-culture produced 250 mg/L of lovastatin
and 593 mg/L monacolin J in bioreactor cultivations using methanol as
substrate. This novel strategy enabled efficient tuning of the synthesis
pathway by controlling the strain abundance, however it was shown to
be dependent on the diffusion of the key intermediate through the cell
membrane.
Metabolic pathway optimization is generally a very challenging
endeavor because of the complex regulation that cells have evolved to
maintain homeostasis and robustness (Nielsen and Keasling, 2016).
Direct relief of native regulation mechanisms can thus be used to im-
prove metabolite production in P. pastoris. In a study, the production of
riboflavin (Vitamin B2) was increased by removing the transcriptional
regulation of each of the six genes in the riboflavin biosynthesis
pathway (Marx et al., 2008). The approach required the stepwise re-
placement of all native promoters with the constitutive GAP promoter,
resulting in a strain accumulating up to 175 mg/L riboflavin in fed-
batch cultivation. Similarly, the native post-translational regulation of
the synthesis pathway of glucaric acid was by-passed by expression of a
myo-inositol oxygenase from mouse, enabling the production of 6.61 g/
10
Metabolic Engineering xxx (xxxx) xxx–xxx
L of glucaric acid in fed-batch cultures (Y. Liu et al., 2016).
Recently, pathway targeting to the peroxisome has attracted interest
as a metabolic engineering strategy in yeasts, because of the possibility
to localize orthogonal metabolic pathways and decrease by-product
formation (DeLoache et al., 2016). Methylotrophic yeasts in particular
have evolved peroxisomal compartments to minimize the toxic effects
of formaldehyde and hydrogen peroxide, which may damage proteins
in the cytosol, and to enhance energy efficiency (van der Klei et al.,
2006). Upon induction with methanol, peroxisomes dominate the cell
volume to contain large amounts of alcohol oxidase and catalase, en-
abling methanol assimilation as described earlier (Russmayer et al.,
2015a). Another interesting native pathway localized in this compart-
ment is the beta-oxidation of fatty acids, which delivers the key pre-
cursors 3-hydroxyacyl-CoA and acetyl-CoA. Poirier et al. (2002) de-
scribed the use of a PHA synthase from Pseudomonas aeruginosa to
convert medium chain length 3-hydroxyacyl-CoA from oleic acid into
polyhydroxyalkanoates (PHAs), bacterial polyesters that can be used as
biodegradable polymers. The expression of the PHA synthase under the
control of the promoter of the native acyl-CoA oxidase gene POX1 en-
abled PHA accumulation up to 1% DCW, depending on the substrate
concentration. Similarly, peroxisomal synthesis of carotenoids, a class
of terpenoids used as food colorants, antioxidant and nutraceuticals has
also been attempted (Bhataya et al., 2009). The authors reported the
introduction of carotenogenic enzymes from Erwinia uredovora, which
correctly localized to the peroxisome (Lee et al., 2009) and resulted in a
yield of 4.6 mg/g DCW lycopene. Exploiting a cytosol-localized version
of the same pathway, (Araya-Garay et al., 2012b) achieved the synth-
esis of 339 μg/g DCW ß-carotene from glucose, by additional expression
of gene crtL from Ficus carica. Furthermore, introduction of the β-car-
otene ketolase and β-carotene hydroxylase from Agrobacterium aur-
antiacum enabled the synthesis of astaxanthin (Araya-Garay et al.,
2012a), a keto-carotenoid used as a feed supplement in aquaculture.
Besides polyhydroxyalcanoates, other high molecular-weight com-
pounds have been produced in P. pastoris. Jeong et al. (2014) reported
the synthesis of hyaluronic acid, a clinically relevant biopolymer by
overproducing the native UDP-N-acetylglucosamine pyrophosphorylase
and phosphoglucose isomerase, in combination with the heterologous
enzymes hyaluronan synthase 2 and UDP-glucose dehydrogenase. Ex-
pression optimization of the latter resulted in a strain producing
0.8–1.7 g/L of large molecular weight hyaluronic acid (2.5 MDa), which
is required for clinical applications.
Considering the ability of P. pastoris to grow on a wide range of
substrates and to secrete functional heterologous enzymes, strategies to
degrade and utilize biomass feedstocks are particularly interesting in
this host. For example, P. pastoris was engineered to grow on cellubiose
and carboxymethyl cellulose by simultaneous production of a fungal
heterologous B-glucosidase, endo and exoglucanase (Kickenweiz et al.,
2018). Even though no growth was observed on cellulose, the produced
enzymes were shown to release glucose at higher temperatures. Fur-
thermore, construction of a mini-cellulosome by co-localizing a xyla-
nase from Clostridium cellulovorans and an endoglucanase from Clos-
tridium thermocellum resulted in a strain producing 1 g/L of ethanol by
direct biomass hydrolysis (Shin et al., 2015).
4. Outlook
The last decade has seen an impressive increase in molecular and
cell biology knowledge of P. pastoris, as well as state of the art cell
engineering and synthetic biology tools. These novel developments will
further support metabolic engineering endeavors to make P. pastoris a
leading host organisms for metabolite production. However, when
forecasting future applications we should first answer the question why
P. pastoris should be used as a metabolic engineering platform at all,
beside other established hosts such as bacteria or S. cerevisiae, among
yeasts. To date, the published applications of P. pastoris to produce
metabolites are still limited and rather scattered, including just a subset
D.A. Peña et al.
of products demonstrated in other cell factories, whereby mainly proof-
of-concept strains have been reported. Titers obtained were lower than
1 g/L in many cases and thus not close to commercialization stage,
indicating that this is still an emerging field. However, P. pastoris has
established in a niche for complex metabolite production. Several fac-
tors could account for this. Firstly, the traditional application of P.
pastoris as a host for heterologous protein production showed its ability
to produce functional bacterial, fungal, and plant enzymes, a key suc-
cess factor in pathway engineering for high value compounds.
Secondly, the Crabtree-negative phenotype of P. pastoris allows pro-
duction of large amounts of biomass and proteins without significant
fermentation, at the expense of a reduced glucose uptake rate.
Connected to this, higher energy efficiency and a higher flux ratio di-
verted into the PPP as compared to S. cerevisiae likely provides for more
resources to synthesize highly reduced complex metabolites. While part
of the disadvantages of Crabtree positive yeasts can be circumvented by
employing fed-batch cultures this increases costs and complexity of
large scale production and still cannot cope with the limitations of
energy efficiency in S. cerevisiae and related species. On the contrary,
efficient and fast production of primary metabolites directly derived
from the central carbon metabolism is potentially less favored in P.
pastoris than in Crabtree positive yeasts, as the central metabolism is
tightly controlled and limited.
Synthetic biology calls for the development of chassis strains as
starting platforms for the development of individual production strains.
Specifically, the synthesis of secondary metabolites initiates from me-
tabolic nodes at key intermediate metabolites. Strains dedicated to
overproduce these intermediates constitute such chassis strains. By this
concept repetitive redesign and optimization of the same pathways can
be avoided, as demonstrated in S. cerevisiae (Jouhten et al., 2016). As a
special feature of P. pastoris, the highly expandable peroxisomal com-
partment could be interesting to localize entire heterologous pathways.
A possible advantage of this strategy would be increased local meta-
bolite concentrations, which improve the thermodynamic efficiency of
pathways. Furthermore, energy production by methanol dissimilation
offers an opportunity to decouple product formation from growth, and
even from energy metabolism as such, possibly implementing com-
pletely orthogonal anabolic pathways (Pandit et al., 2017).
Besides the development of specialized chassis for high value me-
tabolites, decades of experience with heterologous protein production
in P. pastoris enabled the recent successes of metabolic and cell en-
gineering to boost protein productivity. Novel developments of P. pas-
toris systems and synthetic biology allow to anticipate a new generation
of dedicated “protein factory” strains with high productivity, also en-
abling the production of hitherto “difficult to express” proteins which
regularly abscond from entering application tests and clinical trials due
to the inability to produce them in reasonable amounts.
We anticipate that recent developments in synthetic biology, me-
tabolic modeling and analysis will accelerate attaining these goals.
Several tools for genome engineering, modular part assembly, and
stable integration will have a great impact on the speed of strain en-
gineering. Furthermore, as metabolic models improve to include further
constraints including resource allocation and post-translational pro-
cesses, rational cell refactoring including new targets in regulatory and
metabolic genes will become feasible.
Acknowledgements
DAP was funded by the Austrian Science Fund (FWF): Doctoral
Program BioToP—Biomolecular Technology of Proteins (FWF W1224).
Research on P. pastoris in our labs is supported by the Federal Ministry
for Digital and Economic Affairs (bmwd), the Federal Ministry for
Transport, Innovation and Technology (bmvit), the Styrian Business
Promotion Agency SFG, the Standortagentur Tirol, the Government of
Lower Austria and ZIT – Technology Agency of the City of Vienna
through the COMET-Funding Program managed by the Austrian
11
Metabolic Engineering xxx (xxxx) xxx–xxx
Research Promotion Agency FFG (BG, JZ, MGS, DM), and by the
Austrian Federal Ministry for Digital and Economic Affairs (bmwd) and
the National Foundation for Research, Technology and Development
(BG).
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