Biotechnol. J. 2013, 8 DOI 10.1002/biot.201200345 www.biotechnology-journal.

com

Review

Toward systems metabolic engineering of Aspergillus and

Pichia species for the production of chemicals and biofuels

Luis Caspeta1 and Jens Nielsen1,2

1 Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
2 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark

Recently genome sequence data have become available for Aspergillus and Pichia species of indus-
Received 09 OCT 2012
trial interest. This has stimulated the use of systems biology approaches for large-scale analysis of Revised 19 FEB 2013
the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which Accepted 14 MAR 2013
has resulted in much new biological information. Case-specific contextualization of this informa-
tion has been performed using comparative and functional genomic tools. Genomics data are also
the basis for constructing genome-scale metabolic models, and these models have helped in the
contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Fur-
thermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving
from traditional approaches, such as random mutagenesis, to a systems metabolic engineering
approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species,
highlighting the relevance of these developments for systems metabolic engineering of these
organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.

Keywords: Aspergillus and Pichia species · Contextualization · Genome-scale metabolic models (GEMs) · High-throughput ·
Systems metabolic engineering

1 Introduction
Concerns about environmental deterioration, increased
demand and limited supply of fossil resources have moti-
vated the development of alternative routes for produc-
tion of chemicals and fuels. Microbe-based production of
these products is interesting since the associated
processes can rely on renewable biomass, including lig-
nocellulose as the most feasible feedstock [1]. Neverthe-
less, these processes involve two challenging conversion
steps: lignocellulose hydrolysis to fermentable sugars,
and their subsequent conversion to the products by fer-
mentation (Fig.  1). To become economically feasible,
these processes must have high productivities and yields,
Correspondence: Prof. Jens Nielsen, Department of Chemical and Biologi-
cal Engineering, Chalmers University of Technology, Kemivägen 10,
SE-412 96 Gothenburg, Sweden
E-mail: nielsenj@chalmers.se
Abbreviations: AO, alternative respiration; DOT, dissolved oxygen tensions;
EST, expressed sequence tag; GEM, genome-scale metabolic model
and the consolidation of these two steps into a single one,
often referred as simultaneous saccharification and fer-
mentation, is desirable. Although some microbes natural-
ly synthesize and excrete hydrolytic enzymes, and/or
convert sugars to ethanol and/or organic acids, their
performance is often limited for industrial applications.
Current strategies for improving the capabilities of
microbes involve either cycles of mutagenesis, selection,
and analysis, or a more design-oriented approach that
includes modeling, implementation, and analysis, gener-
ally referred to as metabolic engineering [2]. The latter is
of upmost significance since it is the basis for future
design-based engineering of industrial microbes [3]. To
reach this level of precision, models have to incorporate
an accurate description of the fundamental biology. Itera-
tive engineering cycles of genome-scale modeling, imple-
mentation through genetic engineering, synthetic biolo-
gy or evolutionary engineering, and systems-wide analy-
sis using ’omics technologies (jointly termed systems
metabolic engineering [4, 5]) can dramatically increase
the knowledge of the fundamental biology and hereby
improve the engineering process (Fig. 2).

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Figure 1. Summary of key biotechnologi-

cal products relevant as biofuels and
chemicals that are natively and non-
natively produced in Aspergillus and
Pichia species compared with S. cerevi-
siae. The utilization of pentoses and
hexoses released from the hydrolysis of
lignocellulosic materials is also shown.
The required steps for processing the
production of these chemicals from
renewable biomass, and the convenience
of performing the process in one step
(consolidated processing), or simultane-
ous saccharification and fermentation is
shown in the lower part of the figure.

Figure 2. Systems metabolic engineering

of industrial microbes is an interactive
process. ’Omics and fermentation data
accumulated during the analysis of fer-
mentations with native or engineered
microbes is stored for their later use in
modeling with GEMs, previous filtration
with genomic tools and biochemical
databases. Data from modeling is used
to guide manipulation of microbes with
the desired production background.
Titration of the desired product along
with high-throughput analysis of the
robustness of the engineered microbe
within well-defined production condi-
tions may result in the desired fermenta-
tion process for its subsequent transfer
to the industrial production. Generated
data are analyzed and stored with previ-
ous knowledge for subsequent rounds of
engineering.

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Biotechnol. J. 2013, 8
Metabolic engineering strategies have been applied to
design microbial cell factories for the production of bio-
fuels and chemicals from renewable biomass using
Escherichia coli and Saccharomyces cerevisiae [5, 6].
Efforts relying on engineering non-native capabilities,
such as hydrolysis of sugar polymers and pentose metab-
olism, have been made to consolidate the process and to
increase the yield of lignocellulose conversion into the
desired compound (Fig. 1). For instance, displaying non-
native hydrolytic enzymes on the surface of E. coli and
S. cerevisiae has enabled the direct conversion of cellulose
or cellobiose to ethanol [7, 8]. Engineering pentose metab-
olism, which is challenging due to complex cofactor bal-
ance requirements, was guided by genome-scale meta-
bolic models (GEMs) [9]. These tools also allowed the
unscrambling of complex metabolic networks and led to
valuable designs of cellular phenotypes. Hence, target
gene deletions or amplifications for increasing the pro-
duction of the native and non-native biofuel molecules
ethanol and butanol in E. coli and S. cerevisiae [10–13], as
well as the non-native chemical 1,4-butanediol [14], were
accomplished by GEM modeling. By combining modeling
with high-throughput analysis, i.e. genome, transcrip-
tome, proteome, metabolome and fluxome, it has been
possible to gain an increased understanding of basic cel-
lular and metabolic features, and identify gene targets for
metabolic engineering. The usefulness of this strategy in
developing E. coli and S. cerevisiae strains for the produc-
tion of chemicals and biofuels has been well documented
[4, 5, 15, 16]. Strain tolerance to the accumulation of the
desired compound is also challenging due to the complex
interactions among metabolic and regulatory networks.
Evolutionary engineering combined with high-through-
put analysis has shown to be a powerful tool for recogniz-
ing molecular mechanisms that allow cells to cope with
high concentrations of toxic products [4]. Current and
future advances in systems metabolic engineering of
E. coli and S. cerevisiae will support the position of these
species as the main workhorses for development of cell
factories. There is, however, another set of organisms that
has gained lot of interest due to their native abilities to:
perform more than two steps of the lignocellulose-to-prod-
uct conversion process (Fig. 1), tolerate challenging envi-
ronmental conditions, or use lignocellulose directly as a
feedstock. For these organisms, the systems biology era
has recently started.
Microorganisms of the Aspergillus and Pichia genera
have been extensively used as model organisms in basic
science and as cell factories for the production of indus-
trially valuable products (Fig. 1). The Aspergillus species
niger, oryzae and nidulans can produce hydrolytic
enzymes and organic acids from hexoses and pentoses,
and tolerate low pH [17]. P. stipitis can ferment xylose and
arabinose to ethanol [18, 19]. P. pastoris produces and
excretes heterologous enzymes efficiently, and converts
alcohols to aldehydes [20, 21] – formaldehyde from
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methanol is produced at about six thousand tons per year.
P. stipitis and pastoris were recently relocated to the gen-
era Scheffersomyces and Komagataella, respectively, due
to the large physiological and genetic differences within
the Pichia genus [22, 23]. However, the genus Pichia is
used in this manuscript to facilitate the description of pre-
vious results. The applications of Aspergillus and Pichia
have motivated whole genome sequencing projects, and
in turn motivated GEMs reconstructions. There have also
been advances in high-throughput technologies specific
for these microorganisms. Altogether, these advances
have enabled systems-wide analysis and modeling. The
limitation of practical tools for implementing the results
from modeling has been overcome for Aspergillus spp.
and P. stipitis, and mostly for P. pastoris; hence the sys-
tems metabolic engineering cycle (Fig. 2) can be imple-
mented and metabolic engineering of these species is
moving to a systems level. Here we review the contribu-
tion of ’omics technologies to unraveling the fundamental
biology of Aspergillus and Pichia species. We highlight
the potential application of gained knowledge for opti-
mizing the production of biofuels and chemicals from bio-
mass through a systems metabolic engineering approach.
2 Potential contributions from high-
throughput analysis
2.1 Genomics
Metabolic pathway optimization to produce a desired
product typically requires the manipulation of gene
expression. Metabolic engineering efforts are commonly
based on deletion and overexpression of native and het-
erologous genes to provide a desired cell phenotype.
Besides driving holistic-based studies on the cell’s func-
tions, genome sequencing and functional genomics can
also lead to the identification of potential target genes for
engineering metabolic functions and regulatory net-
works.
S. cerevisiae was the first eukaryotic microorganism
to be sequenced in 1996 [24]. Advances in DNA sequenc-
ing and reduction of costs have allowed the sequencing of
many more microbes including Neurospora crassa as the
first filamentous fungi, sequenced in 2003 [25]. The
genomes of A. nidulans [26] and A. oryzae [27] were pub-
lically released in 2005, P. stipitis [28] and A. niger in 2007
[29] and 2011 [30], and P. pastoris was sequenced twice in
2009 [31, 32]. Some characteristics of these fungal
genomes are given in Table 1.
The most visible differences arising after comparing
genome sequences are the higher GC (guanine-cytosine)
content and larger genome sizes of Aspergillus spp. and
N. crassa compared with Pichia spp. and S. cerevisiae.
Higher GC content was also found in bacterial popula-
tions from soil samples compared to those living in watery
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Table 1. Genomes of industrially relevant Aspergillus and Pichia species belonging to the Saccharomycotina and Pezizomycotina subphyla of the kingdom
Fungi.

Subphyla Saccharomycotina Pezizomycotina

Species (strain) S. cerevisiae P. pastoris P. stipitis N. crassa A. nidulans A. niger A. oryzae
(S288c) (GS115) (CBS 6054) (N150) (FGSC A4) (CBS 513.88) (RIB 40)
Chromosomes 16 4 8 7 8 8 8
Genome size (Mb) 12.2 9.7 15.4 38.6 30.1 33.9 37.0
GC content (mole %) 38.3 41.1 41.1 50.0 50.0 50.4 48.0
Predicted genes 5,807 5,313 5,841 10,082 9,541 14,165 12,074
Coding (as % of total) 69.3 79 21.1 43.7 49.0 65.7 37.6
Mean gene length (bp) 1,455 1,442 1,479 1,673 1,547 1,572 1,152
Year of sequencing 1996 2009 2007 2003 2005 2007 2005
Secreted hydrolytic
enzymes (% of total 72 77 73
secreted proteins)
a) Genomes for closely related model organisms are included for comparisons

environments [33, 34]. Hence, it will be interesting to
determine if genes unique to the filamentous lifestyle of
Aspergillus spp. that confer a special trait in its natural
habitat tend to have higher GC content. This lifestyle
probably caused the multiplication of specific fragments
of DNA, duplication of some genes and incorporation of
novel ones with the concomitant enlargement of their
genome [35]. Environmental changes and sugar availabil-
ity and distribution may also have been the reason that
these microorganisms started to move by generating vari-
able hyphal structures [36]. Intricate arrangements of
gene gains and losses are part of these changes [27]. As a
result, Aspergillus spp. produce hydrolytic enzymes when
growing in their native environment consisting of biolog-
ical polymers [37]. The main habitat of P. stipitis is the gut
of larvae, where it feeds on xylose and arabinose previ-
ously hydrolyzed from lignocellulose by other microorgan-
isms [19]. P. pastoris can be isolated from exudates, fruits
and barks of trees in which feeds on methanol mainly pro-
duced by hydrolysis of metoxyl groups found in pectin
and lignin [20]. Methanol metabolism in this yeast
involves the formation of formaldehyde, and this trait has
raised interest in using this microbe for the production of
aldehydes from alcohols [21].
Analysis through comparative and functional
genomics has been performed to assess phylogenetic
relationships, and detect gene orthologs and synteny.
These studies can be used to determine unique metabolic
and regulatory functions, which can then be transferred
to other organisms (e.g. hydrolytic enzymes modules,
pathways and regulatory mechanisms for pentose meta-
bolism). Gene-function-based analysis of hydrolytic
enzymes in Aspergillus revealed that A. nidulans,
A. niger, and A. oryzae have a similar capability for excret-
ing these enzymes (e.g. 70% of total secreted enzymes)
[29]. However, A. oryzae and A. niger contain specific
blocks of DNA without synteny in A. nidulans, which is
enriched of secondary-metabolism genes and transport-
related gene families [27, 29]. A. oryzae may also possess
the most complex regulatory system among these organ-
isms [27], which has to be considered in future metabolic
engineering.
Hydrolytic enzymes can be heterologously produced
with P. pastoris and S. cerevisiae, or overexpressed in
native producers. S. cerevisiae and P. pastoris have simi-
lar codon usage, but P. pastoris has less tRNA coding
genes (123 compared with 274) [31]. Genes involved in
post-translational modification and secretion have been
annotated in the P. pastoris genome [31, 32], which has
been shown to possess a higher ability to secrete proteins
and perform less protein glycosylation. The final stages of
N-glycan synthesis in A. niger and A. oryzae follow the
high mannose pathway, as in S. cerevisiae, but the num-
ber of mannoses is lower [38]. Therefore, reduction of
mannose incorporation in S. cerevisiae proteins may be
necessary for the production of cellulases.
Comparative genomics were used to assess the fea-
tures of pentose consumers. A multispecies comparative-
genomics study was useful, for example, in demonstrat-
ing that xylose consumers are found in the CUG clade of
commensal fungi [39]. Interestingly, this clade marks the
diversification between P. stipitis and S. cerevisiae [23].
Through this study, the authors also identified possible
genes required for metabolic engineering of xylose assim-
ilation in S. cerevisiae, which cannot ferment xylose nat-
urally. Amino acid sequences of enzymes required for
xylose and arabinose metabolism were used to map
homologous genes in 38 fungal genomes, and it was

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found that most of these enzymes are present in A. niger,
A. oryzae and A. nidulans [40]. Interestingly, arabinose-
responsive and xylose-responsive transcriptional activa-
tors were found in the Trichocomaceae order Eurotiales
(e.g. Aspergillus spp. among other lignocellulose con-
sumers), but not in three representatives of Onygenales
(i.e. keratin consumers). Therefore, Aspergillus spp. are
the most suitable microorganisms for naturally perform-
ing both lignocellulose saccharification and fermentation
of sugars, and have become valuable models for engi-
neering those traits in well-known microbes.
2.2 Transcriptomics
Gene transcription is one of the first cell responses to a
change in the extracellular or intracellular environment,
and whole transcriptional analysis (transcriptomics) has
become an essential part of the high-throughput analysis
of cell factories. Changes in gene expression upon accu-
mulation of toxic molecules or metabolic intermediates
have been unraveled by transcriptomics. Hence, this tool
can be used to identify target genes for engineering the
cell’s resistance or redirection of carbon fluxes to a desired
molecule. Pre-genomic studies on the gene expression
pattern of sexual structures in A. nidulans by expressed
sequence tag (EST) sequencing represent the first large-
scale analysis of gene expression among the organisms
under study [41]. EST sequencing was also used to
enhance gene annotation in the GEM of A. oryzae [42].
Various transcriptomics studies have been motivated
by previous knowledge of the phylogenetic relationship
between Aspergillus spp. and the free access to genome
sequences. For instance, Andersen at al. [43] designed an
Affymetrix GeneChip for genes from A. nidulans, A. niger
and A. oryzae, and used this to analyze differentially
expressed genes in fermentations with glucose or xylose.
This DNA array was then used to analyze transcription
patterns of A. niger cultivated at different pH during the
production of organic acids [44], and of A. oryzae produc-
ing alpha-amylase [45]. Microarrays in combination with
functional genomic analysis also helped demonstrate
that the regulation of glycerol and lipid metabolism by
the transcription factor Adr1 represents a co-evolved
core regulatory pathway among A. niger, A. oryzae and
S. cerevisiae [46]. This information must be considered for
further metabolic engineering of glycerol and lipid metab-
olism, e.g. for producing fatty acid ethyl esters (biodiesel).
In another study, A. niger genes involved in the degrada-
tion of pectin, arabinan and arabinogalactan were detect-
ed by genome mining [47], and the expression of those
genes in cultivations with pectin, galacturonic acid,
rhamnose and xylose was followed using DNA arrays,
leading to the reconstruction of a pectin-degrading
enzyme network.
Organisms considered in this review have a high pref-
erence for respiratory metabolism, but large-scale biore-
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actors typically perform under hypoxic conditions.
Whereas A. fumigatus can grow at low oxygen tensions
(0.5%), A. niger and A. nidulans cannot grow at oxygen
tensions below 2.5% [48]. Hypoxic conditions in cultiva-
tions of A. nidulans affected the expression of 27% of the
total genes found in this microbe [49]. Overexpressed
genes included those related to glycolysis, fermentation
and the tricarboxylic acid (TCA) cycle. Integration of tran-
scriptomics and proteomics was used to analyze the
adaptation of P. pastoris to hypoxic conditions during
recombinant protein production using glucose, and
demonstrated the importance of regulatory mechanisms
in glycolysis, the pentose phosphate pathway and the
TCA cycle, as well as for protein modification and secre-
tion pathways [50]. Production of hydrolytic enzymes
under hypoxic conditions can benefit the production of
biofuels using simultaneous saccharification and fermen-
tation, as this process should be carried out under fer-
mentative conditions (Fig. 1).
New technologies have also been added to mRNA
microarrays and EST sequencing for studying transcrip-
tional profiles. Whole transcriptome shotgun sequencing
(RNAseq) was used to study differences in gene expres-
sion of P. stipitis growing in glucose or xylose [51]. In this
study, the authors identified 214 genes differentially
expressed under these conditions. RNAseq was also used
to identify differentially expressed genes in A. niger
exposed to lignocellulose [37]. Expanding the options for
transcription analysis of Aspergillus and Pichia spp. will
ensure the accumulation of primary data on cellular
responses to diverse internal and external stimulus,
which will serve to enhance further metabolic engineer-
ing efforts.
2.3 Proteomics
Screening of useful enzymes and conditions that enhance
the secretion of native and heterologous proteins has
been achieved through proteomics. Various studies on
proteins excretion have been published for A. oryzae,
A. niger, A. fumigatus and P. pastoris [32, 52–54]. For
example, 85 protein spots from the extracellular proteome
of A. oryzae growing in solid-state, and 110 from sub-
merged, cultivations were analyzed [52]. Glucosidases
and xylanases were identified under both conditions,
while alpha-amylase and beta-glucosidase were secreted
in solid-state cultivations but stayed in the cell wall under
the submerged condition. Secretion of glucosidases and
alpha-amylase were also detected in cultivations of
A. niger on liquid media with starch [53]. Alcohol oxidase
and superoxide dismutase, which are abundant intracel-
lular proteins during cultivations with methanol [55], were
found among other proteins in cultivations of P. pastoris
with methanol, suggesting cell lysis [54]. Hence, cultiva-
tion of this microbe for hydrolytic-enzyme production
using glucose instead of methanol will be more useful [32].
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Intracellular protein levels of P. pastoris growing on
glucose, glycerol and methanol at different temperatures
were also analyzed by proteomics [50, 55, 56]. Under nor-
mal and hypoxic conditions, 85 proteins with different
abundance were identified in P. pastoris cultivations in
glucose-limited chemostats [50]. Proteins with higher
abundance on low dissolved oxygen tensions (DOTs)
were related to glycolysis, amino acid metabolism and a
general stress response, while those involved in the TCA
cycle and an oxidative stress response were less abun-
dant. Besides promoting the accumulation of related
metabolic proteins, methanol also induced the over-
expression of proteins related to reactive oxygen species
(ROS) stress, the unfolded protein response and a protein
degradation pathway [55]. Vacuolar proteases and
autophagy-related proteins were also observed in this
analysis. This is in agreement with the higher extracellu-
lar accumulation of some intracellular enzymes during
methanol cultivations [52]. Temperatures below 20°C
enhance recombinant protein production. This benefit
has been related to lower protein production rates allow-
ing the post-translational machinery to better perform
modifications, proper folding and excretion of proteins.
A decrease in the levels of proteins related to the TCA
cycle and oxidative stress response was observed when
the temperature decreased from 30° to 20°C [56]. How-
ever, the lower specific growth rate compared with 20°C
has to be considered.
Proteome analysis of P. stipitis at the exponential and
stationary phases of xylose fermentations under hypoxic
conditions was useful to identify the abundance of
enzymes related to alternative respiration (AO), the gly-
oxylate cycle, galactose metabolizing, and the putative
high-affinity xylose sugar transporter [57]. AO is a way for
NADH oxidation without ATP production that allows the
cell to regenerate NAD+ and avoid oxidative stress, main-
taining redox balances among cytosol and mitochondria.
Therefore, it would be interesting to look at this pathway
for metabolic engineering of xylose metabolism, as redox
balance is challenging when xylose reductase activity is
used.
2.4 Metabolomics and fluxomics
Whole-metabolic profile analysis (metabolomics) can be
used to investigate the chemodiversity of the cell. As
Aspergillus spp. possess larger genome sizes than Pichia
spp. and S. cerevisiae, it would be expected that the for-
mer two have a higher chemodiversity, especially of
metabolites related to the secondary metabolism (e.g.
antibiotics, flavonoids and terpenoids), which can be
used as biofuels and fine chemicals. Comparisons
between the metabolite profiles of species can also lead to
the identification of enzymatic capacities through
metabolite levels, and help in identifying the bottlenecks
in metabolic fluxes.
6 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Large-scale metabolite profiling has been developed
for filamentous fungi [58] and P. pastoris [59, 60]. Compar-
ison between S. cerevisiae and P. pastoris metabolite fin-
gerprinting indicated possible lower enzyme activities in
glycolysis in P. pastoris, which also showed lower levels of
excreted organic acids [60], and high concentrations of
arabitol [59]. The apparent lack of arabitol dehydrogenase
activity, which converts arabitol into xylulose 5-phos-
phate, and the inability of P. pastoris to metabolize xylose
could be the reasons for arabitol accumulation. Hence,
redox balances have to be considered to incorporate pen-
tose metabolism [61]. It was also discovered by metabolic
profiling that the phosphoketolase pathway, through
which xylulose 5-phosphate is converted to acetyl phos-
phate and D-glyceraldehyde 3-phosphate, is present in
A. nidulans [62], but not in S. cerevisiae, P. stipitis and
P. pastoris, which apparently only have the phosphoketo-
lase activity.
Metabolic flux analysis is an essential computational
tool for metabolic engineering [2, 63], as this technique
can be used to quantify the fluxes through a particular
pathway or the entire cell metabolism. This technique is
based on mass balance calculations over the total pool of
metabolites included into a metabolic model given a set
of specific constraints (Fig. 3). Specific rates of glucose
consumption and ethanol, organic acid, CO2 and bio-
mass production can be used as constraints. However,
better estimations have been made by using an addi-
tional set of constraints obtained from 13C-labeling
experiments [2, 63]. For instance, this technique was
employed for measuring metabolic fluxes in A. niger,
A. nidulans, P. stipitis and P. pastoris using a model of the
central carbon metabolism containing 40–60 reactions
[64–67]. These studies helped to identify changes in
metabolic fluxes due to the overproduction of a lipase in
P. pastoris and glucoamylase in A. niger [64], and differ-
ences in central carbon metabolism between S. cere-
visiae and P. stipitis growing on xylose or glucose [66,
67]. Changes in metabolic fluxes between control and
recombinant protein producer strains can help identify
bottlenecks on anabolic-catabolic pathways that deal
with the high amount of energy and amino acids need-
ed for protein production. As expected, P. stipitis has
higher carbon fluxes through the pentose phosphate
pathway [66, 67], and this allows higher turnover of the
NADPH/NADP+ couple that may balance the turnover of
NADH/NAD+ couple in glycolysis with the concomitant
precise balance during xylose consumption. This find-
ing is useful for further metabolic engineering of xylose
metabolism in other yeasts [18].
Metabolic flux analysis can also be constrained by
enzyme activities and data from experiments (Fig.  3).
Using a model consisting of 69 reactions compartmental-
ized into mitochondria and cytosol, and activities of key
enzymes, Pedersen et al. [68] analyzed metabolic fluxes of
A. oryzae producing alpha-amylase and identified the
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localization of such enzymes. Proper enzyme localization
is very important for identifying the transport of organic
acids, which is also coupled with redox balances between
cytosol and mitochondria and with their excretion by
Aspergillus spp. and S. cerevisiae.
The large amount of data collected during the exten-
sive utilization of A. niger as a cell factory allowed the
reconstruction of larger metabolic models that, besides
including gene annotation and reaction localization in
three compartments, also contained a higher number of
reactions and metabolites than previous models (335
and 284, respectively) [69]. These models were used for
characterization of the A. niger’s growth phenotype
upon reaction deletion and growing on different carbon
sources for succinic acid production. The release of
genomic DNA sequences for the microorganisms con-
sidered here has provided the knowledge for starting
with the whole-genome analysis of metabolic fluxes
using GEMs.
2.5 ’Omics data integration for GEM
reconstructions
The increasing amounts of data require sources for col-
lecting and categorizing. For instance, the filamentous
fungal gene expression database (FFGED) is an open
source that provides gene expression data and analysis
[70]. The genome annotation source BOGAS offers tools
and information to validate and correct gene annota-
tions [71]. The Kyoto encyclopedia of genes and
genomes (KEGG) provides systematic knowledge link-
ing genomes to cellular functions categorized in genom-
ic, chemical, and interacting diagrams of gene-reaction-
metabolites networks [72]. The Universal Protein
Resource (Uniprot) is a comprehensive, high-quality and
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Figure 3. Metabolic flux analysis using
models from the central carbon metabo-
lism or GEMs. Traditional methods for
metabolic flux analysis include the incor-
poration of 13C-labeling profiling and
enzyme activities to constrained simula-
tions with a model of the central carbon
metabolism. The current simulations with
GEMs required a larger set of constraints
obtained from high-throughput analyses
and extensive data accumulated in the lit-
erature to narrow down the possible
space of solutions. Through cycles of
comparison between the results from
simulations and experimental work mod-
el predictions can be enhanced.
freely accessible resource of protein sequences and
functional information [73]. Another open source for the
analysis of data and integration of transcriptome and
molecular interactions related to fungi is the bioinfor-
matics and metabolomics (BioMet) Toolbox [74]. This
web-based source also provides a number of mathemat-
ical models and integrative strategies using computa-
tional modeling. GEMs for A. niger, A. oryzae, A. nidu-
lans, P. pastoris and P. stipitis are so far included in this
publicly available repository. These and many other
repositories have served as a source of data for GEM
reconstruction. The reconstruction process and related
applications on modeling Aspergillus and Pichia spp. are
discussed in the following section.
3 Current contributions of GEM modeling
for metabolic engineering of Aspergillus
and Pichia species
GEMs incorporate genome data on gene-reaction-
metabolite interactions and are used for overall metabolic
flux analysis (Fig. 3). Up to now, more than 85 GEMs have
been reconstructed following an interactive process con-
sisting of: (i) initial reconstruction based on gene annota-
tion; (ii) manual curation using data reported in journals,
books and on-line; (iii) mathematical formulation and
debugging (e.g. component balance and constraints
based on metabolic capabilities); and (iv) network evalu-
ations (e.g. tests for known organism capabilities, experi-
mental growth rates, and computed single gene deletion
phenotypes) [75]. Through this reconstruction scheme,
GEMs of good quality can be obtained for further analysis
covering applications that include: (i) biological interpre-
7
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Journal
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tation and discovery; (ii) evolutionary elucidation;
(iii) interrogation of multispecies relationships; and
(iv) guidance for metabolic engineering [76].
3.1 GEMs of Aspergillus and Pichia species
There are currently nine GEMs for the microbes consid-
ered in this review. The A. nidulans iHD666 model was
the first reconstruction of an Aspergillus species [77]. Its
reconstruction relied on reaction networks assembled for
A. niger and S. cerevisiae. Reactions list of iHD666 was
used as a template to reconstruct the A. niger iMA871
model, after prior elimination of redundant reactions [78].
Vongsangnak et al. [42] used reactions listed in S. cere-
visiae, A. nidulans, and A. niger models to accomplish the
first set of reactions for A. oryzae’s GEM iWV1314. To date
three GEMs have been released for P. pastoris: PpaM-
BEL1254 [79], iPP668 [80], and iLC915 [81], and P. stipitis:
iSS884 [81], iBB814 [82], and iTL885 [83].
3.2 Contributions of GEM modeling to biological
interpretation and discovery of metabolic
engineering targets
Once a GEM is reconstructed, it needs to be converted
into a mathematical format, i.e. a numerical matrix (Sij)
containing stoichiometric coefficients (rows, i) from bio-
chemical reactions (columns, j) (Fig. 3). Steady-state bal-
ance in metabolic pools (rate of metabolite conversion
equal to zero) is a common assumption, and this con-
strains the simulations substantially, resulting in a reduc-
tion of the solution space [84]. This treatment is conduct-
ed using computational algorithms [76], which also allow
the use of GEMs in simulations and analysis of ’omics
data to accomplish current applications of large-scale
metabolic modeling, including metabolic engineering.
Most of these algorithms are based on maximizing or min-
imizing metabolic fluxes through a reaction by indicating
input values that permit a set of outputs for the chosen
reaction, combined with chemical or physical constrains
to improve simulations [85]. While most GEMs of
Aspergillus and Pichia species were tested for simulating
maximum growth rate with different carbon sources com-
monly used experimentally, some were also used to ana-
lyze the production of recombinant proteins, ethanol and
different chemicals.
P. pastoris enhances the production of protein at low
DOTs or when methanol and glycerol or sorbitol is used in
the production [50]. Simulations with P. pastoris models
iLC915 and PpaMBEL1254 reproduced the increase of
recombinant protein production under low DOT [79, 81].
Additionally, iLC915 contextualized the benefits of com-
bining methanol with glycerol or sorbitol. This benefit is
related to an efficient use of methanol in energy path-
ways, while glycerol or sorbitol provides carbon interme-
diates for synthesis of amino acids. Chung et al. [80] ana-
8 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2013, 8
lyzed the production of 2,3-butanediol by enzymatic
reduction of acetoin in P. pastoris using the iPP668 mod-
el. Their study was driven by findings from model simula-
tions that predicted a high turnover rate of NADH. This
trait, along with the efficient consumption of alcohols and
the large availability of genetic engineering tools for this
organism, has motivated its use to investigate the pro-
duction of aldehydes.
P. stipitis produces ethanol from pentoses and hexos-
es found in biomass, but its metabolism is sensitive to
oxygen and it cannot grow anaerobically [86]. Thus, its
utilization in industrial processes is limited. The effect of
the oxygen uptake rate on the production of ethanol from
xylose and glucose was successfully reproduced with
iSS884 and iBB814 GEMs [81, 82]. The strong preference
for glucose over xylose, as well as for respiratory growth
was constrained in the iSS884 model to simulate ethanol
production from mixtures of glucose and xylose using
reactors in series [81]. Simulations with the iTL885 mod-
el using the OptKnock algorithm allowed the identifica-
tion of gene knockouts that may increase ethanol produc-
tion with a negligible effect on growth [83]. By constrain-
ing the uptake of sterols and unsaturated fatty acids in
simulations with the iBB814 model, it was possible to
identify 28 target reactions that may enable anaerobic
growth [82].
Analysis of metabolic responses following changes in
oxygen availability and pH has been studied in A. nidu-
lans [49, 87]. Under oxygenated conditions, A. nidulans
contains similar amounts of intracellular NADH and
NAD+, but NAD+ levels decreased under hypoxic condi-
tions. It was suggested that the GABA shunt coupled
with glutamate formation is the preferred way to deal with
NADH excess under hypoxic conditions [49]. By ensuring
well-oxygenated conditions in A. niger cultivations, it
was possible to study the impact of pH on acids produc-
tion [44]. With a new algorithm for GEM modeling of extra-
cellular proton balance, the authors were able to identify
genes for which the transcription level corresponded
directly with change in pH. Detailed analysis of these
genes combined with comparative genomics using
A. nidulans allowed the identification of putative pH-
sensing genes in A. niger.
AO is present in many organisms and seems to be
induced during different forms of stress. Analysis of
A. niger’s metabolism showed that AO in the mitochon-
dria is a means to oxidize NADH without producing ATP,
and to drive an increase in the production of organic acids
[78]. It has been shown that a reduction in the ATP pool
accelerates glycolysis and the subsequent production of
citric acid [88]. In silico and experimental deletion of the
AO in P. pastoris resulted in a decrease in biomass yield
[81, 89]. Decreased production of acids was also observed
in silico and experimentally after impairing AO in A. niger
[78, 90].
Biotechnol. J. 2013, 8
Despite the fact that GEMs for Aspergillus and Pichia
species have only been released within the last five years,
genome-scale modeling has made several contributions
to contextualizing the biological meaning of the data gen-
erated in controlled experimental environments. These
assurance tests have supported the use of GEMs in sim-
ulations to predict maximum yields and titers of proteins
(e.g. hydrolytic enzymes), ethanol and chemicals under
specific culture conditions. Constrained with ’omics data,
GEM modeling has also been useful in assessing insights
into evolutionary traits in Aspergilla species. Incorpora-
tion of future ’omics data generated upon rounds of mod-
eling, implementation, and genome-wide analysis (Fig. 2)
combined with constraint-based modeling (Fig.  3) will
increase the accuracy of model predictions with a con-
comitant success in gene targeting for metabolic engi-
neering of cell factories for the production of biofuels and
chemicals (Fig. 1), moving this task to a systems level.
4 Conclusions and future perspectives
Aspergillus and Pichia spp. A. niger, A. oryzae, A. nidu-
lans, A. fumigatus, P. pastoris and P. stipitis have gained
a lot of attention in industrial applications for production
of heterologous proteins (e.g. hydrolytic enzymes), chem-
icals and biofuels because of their unique native capaci-
ties. The release of their genome sequences has allowed
systematic analysis and modeling to guide metabolic
engineering to improve their native capacities or to
implant new ones required to support a bioeconomy
based on renewable sources.
As current ’omics data have been used in the recon-
struction of present GEMs, it can be expected that the
addition of this new fundamental knowledge will increase
the ability of GEMs to make better predictions of biologi-
cal traits of these microbes, as seen with the E. coli and
S. cerevisiae models. With the set of genetic engineering
tools for P. pastoris and A. niger being expanded, it can be
expected that these organisms may in the future domi-
nate the production of recombinant proteins by fungi. It is
also possible that this fact will drive the utilization of these
microbes for synthesizing chemicals and biofuels along
with E. coli and S. cerevisiae.
We would like to acknowledge European Research Coun-
cil (grant no. 247013), the Knut and Alice Wallenberg
Foundation and the Chalmers Foundation.
The authors declare no conflict of interest.
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
Jens Nielsen has an M.Sc. degree in
chemical engineering and a Ph.D.
degree (1989) in biochemical engineer-
ing from the Danish Technical Univer-
sity (DTU). He then established his
independent research group and was
appointed full Professor in 1998. He
was Fulbright visiting professor at MIT
in 1995–1996. At the DTU he founded
and directed the Center for Microbial
Biotechnology. In 2008, he was recruited as Professor and Director at
the Chalmers University of Technology, Sweden, where he is currently
directing a research group of more than 50 people and the Life Science
Area of Advance, which coordinates over 200 researchers from
5 departments. He has published more than 350 research papers that
have been cited more than 11 000 times (current H-index 52), co-
authored more than 40 books, and is an inventor of more than
50 patents. He has founded several companies that have raised more
than €25 million in venture capital.
Luis Caspeta is a chemical engineer
with experience in control and automa-
tion of industrial processes. He has an
M.Sc. degree in biotechnology from the
Autonomous University of the State of
Morelos, Mexico, and a Ph.D. degree
with honors in biochemical sciences
from the National Autonomous Univer-
sity of Mexico (2009). He is a member
of the National Researchers System of
Mexico, level of candidate, and is currently a postdoctoral fellow at
Chalmers University of Technology, Sweden, where he is working on
different subjects, including genome-scale metabolic modeling, evolu-
tionary engineering, genome-wide analysis of molecular and metabolic
responses, and advanced calculations for assessing technical, eco-
nomic and environmental feasibilities of processes for advanced-bio-
fuel production. He has published 11 research papers and has an
H-index of 4. He has also participated in more than 25 domestic and
international congresses and given some lectures by invitation.
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