Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

Abstract

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and S. cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01µmol.min(-1).mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae.
Copyright © 2015. Published by Elsevier Inc.

Full text

Functional expression of a heterologous nickel-dependent,
ATP-independent urease in Saccharomyces cerevisiae
N. Milne, M.A.H. Luttik, H. Cueto Rojas, A. Wahl, A.J.A. van Maris, J.T. Pronk, J.M. Daran
n
Q1
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
article info
Article history:
Received 26 February 2015
Received in revised form
18 May 2015
Accepted 21 May 2015
Keywords:
Saccharomyces cerevisiae
ATP-independent urease
Ni-dependent enzyme
Q6
ATP conservation
Nitrogen metabolism
Physiology
abstract
In microbial processes for production of proteins, biomass and nitrogen-containing commodity
chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing
substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential
platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium
requires 1 ATP per incorporated NH
3
. Urea assimilation by this yeast is more energy efficient but still
requires 0.5 ATP per NH
3
produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene
encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding
ATP-independent urease (ure2), along with its accessory genes ureD,ureF and ureG. Since S. pombe ure2 is
aNi
2þ
-dependent enzyme and Saccharomyces cerevisiae does not express native Ni
2þ
-dependent
enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of
the S. pombe genes into dur1,2ΔS. cerevisiae yielded an in vitro ATP-independent urease activity of
0.4470.01 mmol min
1
mg protein
1
and restored growth on urea as sole nitrogen source. Functional
expression of the Nic1 transporter was essential for growth on urea at low Ni
2þ
concentrations. The
maximum specific growth rates of the engineered strain on urea and ammonium were lower than those
of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the
engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the
biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-
containing chemicals and demonstrate that Ni
2þ
-dependent enzymes can be functionally expressed
in S. cerevisiae.
&2015 International Metabolic Engineering Society. Published by Elsevier Inc.
1. Introduction
Industrial biotechnology can contribute to the transition to
sustainable production of fuels and chemicals from renewable
agricultural feedstock's by enabling efficient microbial conversion
of carbohydrates into a wide range of economically relevant
compounds (Hong and Nielsen, 2012). To be competitive with
petrochemical processes, it is essential that microbial conversions
are optimized to achieve maximum yields and productivities.
Microbial production of large-volume nitrogen-containing che-
micals such as amino acids (Wu, 2009) and diamines (Lucet et al.,
1998) is currently based on bacteria, and in particular on Coryne-
bacterium glutamicum and Escherichia coli (Hermann, 2003; Ikeda,
2003; Qian et al., 2009; Qian et al., 2011). Efficient export systems
for nitrogen-containing products and high product titres contri-
bute to the popularity of these bacterial hosts (Hermann, 2003;
Ikeda, 2003). Current microbial biotechnology processes for
production on nitrogenous organic compounds are, without
exception, aerobic (Hermann, 2003). Aeration of large industrial
bioreactors is expensive and respiratory conversion of growth
substrates causes reduced product yields. Moreover, aerobic
respiration is strongly exergonic which, in large reactors, leads to
extra cooling costs. Large-scale microbial production processes
should therefore, whenever permitted by thermodynamics and
biochemistry of the metabolic pathways involved, be performed
under anaerobic conditions. To achieve a high product yield on
substrate, the ATP yield from the product pathway should ideally
be low, but sufficient to achieve a situation in which high catabolic
fluxes are needed to meet ATP requirements for maintenance and
growth (de Kok et al., 2012; Weusthuis et al., 2011). Such a
situation is exemplified by anaerobic alcoholic fermentation of
glucose by S. cerevisiae, which yields only 2 mol ATP per mol of
glucose. In contrast, completely respiratory dissimilation of glu-
cose by S. cerevisiae, which has a P/O ratio of 1.0 (Bakker et al.,
2001), yields ca. 16 mol ATP per mol of glucose and, consequently,
results in much higher biomass yields. Engineered S. cerevisiae
strains are intensively studied and applied for production of
organic compounds (Hong and Nielsen, 2012), but its use for
production of nitrogen-containing compounds is so far restricted
to high-value compounds such as proteins and peptides, most
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Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ymben
Metabolic Engineering
http://dx.doi.org/10.1016/j.ymben.2015.05.003
1096-7176/&2015 International Metabolic Engineering Society. Published by Elsevier Inc.
n
Corresponding author.
E-mail address: j.g.daran@tudelft.nl (J.M. Daran).
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i
Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎

notably human insulin (Cousens et al., 1987; Ostergaard et al.,
2000; Walsh, 2005).
Especially in the design of anaerobic processes for production
of low-molecular weight nitrogen-containing compounds (e.g.
amino acids) by S. cerevisiae, indicated ATP costs for uptake and
assimilation of nitrogen sources should be minimized. Ammonium
and urea are two cheap nitrogen sources commonly used in large-
scale fermentation processes (Albright, 2000; Xun Yao, 2014). In S.
cerevisiae, ATP is expended in the initial steps of the assimilation of
both these compounds. Import of NH
4þ
via the Mep1, Mep2 and
Mep3 uniporters (Marini et al., 1997) is followed by its intracellular
dissociation into NH
3
, which is used for biomass/product forma-
tion, and a proton, which must be exported in order to maintain
homoeostasis of the proton motive force (de Kok et al., 2012). In S.
cerevisiae, this process is catalysed by the ATP-dependent plasma-
membrane H
þ
-ATPase Pma1 (Magasanik, 2003
Q2
) with a stoichio-
metry of 1 ATP per proton (de Kok et al., 2012). The energy
dependency of ammonium uptake in S. cerevisiae makes it extre-
mely challenging to improve ATP utilization. Per mole of nitrogen,
urea (NH
2
–CO–NH
2
) is often cheaper than ammonium (Bryce
Knorr, 2015) and, in contrast to ammonium, its assimilation does
not cause medium acidification (Hensing et al., 1995). In urea-
sufficient cultures of S. cerevisiae, urea uptake is not proton-
coupled (Cooper and Sumrada, 1975) but ATP is expended during
its conversion to ammonia by urea amidolyase (urease) encoded
by DUR1,2 (Fig. 1). This urease converts urea into two molecules of
ammonia and one mole of CO
2
in a two-step reaction that involves
ATP hydrolysis (Mobley et al., 1995), resulting in a cost of 0.5 ATP
per mol NH
3
assimilated into product. In many other micro-
organisms, urea can be converted into two molecules of ammonia
in a single, ATP-independent reaction (Genbauffe and Cooper,
1986
Q3
)(Fig. 1).
Although clearly beneficial for the ATP economy of the cell
during growth on urea, the use of ATP-independent urease
introduces the complication that all known ATP-independent
urease enzymes require nickel insertion at the active site for
catalytic activity. A notable exception is the Helicobacter mustelae
urease, which requires iron instead of nickel, presumably to allow
this pathogen to inhabit the low-nickel environment of its host
Mustela putorius furo (Carter et al., 2011). In the fission yeast
Schizosaccharomyces pombe, ATP-independent urea assimilation is
catalysed by the nickel-requiring enzyme Ure2, whose activity
requires the three accessory proteins UreD, UreF and UreG. UreD
and UreF have been proposed to function as chaperones to
incorporate nickel into the Ure2 enzyme active site and to assist
in protein folding, while UreG is thought to deliver nickel from the
cell membrane to the urease enzyme (Bacanamwo et al., 2002).
The high-affinity nickel transporter Nic1 is involved in Ni
2þ
uptake across the S. pombe plasma membrane (Eitinger et al.,
2000). The roles of the proteins involved in ATP-independent urea
assimilation have been predominantly elucidated in Klebsiella
aerogenes and are homologous across distantly related species
(Mobley et al., 1995).
The aim of the present study was to investigate whether the
native ATP-dependent urease of S. cerevisiae can be functionally
replaced by a heterologous ATP-independent and nickel-requiring
urease. To this end we sought to replace the native ATP-dependent
urea assimilation gene DUR1,2 with the complete ATP-inde-
pendent urea assimilation system from S. pombe (Bacanamwo
et al., 2002; Eitinger et al., 2000; Mobley et al., 1995; Navarathna
et al., 2010). The functionality of the heterologous urease was
characterized in vivo and in vitro. To investigate the impact of
these genetic modifications on yeast physiology, growth of engi-
neered strains and a DUR1,2 reference strain was compared during
growth on urea and ammonium-containing media and at different
nickel concentrations in batch and chemostat cultures.
2. Materials and methods
2.1. Media, strains and maintenance
All S. cerevisiae strainsusedinthisstudy(
Table 1) were derived
from the CEN.PK strain family background (Entian and Kötter, 2007;
Nijkamp et al., 2012). The S. pombe CBS7264 strain was obtained from
CBS-KNAW (Utrecht, The Netherlands [http://www.cbs.knaw.nl/]).
Frozen stocks of E. coli and S. cerevisiae were prepared by addition of
glycerol (30% (v/v)) to exponentially growing cells and aseptically
storing 1 mL aliquots at 80 1C. Cultures were grown in synthetic
medium according to the following recipes. Ammonium sulphate
synthetic medium (SMA) was prepared with 3 g/L KH
2
PO
4
,0.5g/L
MgSO
4
7H
2
Oand5g/L(NH
4
)
2
SO
4
(Verduyn et al., 1992). Urea synthetic
medium (SMU) was prepared with 6.6 g/L K
2
SO
4
,3g/LKH
2
PO
4
,0.5g/L
MgSO
4,
7H
2
Oand2.3g/LCO(NH
2
)
2
. Serine synthetic medium (SMS)
was prepared with 6.6 g/L K
2
SO
4
,3g/LKH
2
PO
4
, 0.5 g/L MgSO
4,
7H
2
O
and 5 g/L serine. Histidine synthetic medium was prepared with 6.6 g/
LK
2
SO
4
,3g/LKH
2
PO
4
,0.5g/LMgSO
4,
7H
2
O and 10 mg/L histidine. In
all cases unless stated otherwise 20 g/L glucose and appropriate
growth factors were added according to (Pronk, 2002), and the pH
adjusted to 5.0. If required for anaerobic growth Tween-80 (420 mg/L)
and ergosterol (10 mg/L) were added. Synthetic medium agar plates
were prepared as described above but with the addition of 20 g/L agar
(Becton Dickinson B.V. Breda, The Netherlands)
2.2. Strain construction
S. cerevisiae strains were transformed using the lithium acetate
method according to (Gietz and Woods, 2002). The DUR1,2 dele-
tion cassette was constructed by amplifying the KanMX4 cassette
from the vector pUG6 (Guldener et al., 1996) using primers with
added homology to the upstream and downstream regions of
DUR1,2 (DUR1,2 KO Fwd/DUR1,2 KO Rev) (Table 3). PCR amplifica-
tion was performed using Phusion
s
Hot Start II High Fidelity
Polymerase (Thermo scientific, Waltham, MA) according to the
manufactures instructions using HPLC or PAGE purified, custom
synthesized oligonucleotide primers (Sigma Aldrich, Zwijndrecht,
The Netherlands) in a Biometra TGradient Thermocycler (Biome-
tra, Gottingen, Germany). The KanMX4 deletion cassette was
transformed into CEN.PK113-5D (ura3-52) yielding strain IMK504
(ura3-52, dur1,2Δ) and transformants were selected on SMA agar
with 200 mg/L G418 (Sigma Aldrich) and 150 mg/L uracil. Deletion
of DUR1,2was confirmed by PCR on genomic DNA preparations
using the diagnostic primers listed under “Primers for verification
of knockout cassettes”in Table 3. Diagnostic PCR was performed
using DreamTaq (Thermo scientific) and desalted primers (Sigma
Aldrich) in a Biometra TGradient Thermocycler (Biometra). Geno-
mic DNA was prepared using a YeaStar Genomic DNA kit (Zymo
Research, Orange, CA).The prototrophic dur1,2Δstrain IME184 was
constructed by transformation of IMK504 with plasmid p426GPD
(2 μm, URA3)(
Mumberg et al., 1995). Transformants were selected
on SMA agar.
Construction of the ATP-independent urease strain IMY082 was
achieved using in vivo vector assembly by homologous recombi-
nation according to (Kuijpers et al., 2013). DNA coding sequences
of S. pombe ure2 (NM_001020242), ureD (NM_001018767.2), ureF
(NM_001020298.2), ureG (NM_001023020.2) and nic1 (NM_00-
1022671.2) were codon optimized for S. cerevisiae using the JCat
algorithm (Grote et al., 2005). Custom synthesized cassettes
cloned into the vector pUC57 (GenBank accession number:
Y14837.1) were provided by BaseClear (Leiden, The Netherlands)
containing the codon optimized genes, flanked by strong consti-
tutive promoters and terminators from the S. cerevisiae glycolytic
pathway. Each cassette was further flanked with 60 bp tags
(labelled A through I) with homology to an adjacent cassette.
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N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎2
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

These tags have no significant homology to the S. cerevisiae
genome ensuring that each cassette can only recombine with an
adjacent cassette using homologous recombination (Kuijpers et al.,
2013). Custom synthesis resulted in plasmids pUD215 (B-TDH3
p
-
ure2-CYC1
t
-C), pUD216 (G-PGK1
p
-nic1-TEF1
t
-I), pUD217 (D-TEF1
p
-
ureD-PGK1
t
-E), pUD218 (E-ADH1
p
-ureF-PYK1
t
-F) and pUD219 (C-
TPI1
p
-ureG-ADH1
t
-D) (Table 2). Each plasmid was transformed into
chemically competent E. coli (T3001, Zymo Research) according to
the manufacturer's instructions, and the gene sequences con-
firmed by Sanger sequencing (BaseClear). Also included were
plasmids with cassettes encoding a URA3 yeast selection marker
(pUD192: A-URA3-B), a CEN6-ARS4 yeast replicon (pUD193: F-
CEN6-ARS4-G), and a fragment containing an AmpR ampicillin
resistance marker and E. coli origin of replication (pUD195:
I-AmpR-A) to allow selection and propagation in both S. cerevisiae
and E. coli. Plasmids propagated in E. coli were isolated with Sigma
GenElute Plasmid Kit (Sigma Aldrich). Each cassette was flanked
by unique restriction sites allowing them to be excised from the
plasmid backbone. For digestion of each plasmid, high fidelity
restriction endonucleases (Thermo Scientific) were used according
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Fig. 1. Overview of native urea and ammonium assimilation in S. cerevisiae and proposed strategy for engineering ATP-independent urea assimilation into this yeast.
(A) Native ATP-dependent urea assimilation involves urea crossing the cell membrane by either passive/facilitated diffusion or via the Dur3 urea active transporter. In the
cytoplasm the urea carboxylase activity of the bi-functional enzyme Dur1,2 converts urea to allophanic acid at the expense of 1 ATP. The allophonic acid is then converted to
2 molecules of NH
3
by the allophanate hydrolase activity of Dur1,2. (B) Heterologous ATP-independent urea assimilation involves urea entering the cytoplasm as previously
described then being converted to 2 molecules of NH
3
in an ATP-independent manner. In both cases CO
2
is produced as a by-product. C. Ammonium assimilation involves the
import of the charged ammonium molecule (NH
4þ
) into the cell by one of three ammonium permeases (Mep1/2/3). At the close to neutral pH in the cytoplasm, NH
4þ
dissociates forming NH
3
and the corresponding H
þ
. In order to maintain pH homoeostasis and proton motive force the proton generated is exported from the cell by the H
þ
-
ATPase Pma1 at the expensive of 1 ATP. In all cases, the resulting NH
3
molecules can then be incorporated into amino acids, via reductive amination of α-ketoglutarate,
yielding glutamate.
Table 1
Strains used in this study.
Name Relevant genotype Origin
Saccharomyces cerevisiae
CEN.PK113-7D MATa URA3 HIS3 LEU2 TRP1 MAL2-8c SUC2 Entian and Kötter (2007) and Nijkamp et al. (2012)
CEN.PK113-5D MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 Entian and Kötter (2007) and Nijkamp et al. (2012)
IME140 MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2þp426GPD (2 mmURA3)Kozak et al., (2014b) and Nijkamp et al. (2012)
IMK504 MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 dur1,2Δ: loxP-KanMX4-loxP This study
IME184 MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 dur1,2Δ:loxP-KanMX4-loxP þp426GPD (2mmURA3) This study
IMY082 MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 dur1,2Δ:loxP-KanMX4-loxP þpUDC121 This study
IMZ459 MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2 dur1,2Δ:loxP-KanMX4-loxP þpUDE266 This study
Schizosaccharomyces pombe
S. pombe 7264 Schizosaccharomyces pombe wild-type strain CBS-KNAW
a
a
Utrecht, The Netherlands [http://www.cbs.knaw.nl/].
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎ 3
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

to the manufacturer's instructions. pUD215 and pUD219 were
digested with ApaI and EcoRV, pUD217 and pUD218 were digested
with ApaI and BamHI, pUD216 was digested with SalI and SphI,
pUD192 was digested with XhoI, pUD193 was digested with SacII,
and pUD195 was digested with NotI. After digestion each fragment
was purified by gel electrophoresis using 1% (w/v) agarose (Sigma
Aldrich) in TAE buffer (40 mM Tris-acetate pH 8.0 and 1 mM
EDTA). Isolation of agarose trapped DNA fragments was performed
using Zymoclean Gel DNA Recovery Kit (Zymo Research).
Equimolar amounts of each fragment were transformed into
IMK504 (dur1,2Δ) allowing for in vivo vector assembly of pUDC121
and pUDE266 by homologous recombination. Correctly assembled
transformants were first selected on SMA agar, single colonies
were then streaked onto SMU agar containing 20 nM NiCl
2
.A
single colony isolate with restored growth on urea was stocked
and labelled as IMY082. Correct plasmid assembly was verified
using primer pairs which bound in each of the gene cassettes and
amplified the 60 bp homologous tags (“Primers for verification of
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Table 2
Plasmids used in this study. CO: codon optimized.
Name Characteristics Origin
pUC57 Delivery vector used for blunt cloning of custom synthesized gene cassettes BaseClear, Leiden, The Netherlands
pUD215 pUC57þTDH3
p
-COure2-CYC1
t
This study
pUD216 pUC57þPGK1
p
-COnic1-TEF1
t
This study
pUD217 pUC57þTEF1
p
-COureD-PGK1
t
This study
pUD218 pUC57þADH1
p
-COureF-PYK1
t
This study
pUD219 pUC57þTPI1
p
-COureG-ADH1
t
This study
pU
Q5
D192 pUC57þURA3 Kozak et al. 2014b and Kozak et al.
(2014a)
pUD193 pUC57þCEN6-ARS4 Kozak et al. (2014b)
pUD195 AmpR, E. coli replicon Kozak et al. (2014a)
p426GPD 2 mm ori, URA3, TDH3
p
-CYC1
t
Mumberg et al. (1995)
pUG6 PCR template for loxP-KanMX4-loxP cassette Guldener et al. (1996)
pUDC121 CEN6-ARS4 ori, AmpR,URA3, TDH3
p
-COure2-CYC1t, TPI1
p
-COureG-ADH1
t
, TEF1
p
-COureD-PGK1
tt
, ADH1
p
-COureF-PYK1
t
,
PGK1
p
-COnic1-TEF1
t
This study
pUDE266 CEN6-ARS4 ori, AmpR,URA3, TDH3
p
-COure2-CYC1t, TPI1
p
-COureG-ADH1
t
, TEF1
p
-COureD-PGK1
t
, ADH1
p
-COureF-PYK1
t
This study
Table 3
Oligonucleotide primers used in this study.
Name Sequence (5
0
-3
0
)
Primers for knockout cassettes
DUR1,2 KO Fwd GTCACAATAAATTTCAGTTTTGATTAAAAAATGACAGTTAGTTCCGATACAACTGAGATCGAGGTTTAGCCATGTCAGCT-
GAAGCTTCGTACGC
DUR1,2 KO Rev GTCACAATAAATTTCAGTTTTGATTAAAAAATGACAGTTAGTTCCGATACAACTGAGATCGAGGTTTAGCCATGTCAGCT-
GAAGCTTCGTACGC
Primers for verification of knockout cassettes
DUR1,2 Upstream Fwd CGCCACGCATCTTTGGCTGCATTTCG
DUR1,2 Downstream Rev ATGCCTTGTAGTCGCCACCTGCTTCCTC
DUR1,2 Internal Fwd GTCTGGCCGCATCTTCTGAGGTTCC
DUR1,2 Internal Rev TCTACCAGAACCTGCTGTATCAGTA
KanMX4 Internal Fwd CGAGGCCGCGATTAAATTC
KanMX4Internal Rev AAACTCACCGAGGCAGTTC
Primers for plasmid construction
G-I Linker Upper GCCAGAGGTATAGACATAGCCAGACCTACCTAATTGGTGCATCAGGTGGTCATGGCCCTTTATTCACGTAGACGGATAGG-
TATAGCCAGACATCAGCAGCATACTTCGGGAACCGTAGGC
G-I Linker Lower GCCTACGGTTCCCGAAGTATGCTGCTGATGTCTGGCTATACCTATCCGTCTACGTGAATAAAGGGCCATGACCACCTGATG-
CACCAATTAGGTAGGTCTGGCTATGTCTATACCTCTGGC
Primers for verification of plasmid assembly
Tag A amp Fwd ATTATTGAAGCATTTATCAGGGTTATTGTCTCATG
Tag A amp Rev GAAATGCTGGATGGGAAGCG
Tag B amp Fwd GGCCCAATCACAACCACATC
Tag B amp Rev GCATGTACGGGTTACAGCAGAATTAAAAG
Tag C amp Fwd TGTACAAACGCGTGTACGCATG
Tag C amp Rev CAGGTTGCTTTCTCAGGTATAGCATG
Tag D amp Fwd ACTCTGTCATATACATCTGCCGCAC
Tag D amp Rev GCTAAATGTACGGGCGACAG
Tag E amp Fwd TTTCTCTTTCCCCATCCTTTACG
Tag E amp Rev GTCGTCATAACGATGAGGTGTTGC
Tag F amp Fwd GCCTTCATGCTCCTTGATTTCC
Tag F amp Rev GGCGATCCCCCTAGAGTC
Tag G amp Fwd AAAAGATACGAGGCGCGTGTAAG
Tag G amp Rev CGCCTCGACATCATCTGCCCAG
Tag I amp Fwd TGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCC
Tag I amp Rev AGTCAGTGAGCGAGGAAGC
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎4
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

plasmid assembly”,Table 3). The plasmid was extracted from
IMY082, named as pUDC121 and transformed into E. coli DH5α
by electroporation in 2 mm cuvettes (1652086, BioRad, Hercules,
CA) using a Gene PulserXcell electroporation system (BioRad)
following the manufacturer's protocol and stocked in the E.
coli host.
IMZ459 was constructed in the exact same manner as
described for IMY082. However in place of a fragment containing
the nic1 gene cassette a 120 bp fragment with 60 bp homology to
each adjacent cassette was used (“G-I linker upper and lower”,
Table 3). The resulting in vivo assembled plasmid was transformed
into E. coli DH5αby electroporation and labelled as pUDE266.
2.3. Shake flask and chemostat cultivation
S. cerevisiae and S. pombe were grown in either SMA (Verduyn
et al., 1990), SMU or SMS. When required, 20 nM NiCl
2
was added.
If required, 150 mg/L uracil was added to the media. Cultures were
grown in either 500 mL or 250 mL shake flasks containing 100 mL
or 50 mL of synthetic medium and incubation at 30 1C in an Innova
incubator shaker (New Brunswick Scientific, Edison, NJ) at
200 rpm. Optical density at 660 nm was measured at regular
intervals using a Libra S11 spectrophotometer (Biochrom, Cam-
bridge, United Kingdom). Controlled aerobic, carbon-limited che-
mostat cultivation was carried out at 30 1C in 2 L bioreactors
(Applikon, Schiedam, The Netherlands) with a working volume
of 1 L. Chemostat cultivation was preceded by a batch phase under
the same conditions. When a rapid decrease in CO
2
production
was observed (indicating glucose depletion), continuous cultiva-
tion at a dilution rate of 0.1 h
1
was initiated. Synthetic medium
was supplemented with 7.5 g/L glucose and 0.2 g/L of Pluronic
antifoam (BASF, Ludwigshaven, Germany). The pH was maintained
constant at pH 5.0 by automatic addition of 2 M KOH and 2 M
H
2
SO
4
. The stirrer speed was constant at 800 rpm and the aeration
rate kept at 500 mL/min. Chemostat cultures were determined to
be in steady state when after at least 5 volume changes the CO
2
production rate, O
2
consumption rate and cell dry weight had all
varied by less than 2% over a period of 2 volume changes. Steady-
state samples were taken between 12 and 15 volume changes after
inoculation.
2.4. Analytical methods
96 well plate assays were prepared by adding 100 μL of SMA or
SMU with 20 g/L glucose, 20 nM NiCl
2
, Tween-80 (420 mg/L) and
ergosterol (10 mg/L). If required, 10 mg/L histidine was added.
Optical density was regularly measured at a wavelength of 660 nm
in a GENios pro plate reader (Tecan Benelux, Giessen, The Nether-
lands). Cells were inoculated in each well to a starting OD
660 nm
of
0.1. Plates were then covered with Nunc™sealing tape (Thermo
Scientific) and incubated at 30 ˚C with constant shaking at
200 rpm.
Biomass dry weight from bioreactors was determined by
filtration of 10 mL broth over pre-dried and weighed 0.45 mm
nitrocellulose filters (Gelman Laboratory, Ann Arbor, MI). After
filtration the filters were dried for 20 min in a microwave at
350 W.
To determine extracellular glucose, urea and ammonium con-
centrations, samples were taken with the stainless steel bead
method for rapid quenching of metabolism according to (Mashego
et al., 2003). Culture samples were spun down at 3500gRPM and
the supernatant was collected. Extracellular metabolites were
analysed using a Waters Alliance 2695 HPLC (Waters Chromato-
graphy B.V, Etten-Leur, The Netherlands) with an Aminex HPX-
87H ion exchange column (BioRad) operated at 60 1C with a
mobile phase of 5 mM H
2
SO
4
and a flow rate of 0.6 mL/min.
Extracellular urea concentrations were determined using GC–MS
according to (de Jonge et al., 2011). Extracellular ammonium
concentrations were determined using an Ammonium Cuvette
test kit (Hach-Lange, Tiel, The Netherlands) according to the
manufacturer's instructions.
The nitrogen content of the biomass was determined using
Elemental Biomass Composition Analysis (EBCA). For this 150 mg
of biomass were prepared by washing twice in MilliQ water
(MilliQ) and resuspended in a total volume of 1 mL. After 48 h
freeze drying, the biomass in the sample was crushed into a fine
powder using a pestle and mortar. The pestle and mortar were
prepared by autoclaving at 121 ˚C and thoroughly washed with
subsequently 2 M H
2
SO
4
, 2 M KOH, MilliQ water (MilliQ) and
acetone. Finally the pestle and mortar were dried at 100 ˚C for
24 h before use. The fine dried powder was then sent for analysis
(ECN, Petten, The Netherlands) Total nitrogen was determined
using a TOC-L CPH analyser (Shimadzu, ‘s-Hertogenbosch, The
Netherlands) according to the manufacturer's instructions. In this
method all nitrogen is first combusted to nitrogen monoxide,
which is then detected by chemiluminescence using a non-
dispersive infrared gas analyser.
2.5. Enzyme-activity assays
ATP-independent urease activity and urea amidolyase (ATP-
dependent) activity was determined in two separate enzyme
assays. Cell extracts were prepared by harvesting 62.5 mg of
biomass dry weight by centrifugation at 4600gfor 5 min. Cell
pellets were washed with 10 mM potassium phosphate buffer
containing 2 mM EDTA at pH 7.5, then washed again and resus-
pended in 100 mM potassium phosphate buffer at pH 7.5 contain-
ing 2 mM MgCl
2
and 2 mM dithiothreitol. Extracts were prepared
using Fast Prep FP120 (Thermo Scientific) with 0.7 mm glass
beads. Cells were desintegrated in 4 bursts of 20 s at speed 6 with
30 s of cooling on ice between each run. Cellular debris was
removed by centrifugation at 47,000gfor 20 min at 4 1C. The
purified cell free extract was then used immediately for enzyme
assays. Protein concentration of the cell extract was determined
using the Lowry method (Lowry et al., 1951). Enzymatic assays
were performed at 30 1C in a Hitachi U-3010 spectrophotometer.
In both cases, activity of the urease enzyme was coupled to the
conversion of ammonia to glutamate by glutamate dehydrogenase
and the accompanying NADPH oxidation was monitored over time
by a decrease in absorbance at 340 nm. ATP-independent urease
activity was measured using a Urea/Ammonia rapid assay kit
(Megazyme International Ireland, Wicklow, Ireland) with a mod-
ified protocol. The assay mixture contained in a total volume of
1 mL; 130 mL buffer solution, 80 mL NADPH solution, 20 mL gluta-
mate dehydrogenase solution and cell extract. After allowing for
residual enzymatic activity to subside, the reaction was initiated
by addition of 50 mM urea. For determining ATP-dependent
urease activity (urea amidolyase), the assay mixture contained in
a total volume of 1 mL; 50 mM Tris–HCl, 20 mM KHCO
3
,50mM
urea, 8 mM α-ketoglutarate, 15 mM KCl, 0.15 mM NADPH, 2.5 mM
MgCl
2
, 0.02 mM EDTA and 5 mL glutamate dehydrogenase solution
from the urea/ammonia rapid assay kit (Megazyme). After allow-
ing for residual activity to subside, the reaction was initiated by
addition of 2 mM ATP.
2.6. Stoichiometric-model based prediction of the impact of urea
assimilation on the biomass yield
The expected influence of the two different nitrogen sources
and their uptake and utilization mechanisms were quantified by
metabolic network analysis. In particular, the expected biomass
yields were determined using a stoichiometric model containing
1
2
3
4
5
6
7
8
9
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N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎ 5
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

56 reactions (Supplementary information) and 52 balanced meta-
bolites by optimization (linear programming) for a fixed substrate
uptake rate
ðv
glc
¼1Þ:
^
v¼argmaxð
v
v
biomass
Þsubject to
Sv ¼0
v
glc
¼0
v
irr
Z0
8
>
<
>
:
9
>
=
>
;
Where vis the vector of fluxes, Sis the stoichiometric matrix of
the model and v
irr
is the set of irreversible fluxes. Urea was
assumed to be transported passively on the basis that active
transport is present only under urea-limiting conditions (Cooper
and Sumrada, 1975). All calculations were performed using the
tool CellNetAnalyzer (Klamt et al., 2007) using MATLAB linprog
(MathWorks, Eindhoven, The Netherlands).
3. Results
3.1. Construction and selection of ATP-independent urea assimilation
in S. cerevisiae
To engineer ATP-independent urea assimilation, the S. cerevi-
siae DUR1,2 gene, which encodes an ATP-dependent urea
amidolyase, was first deleted. The resulting strain IMK504 (ura3-
52, dur1,2Δ) and the corresponding prototrophic strain IME184
(ura3-52, dur1,2Δp426GPD) were unable to grow on urea as sole
nitrogen source (Fig. 2A), thereby confirming that Dur1,2 is the
only urea assimilation enzyme in S. cerevisiae (Cooper et al., 1980).
Moreover, absence of growth on urea validated the strain as a
suitable platform for a functional complementation study.
The coding sequences of the five S. pombe genes involved in
functional expression of its ATP-independent urease were codon
optimized for expression in S. cerevisiae using the JCat algorithm
(Grote et al., 2005). These five genes comprised the urease
structural gene ure2, as well as three urease accessory genes ureD,
ureF, and ureG, and nic1, which encodes a high-affinity nickel
transporter. While Ure2 is solely responsible for catalysing the
conversion of urea to ammonia, ureD, ureF, and ureG are essential
for its functional expression (Mobley et al., 1995). Although S.
cerevisiae is not known to harbour nickel-dependent enzymes or a
specific nickel transporter, it is well known that nickel ions can
enter S. cerevisiae cells when present at high concentrations
(45mM) (Joho et al., 1995). However, since addition of high
concentrations of Ni
2þ
to growth media is undesirable and would
likely result in nickel toxicity (Joho et al., 1995), S. pombe nic1
(Eitinger et al., 2000) was co-expressed with the urease complex.
Construction of the ATP-independent urease strain IMY082 was
achieved by in vivo vector assembly by homologous
1
2
3
4
5
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Fig. 2. (A) Complementation of S. cerevisiae IMK504 with pUDC121. Cells were plated on SMA or SMU agar with 20 nM NiCl
2
, 0.150 g/L uracil (Ura) and 1 g/L 5-fluoroorotic
acid (5
0
FOA) as indicated. Cells were pre-cultured in liquid synthetic medium with ammonium sulphate and washed twice in MilliQ water prior to plating. Plates were
incubated aerobically for 72 h at 30 ˚C. (B) Outline of the assembly of pUDC121 using vector assembly by in vivo homologous recombination using 60 bp overlapping tags.
(C) PCR analysis of the resulting plasmid pUDC121. PCR bands of the overlapping homologous tags were generated using primers which bound in each of the gene cassettes
with the sizes indicated. L: DNA ladder.
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎6
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

recombination (Kuijpers et al., 2013). In this approach, each
heterologous gene cassette, comprising a strong constitutive
promoter, a codon-optimized coding sequence and a terminator
was flanked by 60 bp homologous tags which allowed adjacent
cassettes with the same homologous tag to be assembled via
in vivo homologous recombination (Kuijpers et al., 2013). With the
addition of a cassette for the URA3 marker, the CEN6ARS4 yeast
replicon and an E. coli fragment including the ampicillin resistance
gene (amp
r
) and a bacterial origin of replication, the yeast expres-
sion plasmid pUDC121 was assembled in vivo in the strain IMK504
(ura3-52,dur1,2Δ) resulting in strain IMY082 (dur1,2Δ,ure2,D,F,G
nic1). Correct assembly of the corresponding plasmid (pUDC121)
(Fig. 2B) was confirmed by PCR (Fig. 2C).
To assess the ability of the ATP-independent urease construct to
support growth of the resulting strain IMY082 (dur1,2Δ,ure2,D,F,G
nic1) on urea, it was initially plated on synthetic medium with
ammonium and, subsequently, replica plated onto synthetic med-
ium containing urea as the sole nitrogen source and supplemented
with 20 nM NiCl
2
(Fig. 2A). IMY082 grew on media supplemented
with urea and NiCl
2
, while the negative control strain IME184
(dur1,2Δ,p426GPD) grew on synthetic medium with ammonium,
but not with urea as the nitrogen source. The reference strains
CEN.PK113-7D (DUR1,2) and IME140 (DUR1,2 ura3-52 p426GPD
(URA3)) grew normally on both ammonium and urea media. Strain
IMY082 did not grow on SMU agar plates that were supplemented
with 5
0
fluoroorotic acid (5FOA) to induce plasmid loss. This
observation further confirmed that, indeed, the heterologous
plasmid was responsible for growth on urea and functionally
complemented the dur1,2 deletion (Fig. 2A).
3.2. Nickel dependency of S. cerevisiae IMY082 (dur1,2Δ, ure2,D,F,G
nic1)
The engineered S. cerevisiae strain IMY082 (dur1,2Δ,ure2,D,F,G
nic1) grew on urea without addition of Ni
2þ
to the growth
medium (Fig. 2A) and continued to grow on urea after 10
successive serial transfers in media that had not been supplemen-
ted with Ni
2þ
. The introduced urea assimilation system requires
only one nickel atom per urease enzyme to become catalytically
active (Mobley et al., 1995) suggesting that only trace amounts of
Ni
2þ
are required for urease activity. This raised the possibility
that nickel contamination from glassware and medium compo-
nents might have been sufficient to support growth of the
engineered strain. To test this hypothesis, a series of growth assays
were performed in the presence of histidine. Histidine is a strong
chelator of nickel (histidine/Ni
2þ
dissociation constant K
D
¼
14 71 nM) (Knecht et al., 2009) and is involved in nickel detox-
ification in S. cerevisiae (Joho et al., 1995). Growth rates of the ATP-
independent urease strain (IMY082) and the ATP-dependent
urease reference strain (IME140) were compared by monitoring
OD
660nm
in 96-well plates in synthetic media (SMA and SMU)
supplemented with anaerobic growth factors Tween-80/ergosterol
and 20 nM NiCl
2
in the presence (10 mg/L) and absence of
histidine. Both strains showed comparable growth rates with
ammonium sulphate as the sole nitrogen source at both histidine
concentrations tested (Fig. 3). During growth on urea the control
strain IME140 showed comparable growth rates at both histidine
concentrations tested. However, for IMY082, growth on urea was
only observed in the cultures to which no histidine had been
added and 10 mg/L histidine completely abolished growth (Fig. 3).
This result confirmed that, also after expression in S. cerevisiae, the
ATP-independent urease from S. pombe has a strict requirement
for nickel. Additionally, this indicates that even without supple-
mentation of NiCl
2
, the synthetic medium used in this study
already contains sufficient nickel to support growth of the engi-
neered strain on urea.
3.3. Functionality of the Nic1 transporter
In S. pombe, Nic1 transports nickel across the plasma mem-
brane and into the cytosol with high affinity. However in the
absence of the Nic1 transporter, nickel is still able to cross the
membrane via non-specific metal uptake systems, particularly via
magnesium transporters (Eitinger et al., 2000). A similar situation
is observed in S. cerevisiae where nickel can enter the cell through
non-specific metal uptake systems, particularly via the magnesium
1
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Fig. 3. Relative maximum specific growth rates of IME140 (DUR1,2p426GPD)
(black bars) and IMY082 (dur1,2Δure2,D,F,G nic1) (white bars) in SMA (NH
4
)or
SMU (Urea) with 20 nM NiCl
2
, Tween-80 (420 mg/L), ergosterol (10 mg/L) with
0 mg/L and 10 mg/L histidine. Cells were cultured in 100 ml volumes in a 96 well
plate and incubated at 30 ˚C with OD660 measured at 15 min intervals. Data are
presented as averages and standard deviations of triplicate experiments, relative to
the average maximum specific growth rate of IME140 under each condition.
Table 4
Aerobic maximum specific growth rates in shake-flask cultivations containing 100 mL SMA and SMU with NiCl
2
supplementation as indicated. Shown are averages and mean
deviations from two replicates. NG: No growth, ND: Not determined.
Media Strains
IME140 IMY082 IMZ459 IME184 S. pombe 7264
(DUR1,2 p426GPD) (dur1,2Δure2,D,F,G nic1)(dur1,2Δure2,D,F,G)(dur1,2Δp426GPD) (ure2,D,F,G nic1
a
NH
4þ
0.3370.01 0.1670.01 0.1470.01 ND ND
NH
4þ
þ20 nM NiCl
2
0.3070.02 0.1970.01 0.1470.01 ND ND
NH
4þ
þ20 mM NiCl
2
0.3070.02 0.1870.01 0.1670.01 ND ND
Urea 0.3170.02 0.1870.00 NG NG ND
Ureaþ20 nM NiCl
2
0.3270.0 0 0.1870.0 0 NG NG 0.1570.00
Ureaþ20 mM NiCl
2
0.2870.01 0.20 70.00 0.1670.00 NG ND
a
Genes expressed under their own native promoter.
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎ 7
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

transporters Alr1 and Alr2 (MacDiarmid and Gardner, 1998). In S.
cerevisiae, wild-type cells have been reported to accumulate
19.9 nmol Ni
2þ
/mg biomass in the presence of 0.1 mM NiCl
2
(Nishimura et al., 1998).
To study the functionality of nic1 in the engineered strain,
especially at low concentrations of Ni
2þ
in growth media, an ATP-
independent urease strain was constructed which lacked the nic1
expression cassette. Analogous to the construction of strain
IMY082, this strain was built using vector assembly by homo-
logous recombination (Kuijpers et al., 2013). The expression
cassettes of the S. pombe urease complex (but lacking the nic1
cassette) were assembled in vivo in the dur1,2Δstrain IMK504,
yielding in plasmid pUDE266. In place of the nic1 cassette, a
120 bp fragment with 60 bp homology to each adjacent cassette
was used, yielding the strain IMZ459 (dur1,2Δ,ure2,D,F,G). After
confirmation of correct assembly by PCR, growth of strain IMZ459
was analysed on synthetic medium with urea as the sole nitrogen
source and with different concentrations of NiCl
2
. In media with
added NiCl
2
concentrations ranging from 0 nM to 1 mM, no growth
was observed. However, at a concentration of 20 mM NiCl
2
, the
strain grew on urea medium with a specific growth rate of
0.1670.00 (Table 4).
To quantitatively determine the effect and potential benefitof
the Nic1 transporter, specific growth rates of IMY082 (with nic1)
and IMZ459 (no Ni-transporter), as well as an ATP-dependent
urease control (IME140) were determined from cultures growing
in SMU and SMA in the presence of 0 nM, 20 nM, and 20 mMof
added NiCl
2
(Table 4). While both IME140 and IMY082 were able
to grow under all conditions tested, IMZ459 (no Ni-transporter)
could only grow on urea in the presence of 20 mM NiCl
2
. The ability
of the strain containing the Nic1 transporter to grow at 41000
fold lower concentrations of NiCl2 compared to the negative
control strain IMZ459 confirmed the functional expression of the
Nic1 transporter.
3.4. ATP-independent urease enzyme activity
After confirmation of the activity and Ni
2þ
-dependency of S.
pombe ure2 expressed in S. cerevisiae, the ATP-(in)dependency and
enzyme activities of both the native and heterologous urease enzyme
were investigated in cell extracts of different strains. In these experi-
ments, strains were pre-grown in synthetic medium containing either
urea or serine as the sole nitrogen source to determine the impact of
nitrogen source on urease activity. Serine was chosen as an alternative
nitrogen source instead of ammonium sulphate so that trace amounts
of ammonium remaining in the cell free extract would not interfere
with the enzyme assays. Irrespective of the nitrogen source used for
growth, ATP-independent urease activity was measured in cell-free
extracts of the engineered strain IMY082 (dur1,2Δ,ure2,D,F,G nic1).
ATP-independent urease activities in cell extracts of urea- and serine-
grown of this strain were comparable (0.44 70.01 and 0.32 7
0.02 mmol min
1
mg protein
1
, respectively) (Table 5). Activity of
the heterologously expressed S. pombe urease in S. cerevisiae was ca.
six-fold higher than the enzyme activity observed in urea-grown
cultures of S. pombe (0.0670.00 mmol min
1
mg protein
1
). Consis-
tent with the deletion of the native S. cerevisiae urease gene DUR1,2,
no ATP-dependent urease activity was detected in cell extracts of
strain IMY082, irrespective of the nitrogen source. Cell extracts of the
ATP-dependent urease control strain (IME140, DUR1,2) only exhibited
activity in the presence of ATP, with a specificactivityof
0.0570.01 mmol min
1
mg protein
1
(Tabl e 5).
3.5. Physiological characterisation
To study the quantitative impact of the replacement of the
native S. cerevisiae ATP-dependent urease with a heterologous
ATP-independent urease, the physiology of strains IMY082
(dur1,2Δ,ure2,D,F,G nic1) and IME140 (DUR1,2) were compared in
glucose-grown shake-flask and chemostat cultures with urea or
ammonium as the sole nitrogen sources.
Irrespective of the nitrogen source, the engineered strain
IMY082 grew 30–50% slower than the control strain in shake flask
cultures (Table 4). Increasing the concentration of NiCl
2
(up to
20 mM) had no significant impact on the specific growth rate on
urea or ammonium as nitrogen sources.
Chemostat cultivation allows for physiological comparison of
strains with different maximum specific growth rates at identical
sub-maximal specific growth rates (dilution rates) determined by
the operator (Tai et al., 2005). Aerobic glucose-limited chemostat
cultures of strains IMY082 (dur1,2Δ,ure2,D,F,G nic1) and IME140
(DUR1,2) were run at a dilution rate of 0.10 h
1
in synthetic
medium with either ammonium or urea as the nitrogen source,
in all cases supplemented with 20 nM NiCl
2
. ATP conservation
during urea assimilation should theoretically result in an increase
in biomass yield as the conserved ATP can be used for biomass
production. While cultivation under anaerobic conditions would
theoretically result in the largest relative increase in biomass yield
(2.6% as compared to 2.1% under aerobic conditions) (Table 6),
both values are within the error limit of the biomass determina-
tions in chemostat cultures. In ammonium-grown, glucose-limited
chemostat cultures, no statistically significant differences were
observed in relevant physiological parameters between IMY082
(dur1,2Δ,ure2,D,F,G nic1) and IME140 (DUR1,2)(
Table 7). Also on
urea, biomass-specificfluxes and biomass yields of IMY082 and
IME140 were not significantly different. However, whilst both
strains released some ammonium after intracellular conversion
of urea, this ammonia release was ca. 70% higher in strain IMY082
than in IME140 (17.2070.21 mM vs 10.3870.2 mM). Additionally,
a lower nitrogen content of the biomass was measured for the
engineered strain (56.071.0 mg/g biomass) than for IME140
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Table 5
Specific enzyme activities for ATP-dependent and ATP-independent ureases in S.
cerevisiae and S. pombe cell extracts grown on SMA and SMS supplemented with
20 nM NiCl
2
. Specific activity is expressed in mmol min
1
mg protein
1
. Data are
presented as averages and mean deviations from two biological replicates. NA: not
applicable (strain does not grow on urea), BD: below detection limit of
0.01 mmol min
1
mg protein
1
).
Strain ATP-dependent ATP-independent
Urea Serine Urea Serine
IME184 (dur1,2Δp426GPD) NA BD NA BD
IME140 (DUR1,2 p426GPD) 0.0570.01 BD BD BD
S. pombe 7264 (ure2,D,F,G nic1
a
) BD BD 0.0670.00 BD
IMY082 (dur1,2Δure2,D,F,G nic1) BD BD 0.4470.01 0.3270.02
a
Genes expressed from their native promoter.
Table 6
Predicted biomass yields of the ATP-dependent urease control strain IME140
(DUR1,2p426GPD) and ATP-independent urease strain IMY082 (dur1,2Δure2,D,F,
G nic1) under aerobic and anaerobic glucose limited chemostat conditions with
both NH
4þ
and urea as the sole nitrogen source. Values were calculated based on a
stoichiometric model optimized for maximal biomass production. All yield values
are expressed as g biomass/g glucose.
Condition N-source Strain Improvement (%)
IME140 IMY082
Aerobic NH
4þ
0.5364 0.5364 0
Urea 0.5360 0.5471 2.1
Anaerobic NH
4þ
0.1035 0.1035 0
Urea 0.1060 0.1088 2.6
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎8
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

(67.570.5 mg/g biomass). Given the differences in nitrogen dis-
tribution observed for IME140 and IMY082 at steady state, an
accurate account of nitrogen distribution was made. The nitrogen
balances of the chemostat cultivations (Supplementary data 2)
nearly closed to 100% when extracellular urea, ammonium, and
nitrogen in the biomass were compared with the urea fed to the
cultures. A closed nitrogen balance was also observed when total
nitrogen analyses on the reservoir medium at the time of steady-
state sampling, the supernatant at steady state, and the whole cell
broth at steady state were compared.
4. Discussion
4.1. Expression of a Ni-dependent ATP-independent urease in S.
cerevisiae
In this study, we demonstrate the functional replacement of the
native S. cerevisiae ATP-dependent urea assimilation enzyme
(Dur1,2) by an ATP-independent enzyme from S. pombe (Ure2)
and three accessory proteins (UreF, UreG and UreD; Bacanamwo
et al., 2002), which were previously shown to be essential for
functional enzymatic activity in organisms expressing ATP-
independent urease (Lee et al., 1992; Park et al., 20 05).
Although the catalytic mechanisms of urea hydrolysis in S.
cerevisiae and S. pombe differ significantly, both systems lead to
ammonia and carbon dioxide formation (Fig. 1).
In fungi there is an evolutionary divergence between organ-
isms that assimilate urea at the expense of ATP and those that do
not. Yeasts belonging to the subphylum Saccharomycotina (e.g. S.
cerevisiae)(
Kurtzman and Robnett, 2013; Weiss et al., 2013)
harbour the DUR1,2 gene encoding ATP-dependent urease. It has
been hypothesized that the evolutionary advantage of expending
ATP was to allow yeasts such as S. cerevisiae to eliminate all nickel-
requiring reactions, thus reducing the number of transition metals
for which cellular homoeostasis and regulation is required
(Navarathna et al., 2010). In this study, we demonstrate that co-
expression of the S. pombe high affinity Ni
2þ
-transporter gene
(Nic1; Eitinger et al., 2000) was required for Ure2-dependent
growth of S. cerevisiae at low Ni
2þ
concentrations. This is in line
with phylogenetic research, which indicates that acquisition of the
bi-functional ATP-dependent Dur1,2 and loss of ATP-independent
urease in the ancestor of Saccharomycotina yeasts coincided with
the loss of the high affinity nickel transporter (Navarathna et al.,
2010; Zhang et al., 2009).
Although the growth of a dur1,2ΔS. cerevisiae strain on urea
could be restored by complementation of the S. pombe urease
system, the engineered strain (IMY082) exhibited a growth rate
decrease ranging from ca. 30% to 50% depending on the growth
conditions relative to a DUR1,2 reference strain (Table 4). Surpris-
ingly, this decreased growth rate was not only observed during
growth on urea, but also on ammonium, a condition in which
urease is not expected to be involved in nitrogen assimilation.
Overexpression of the native DUR1,2 gene, leading to enzyme
activities comparable to those measured with the S. pombe
enzyme in our study, did not result in a reduction of the growth
rate (Coulon et al., 2006) suggesting that increased urease activity
is not the cause for the suboptimal growth. Moreover, release of
ammonia by the cultures indicates that the capacity of the
heterologous enzyme was sufficient to sustain the ammonia
requirement for growth.
In the current metabolic engineering design, heterologous
genes were placed under the control of highly active glycolytic
promoters (Knijnenburg et al., 2009). While placing ure2, the
catalytic enzyme behind such a promoter may be useful to enable
high in vivo fluxes, the high expression of the accessory enzymes
might not be necessary and, in contrast, may have led to an
increased general protein burden (Sauer et al., 2014) and/or
interference with metal metabolism and homoeostasis or protein
folding. Future strain designs should take this possibility into
account by either fine tuning individual gene expression by
selecting appropriate promoters (Blazeck et al., 2012; Nevoigt
et al., 2006) or by evolutionary and reverse engineering to select
strains with recovered growth rates (Oud et al., 2012).
4.2. Functional expression of nickel dependent enzymes in S.
cerevisiae
The present study demonstrates that functional expression of a
heterologous Ni-dependent activity in the S. cerevisiae cytosol is
possible and not precluded by, for example, binding of Ni by
cytosolic histidine. This result represents an innovation in the
metabolic engineering of this yeast with possible implications
beyond engineering of urea metabolism. Ni-containing enzymes
play critical roles in bacteria, archaea, fungi, algae, and higher
plants (Mulrooney and Hausinger, 2003), but encompass a limited
range of activities (i.e. glyoxalase I, acidreductone dioxygenase,
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Table 7
Physiology of the ATP-dependent urease strain IME140 (DUR1,2 p426GPD) and the ATP-independent urease strain IMY082 (dur1,2Δure2,D,F,G nic1) in aerobic glucose limited
chemostat cultures in SMA and SMU with 7.5 g/L glucose and 20 nM NiCl
2
maintained at pH 5.0 and a dilution rate of 0.1 h
1
.
Parameters UreaþNi NH
4þ
þNi
IME140
a
IMY082
a
IME140
b
IMY082
b
Dilution rate (h
1
)0.1070.00 0.1070.0 0 0.1070.0 0 0.1070.01
Yx/s (g/g glucose) 0.5070.01 0.50 70.01 0.49 70.00 0.49 70.00
qO
2
(mmol/g h) 2.7670.07 2.78 70.13 2.7770.15 2.5970.05
qCO
2
(mmol/g h) 3.2470.02 3.30 70.10 2.7570.25 2.64 70.12
qGlucose (mmol/g h) 1.0770.03 1.1070.03 1.1270.03 1.0570.03
qUrea/NH
4þ
(mmol/g/h) 0.4170.01 0.4670.01
c
0.3570.05 0.36 70.09
Residual NH
4þ
(mM) 10.3870.20 17.2070.21 NA NA
Nitrogen in biomass (mg/g biomass) 67.570.5 56.071.0 72.0
d
ND
C recovery (%) 10471. 7 103 70.8 100 73.0 10371. 5
N recovery (%)
e
9670.8 97 70.3 10575.6 106 73.7
NA: not applicable; ND: not determined.
a
Averages and mean deviations from two replicates.
b
Averages and standard deviations from three replicates.
c
Calculated from residual urea values from one chemostat.
d
Based on CEN.PK113-7D data from (Lange and Heijnen, 2001).
e
Calculated from nitrogen balance presented in Supplementary data 2.
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎ 9
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

urease, superoxide dismutase, [NiFe]-hydrogenase, carbon mon-
oxide dehydrogenase, acetyl-coenzyme A synthase/decarbonylase,
methyl-coenzyme M reductase and lactase racemase) (Boer et al.,
2014). In addition, as reported in this study for the S. pombe
urease, the Ni-dependent enzymes require auxiliary proteins that
participate in Ni delivery, metallocenter assembly, or organome-
tallic cofactor synthesis and a dedicated transport system (Higgins
et al., 2012). Having demonstrated the successful expression of a
functionally active Ni-dependent urease, other Ni-dependent
enzymes might also be functionally expressed in S. cerevisiae.As
an example, the optimization of the formation of cytosolic acetyl-
CoA as a precursor for many industrially produced chemicals
(isoprenoids, lipids, butanol, flavonoids) in S. cerevisiae has
recently received a lot of attention (Krivoruchko et al., 2015). This
includes the successful replacement of yeast acetyl-CoA synthases
by several ATP-independent solutions encompassing the cytosolic
expression of the ATP-independent pyruvate dehydrogenase com-
plex (PDH) from Enterococcus faecalis (Kozak et al., 2014b), and the
expression of an acetylating acetaldehyde dehydrogenase and the
expression of a pyruvate-formate lyase (Kozak et al., 2014a). In all
these cases acetyl-CoA is formed from intermediates of central
metabolism, acetate or pyruvate. In contrast, acetogenic micro-
organisms such as Moorella thermoacetica use the Ni
2þ
-dependent
acetyl-CoA decarboxylase/synthase (Mulrooney and Hausinger,
2003) to catalyse the reversible formation of acetyl-CoA from
CO
2
, Co-enzymeA and a corrinoid-bound methyl group (Maynard
and Lindahl, 1999), resulting in net CO
2
fixation. While implemen-
tation of this pathway into S. cerevisiae will involve major other
challenges –e.g. engineering of vitamin B12 biosynthesis into this
eukaryote –the demonstration that Ni-dependent enzymes can be
expressed in this yeast eliminates at least one potential hurdle.
4.3. Decreasing the ATP requirement for nitrogen-containing
products
The elimination of the ATP requirement for urea assimilation
decreases the ATP requirement by 0.5 mol of ATP per mol of
nitrogen assimilated. In this study, urea was solely used as a
nitrogen source for the formation of biomass. Based on stoichio-
metric model-based predictions, the decreased requirement for
ATP in urea assimilation under aerobic conditions could result in
an increase of the biomass yield on glucose of 2.1%. Although
potentially relevant for large scale yeast biomass production, this
small predicted increase is within the error margin of the biomass
yield determinations on glucose in our laboratory chemostat
cultures (Table 7). Additionally, excretion of ammonia into the
extracellular space, which has previously been reported for urea-
grown wild-type cultures of S. cerevisiae (Marini et al., 1997), may
decrease the positive impact of ATP-independent urease. If the
exported ammonia re-associates with a proton to form ammo-
nium and is then take up again, this might cause a futile cycle due
to the energy costs of ammonium uptake (Fig. 1). These results
indicate that, in order to fully benefit from the ATP savings during
urea assimilation in strains expressing ATP-independent urease, its
expression should be tuned to prevent ammonia release into the
medium.
The potential benefit of ATP-independent nitrogen assimilation
can be much larger in strains that not only require nitrogen for
biomass formation, but also for the formation of nitrogen contain-
ing products. The yeast S. cerevisiae has widely been used as a host
for the production of heterologous proteins (e.g. human insulin)
(Cousens et al., 1987; Kazemi et al., 2013a; Kazemi et al., 2013b;
Walsh, 2005). Considering that the production of 1 mol of hetero-
logous human insulin requires approximately 66 mol of NH
3
(based on the total amino acid sequence), the conservation of a
corresponding 33 mol of ATP (amount of ATP required to produce
66 mol of NH
3
from urea using ATP-dependent Dur1,2) would
result in a reduction of 2.06 mol of glucose consumed per mol of
human insulin produced (assuming aerobic conditions and a P/O-
ratio of 1.0; Bakker et al., 2001). An even more drastic impact on
product formation is expected for anaerobic production of
nitrogen-containing low-molecular-weight compounds such as
amino acids. For example, homofermentative production, by an
engineered S. cerevisiae strain, of alanine from glucose and
ammonium is expected to have a net ATP yield of zero, since the
ATP costs for ammonium uptake would exactly cancel out ATP
synthesis in glycolysis. Since ATP is needed for growth and
maintenance, this would preclude an anaerobic production pro-
cess. Conversely, ATP independent-urea assimilation would result
in a net ATP yield of 1 mol per mol alanine, which is equivalent to
the ATP yield from alcoholic fermentation and should therefore
enable a robust anaerobic process. Altering the energetics of
nitrogen assimilation represents a first step in engineering S.
cerevisiae as a metabolic engineering platform for energy-
efficient production of nitrogen containing commodity chemicals
such as diamines or amino acids.
Acknowledgments
This work was performed within the BE-Basic R&D Program
(http://www.be-basic.org/), which was granted an FES subsidy
from the Dutch Ministry of Economic Affairs, Agriculture and
Innovation (EL&I). The authors wish to thank Erik de Hulster,
Jeroen Koendjbiharie, Angela ten Pierick and Patricia van Dam for
their assistance on this project.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ymben.2015.05.003.
References
Albright, E.D., 2000. Encyclopedia of food microbiology, 3 vols. Library J. 125 (90-90).
Bacanamwo, M., Witte, C.P., Lubbers, M.W., Polacco, J.C., 2002. Activation of the
urease of Schizosaccharomyces pombe by the UreF accessory protein from
soybean. Mol. Genet. Genomics 268, 525–534.
Bakker, B.M., Overkamp, K.M., Maris, A.J., Kötter, P., Luttik, M.A., Dijken, J.P., Pronk, J.T.,
2001. Stoichiometry and compartmentation of NADH metabolism in Sacchar-
omyces cerevisiae.FEMSMicrobiol.Rev.25,15–37.
Blazeck, J., Garg, R., Reed, B., Alper, H.S., 2012. Controlling promoter strength and
regulation in Saccharomyces cerevisiae using synthetic hybrid promoters.
Biotechnol. Bioeng. 109, 2884–2895.
Boer, J.L., Mulrooney, S.B., Hausinger, R.P., 2014. Nickel-dependent metalloenzymes.
Arch. Biochem. Biophys. 544, 142–152.
Carter, E.L., Tronrud, D.E., Taber, S.R., Karplus, P.A., Hausinger, R.P., 2011. Iron-
containing urease in a pathogenic bacterium. Proc. Natl. Acad. Sci. USA 108,
13095–13099.
Cooper, T.G., Lam, C., Turoscy, V., 1980. Structural analysis of the dur loci in S.
cerevisiae: two domains of a single multifunctional gene. Genetics 94, 555–580.
Cooper, T.G., Sumrada, R., 1975. Urea transport in Saccharomyces cerevisiae.
J. Bacteriol. 121, 571–576.
Coulon, J., Husnik, J., Inglis, D., van der Merwe, G., Lonvaud, A., Erasmus, D.,
vanVuuren, H., 2006. Metabolic engineering of Saccharomyces cerevisiae to
minimize the production of ethyl carbamate in wine. Am. J. Enol. Vitic. 57 ,
113 –124.
Cousens, L.S., Shuster, J.R., Gallegos, C., Ku, L.L., Stempien, M.M., Urdea, M.S.,
Sanchez-Pescador, R., Taylor, A., Tekamp-Olson, P., 1987. High level expression
of proinsulin in the yeast, Saccharomyces cerevisiae. Gene 61, 265–275.
de Jonge, L.P., Buijs, N.A.A., ten Pierick, A., Deshmukh, A., Zhao, Z., JAKW, Kiel,
Heijnen, J.J., Gulik, W.M., 2011. Scale-down of penicillin production in Penicil-
lium chrysogenum. Biotechnol. J. 6,944–958.
de Kok, S., Kozak, B.U., Pronk, J.T., van Maris, A.J., 2012. Energy coupling in
Saccharomyces cerevisiae: selected opportunities for metabolic engineering.
FEMS Yeast Res. 12, 387–397.
Eitinger, T., Degen, O., Bohnke, U., Muller, M., 2000. Nic1p, a relative of bacterial
transition metal permeases in Schizosaccharomyces pombe, provides nickel ion
for urease biosynthesis. J. Biol. Chem. 275, 18029–18033.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎10
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i

Entian, K.D., Kötter, P., 2007. Yeast genetic strain and plasmid collections. Method
Microbiol. 36, 629–666.
Gietz, R.D., Woods, R.A., 2002. Transformation of yeast by lithium acetate/single-
stranded carrier DNA/polyethylene glycol method. Method Enzymol. 350,
87–96.
Grote, A., Hiller, K., Scheer, M., Munch, R., Nortemann, B., Hempel, D.C., Jahn, D.,
2005. JCat: a novel tool to adapt codon usage of a target gene to its potential
expression host. Nucleic Acids Res. 33, W526–W531.
Guldener, U., Heck, S., Fielder, T., Beinhauer, J., Hegemann, J.H., 1996. A new efficient
gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res.
24, 2519–2524.
Hensing, M.C.M., Bangma, K.A., Raamsdonk, L.M., Dehulster, E., Vandijken, J.P.,
Pronk, J.T., 1995. Effects of cultivation conditions on the production of hetero-
logous alpha-galactosidase by Kluyveromyces lactis. Appl. Microbiol. Biotechnol.
43, 58–64.
Hermann, T., 2003. Industrial production of amino acids by coryneform bacteria.
J. Biotechnol. 104, 155–172.
Higgins, K.A., Carr, C.E., Maroney, M.J., 2012. Specific metal recognition in nickel
trafficking. Biochemistry 51, 7816–7832.
Hong, K.K., Nielsen, J., 2012. Metabolic engineering of Saccharomyces cerevisiae:a
key cell factory platform for future biorefineries. Cell Mol. Life Sci. 69,
2671–2690.
Ikeda, M., 2003. Amino acid production processes. Adv. Biochem. Eng. Biotechnol.
79, 1–35.
Joho, M., Inouhe, M., Tohoyama, H., Murayama, T., 1995. Nickel resistance mechan-
isms in yeasts and other fungi. J. Ind. Microbiol. 14, 164–168 .
Kazemi, S.A., Cruz, A.L., de, H.E., Hebly, M., Palmqvist, E.A., van, G.W., Daran, J.M.,
Pronk, J., Olsson, L., 2013a. Long-term adaptation of Saccharomyces cerevisiae to
the burden of recombinant insulin production. Biotechnol. Bioeng. 110,
2749–2763.
Kazemi, S.A., Palmqvist, E.A., Schluckebier, G., Pettersson, I., Olsson, L., 2013b. The
challenge of improved secretory production of active pharmaceutical ingredi-
ents in Saccharomyces cerevisiae: a case study on human insulin analogs.
Biotechnol. Bioeng. 110, 2764–2774.
Klamt, S., Saez-Rodriguez, J., Gilles, E.D., 2007. Structural and functional analysis of
cellular networks with CellNetAnalyzer. BMC Syst. Biol. 1 (2), 2–8.
Knecht, S., Ricklin, D., Eberle, A.N., Ernst, B., 2009. Oligohis-tags: mechanisms of
binding to Ni
2þ
–NTA surfaces. J. Mol. Recognit. 22, 270–279.
Knijnenburg, T.A., Daran, J.M., van den Broek, M.A., Daran-Lapujade, P.A ., de Winde,
J.H., Pronk, J.T., Reinders, M.J., Wessels, L.F., 2009. Combinatoria
Q4
l effects of
environmental parameters on transcriptional regulation in Saccharomyces
cerevisiae: a quantitative analysis of a compendium of chemostat-based
transcriptome data. BMC Genomics 1053
Kozak, B.U., van Rossum, H.M., Benjamin, K.R., Wu, L., Daran, J.M., Pronk, J.T., van
Maris, A.J., 2014a. Replacement of the Saccharomyces cerevisiae acetyl-CoA
synthetases by alternative pathways for cytosolic acetyl-CoA synthesis. Metab.
Eng. 21, 46–59.
Kozak, B.U., van Rossum, H.M., Luttik, M.A., Akeroyd, M., Benjamin, K.R., Wu, L., de
VS, Daran JM, Pronk, J.T., van Maris, A.J., 2014b. Engineering acetyl coenzyme A
supply: functional expression of a bacterial pyruvate dehydrogenase complex
in the cytosol of Saccharomyces cerevisiae. mBio 5,8–14 .
Krivoruchko, A., Zhang, Y., Siewers, V., Chen, Y., Nielsen, J., 2015. Microbial acetyl-
CoA metabolism and metabolic engineering. Metab. Eng. 28, 28–42.
Kuijpers, N.G., Solis-Escalante, D., Bosman, L., van den Broek, M., Pronk, J.T., Daran, J.M.,
Daran-Lapujade,P.,2013.Aversatile,efficient strategy for assembly of multi-
fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic
recombination sequences. Microb. Cell Fact. 12 (47-12).
Kurtzman, C.P., Robnett, C.J., 2013. Relationships among genera of the Saccharomy-
cotina (Ascomycota) from multigene phylogenetic analysis of type species. FEMS
Yeast Res. 13, 23–33.
Lange, H.C., Heijnen, J.J., 2001. Statistical reconciliation of the elemental and
molecular biomass composition of Saccharomyces cerevisiae. Biotechnol. Bioeng.
75, 334–344.
Lee, M.H., Mulrooney, S.B., Renner, M.J., Markowicz, Y., Hausinger, R.P., 1992.
Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration
that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel
metallocenter biosynthesis. J. Bacteriol. 174, 4324–4330.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
Lucet, D., Le Gall, T., Mioskowski, C., 1998. The chemistry of vicinal diamines.
Angew. Chem. Int. Edit. 37, 2581–2627.
MacDiarmid, C.W., Gardner, R.C., 1998. Overexpression of the Saccharomyces
cerevisiae magnesium transport system confers resistance to aluminum ion.
J. Biol. Chem. 273, 1727–1732.
Marini, A.M., Soussi-Boudekou, S., Vissers, S., Andre, B., 1997. A family of ammo-
nium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4282–4293.
Mashego, M.R., van Gulik, W.M., Vinke, J.L., Heijnen, J.J., 2003. Critical evaluation of
sampling techniques for residual glucose determination in carbon-limited
chemostat culture of Saccharomyces cerevisiae. Biotechnol. Bioeng. 20, 395–399.
Maynard, E., Lindahl, J., 1999. Evidence of a molecular tunnel connecting the active
sites for CO2 reduction and acetyl-CoA synthesis in acetyl-CoA synthase from
Clostridium thermoaceticum. J. Am. Chem. Soc. 121, 9221–9222.
Mobley, H.L., Island, M.D., Hausinger, R.P., 1995. Molecular biology of microbial
ureases. Microbiol. Rev. 59, 451–480.
Mulrooney, S.B., Hausinger, R.P., 2003. Nickel uptake and utilization by micro-
organisms. FEMS Microbiol. Rev. 27, 239–261.
Mumberg, D., Muller, R., Funk, M., 1995. Yeast vectors for the controlled expression
of heterologous proteins in different genetic backgrounds. Gene 156, 119–122.
Navarathna, D.H., Harris, S.D., Roberts, D.D., Nickerson, K.W., 2010. Evolutionary
aspects of urea utilization by fungi. FEMS Yeast Res. 10, 209–213.
Nevoigt, E., Kohnke, J., Fischer, C.R., Alper, H., Stahl, U., Stephanopoulos, G., 2006.
Engineering of promoter replacement cassettes for fine-tuning of gene expres-
sion in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 72, 5266–5273.
Nijkamp, J.F., van den Broek, M.A., Datema, E., et al., 2012. De novo sequencing,
assembly and analysis of the genome of the laboratory strain Saccharomyces
cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology. Microb.
Cell Fact. 11, 36–42.
Nishimura, K., Igarashi, K., Kakinuma, Y., 1998. Proton gradient-driven nickel uptake
by vacuolar membrane vesicles of Saccharomyces cerevisiae. J. Bacteriol. 180,
1962–1964.
Ostergaard, S., Olsson, L., Nielsen, J., 2000. Metabolic engineering of Saccharomyces
cerevisiae. Microbiol. Mol. Biol. Rev. 64, 34–50.
Oud, B., van Maris, A.J., Daran, J.M., Pronk, J.T., 2012. Genome-wide analytical
approaches for reverse metabolic engineering of industrially relevant pheno-
types in yeast. FEMS Yeast Res. 12, 183–196.
Park, J.U., Song, J.Y., Kwon, Y.C., et al., 2005. Effect of the urease accessory genes on
activation of the Helicobacter pylori urease apoprotein. Mol. Cells 20,371–377.
Pronk, J.T., 2002. Auxotrophic yeast strains in fundamental and applied research.
Appl. Environ. Microbiol. 68, 2095–2100.
Qian, Z.G., Xia, X.X., Lee, S.Y., 2009. Metabolic engineering of Escherichia coli for the
production of putrescine: a four carbon diamine. Biotechnol. Bioeng. 104,
651–662.
Qian, Z.G., Xia, X.X., Lee, S.Y., 2011. Metabolic engineering of Escherichia coli for the
production of cadaverine: a five carbon diamine. Biotechnol. Bioeng. 108,
93–103 .
Sauer, M., Branduardi, P., Rußmayer, H., Marx, H., Porro, D., Mattanovich, D., 2014.
Production of Metabolites and Heterologous Proteins. In: Piskur, J., Compagno,
C. (Eds.), Springer-Verlag, Berlin Heidelberg, pp. 299–321.
Tai, S.L., Boer, V.M., Daran-Lapujade, P., Walsh, M.C., de Winde, J.H., Daran, J.M.,
Pronk, J.T., 2005. Two-dimensional transcriptome analysis in chemostat cul-
tures - Combinatorial effects of oxygen availability and macronutrient limita-
tion in Saccharomyces cerevisiae. J. Biol. Chem. 280, 437–447.
Verduyn, C., Postma, E., Scheffers, W.A., van Dijken, J.P., 1992. Effect of benzoic acid
on metabolic fluxes in yeasts: a continuous-culture study on the regulation of
respiration and alcoholic fermentation. Yeast 8, 501–517.
Verduyn, C., Postma, E., Scheffers, W.A., Vandijken, J.P., 1990. Physiology of
Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures.
J. Gen. Microbiol. 136, 395–403.
Walsh, G., 2005. Therapeutic insulins and their large-scale manufacture. Appl.
Microbiol. Biotechnol. 67, 151–159.
Weiss, S., Samson, F., Navarro, D., Casaregola, S., 2013. YeastIP: a database for
identification and phylogeny of Saccharomycotina yeasts. FEMS Yeast Res. 13,
117 –125.
Weusthuis, R.A., Lamot, I., van der Oost, J., Sanders, J.P.M., 2011. Microbial
production of bulk chemicals: development of anaerobic processes. Trends
Biotechnol. 29, 153–158.
Wu, G., 2009. Amino acids: metabolism, functions, and nutrition. Amino Acids 37,
1–17.
Xun Yao Chen, 2014. An investor's guide to nitrogen fertilizers: key 2014 drivers,
〈http://marketrealist.com/2014/03/investors-guide-to-nitrogen-fertilizers-key-
drivers-1q2014/〉.
Zhang, Y., Rodionov, D.A., Gelfand, M.S., Gladyshev, V.N., 2009. Comparative
genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC Genomics
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N. Milne et al. / Metabolic Engineering ∎(∎∎∎∎)∎∎∎–∎∎∎ 11
Please cite this article as: Milne, N., et al., Functional expression of a heterologous nickel-dependent, ATP-independent urease in
Saccharomyces cerevisiae. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.003i