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Applied and Environmental Microbiology, January 2000, p. 98-104, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression of Alcaligenes eutrophus
Flavohemoprotein and Engineered Vitreoscilla
Hemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth of
Escherichia coli
Alexander D.
Frey,
James E.
Bailey, and
Pauli T.
Kallio*
Institute of Biotechnology, ETH-Zürich,
CH-8093 Zürich, Switzerland
Received 19 July 1999/Accepted 20 October 1999
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ABSTRACT |
Expression of the vhb gene encoding hemoglobin from
Vitreoscilla sp. (VHb) in several organisms has been shown
to improve microaerobic cell growth and enhance oxygen-dependent
product formation. The amino-terminal hemoglobin domain of the
flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing
bacterium Alcaligenes eutrophus has 51% sequence homology
with VHb. However, like other flavohemoglobins and unlike VHb, FHP
possesses a second (carboxy-terminal) domain with NAD(P)H and flavin
adenine dinucleotide (FAD) reductase activities. To examine whether the
carboxy-terminal redox-active site of flavohemoproteins can be used to
improve the positive effects of VHb in microaerobic Escherichia
coli cells, we fused sequences encoding NAD(P)H, FAD, or
NAD(P)H-FAD reductase activities of A. eutrophus in frame
after the vhb gene. Similarly, the gene for FHP was
modified, and expression cassettes encoding amino-terminal hemoglobin
(FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed.
Biochemically active heme proteins were produced from all of these
constructions in Escherichia coli, as indicated by their
ability to scavenge carbon monoxide. The presence of FHP or of
VHb-FAD-NAD reductase increased the final cell density of transformed
wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were
achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion
proteins reduced the final cell densities approximately 20% relative
to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant
-lactamase relative to the VHb-expressing control.
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INTRODUCTION |
One of the foremost examples of
inverse metabolic engineering (the genetic transfer of useful
phenotypes to heterologous organisms) is expression of
Vitreoscilla hemoglobin (VHb) in aerobic bacteria, yeast,
fungi, and plants to enhance their growth and productivity (2, 6,
14, 25). Although the exact mechanism by which VHb causes these
effects is unknown, it has been hypothesized that due to its unusual
kinetic parameters for oxygen binding and release
(KD = 72 µM) (45), VHb is able
to scavenge oxygen molecules from solution and provide them for
cellular activities in heterologous organisms (18, 45, 49).
More detailed observations of biochemical and physiological changes
accompanying VHb expression in Escherichia coli indicate
increased overall ATP production and turnover rates, increase in
cytochrome o expression and specific activity, decreased
levels of reduced pyridine nucleotides, and changes in central carbon
metabolism (7, 18, 27, 38-40). Experiments were conducted
with engineered E. coli to determine if globins besides VHb
can be applied to enhance hypoxic growth and protein production levels.
E. coli cells expressing either of two different
hemoglobin-like proteins, horse heart myoglobin and yeast
flavohemoglobin, each having some sequence homology with VHb, grew to
lower final cell densities than did VHb-expressing E. coli
in oxygen-limited fed-batch cultivations (19).
Recently, a family of two-domain globins containing N-terminal oxygen
binding and C-terminal reductase activities, termed FNR (ferredoxin
NADP+ reductase)-like proteins has been identified. The
FNR-like proteins are not identical with the well-characterized global
transcriptional regulator FNR (fumarate nitrate reduction), which
controls the expression of genes required for anaerobic metabolism in
E. coli (42). FNR-like proteins have been
identified in both prokaryotic and eukaryotic organisms such as
E. coli (43), Erwinia chrysanthemi (12), Bacillus subtilis (23),
Candida norvegensis (15), Fusarium
oxysporum (37), and Saccharomyces cerevisiae
(51). One such protein, a megaplasmid-encoded cytoplasmic
hemoglobin-like protein (FHP [flavohemoglobin protein]), was also
identified in a facultatively lithoautotrophic, hydrogen-oxidizing,
gram-negative bacterium, Alcaligenes eutrophus (4, 30,
48). A. eutrophus grows, like Vitreoscilla,
in oxygen-scarce environments and has respiratory-type metabolism, but
A. eutrophus can also grow without oxygen if either nitrate
or nitrite is present as the terminal electron acceptor (4,
30). Limited oxygen supply causes an approximately 20-fold
increase in FHP content, suggesting that expression of FHP in A. eutrophus, like that of VHb in Vitreoscilla, is
regulated by oxygen (3, 31).
The N-terminal hemoglobin domain of FHP (termed FHPg in this report)
shows high sequence homology (51%) with VHb (8, 44). FHP is
a monomeric polypeptide of 403 amino acids (44.8 kDa) consisting of two
different protein modules: an N-terminal hemoglobin (FHPg; residues 1 to 147) domain and a C-terminal redox-active domain with binding sites
for flavin adenine dinucleotide (FAD) (residues 153 to 258) and for
NAD(P)H (residues 266 to 403). The reductase domain is able to reduce
several artificial electron acceptors as well as cytochrome
c. Furthermore, the b-type heme-iron complex of
the FHPg domain is capable of reversibly binding oxygen in its reduced
state (30, 31), confirming that FHP is a member of the
FNR-like protein family (1, 20). The different domains of
the FHP polypeptide are connected by short sequences of five to
seven amino acids, Pro148-Gly-Gly-Trp-Lys152 and
Phe259-His-Ile-Asp-Val-Asp-Ala265, between FHPg-FAD and FAD-NAD
domains, respectively (8). The three-dimensional structure
of the FHP has also been resolved by X-ray crystallography
(10).
The goal of this research was to study potential biotechnological
applications of the expression of FHPg, FHP, and fused parts thereof
(NAD-FAD domains) in E. coli under microaerobic culture conditions in a bioreactor. In addition, the vhb gene was
fused with sequences encoding NAD, FAD, and FAD-NAD activities of the C-terminal domain of FHP. Hypoxic growth of E. coli
constructs expressing these different heterologous and fusion proteins
was compared with growth of VHb-expressing E. coli, reaching
higher final cell densities relative to plasmid carrying VHb-negative E. coli (21), in a controlled hypoxic bioreactor.
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MATERIALS AND METHODS |
E. coli strains and plasmids.
E. coli
DH5
[F
endA1 hsdR17
(rk mk+) supE44
thi-1 
recA1 gryA96 relA1
(
80dlacZ
M15) (lacZYA-argF)U169; Gibco
BRL Life Technologies] was used as a host during the subcloning steps.
E. coli MG1655 ( F; Cold
Spring Harbor Laboratory) was used to analyze the effect of VHb or of
other globin and globin-reductase constructions on growth of cells in a
microaerobic bioreactor. pRED2 carrying the Vitreoscilla vhb
gene has been described elsewhere (21, 22). pUC19
(50) was used for subcloning, and pKQV4 (36) was
used as an isopropyl--D-thiogalactopyranoside
(IPTG)-inducible expression vector. pPPC1, a derivative of pKQV4
containing the vhb gene, has been described by Kallio et al.
(19). pGE276 (8), containing the fhp
gene of A. eutrophus, was a generous gift from B. Friedrich (Humboldt University of Berlin, Berlin, Germany).
DNA manipulations.
All restriction endonucleases were
purchased from commercial suppliers and used according to the
recommended protocols. DNA manipulations were performed according to
standard protocols (33).
PCR (32) was used to amplify the genes encoding VHb (GenBank
accession no. X13516) (22) and FHP (GenBank accession no.
X74334) (8) and open reading frames coding for FHPg, NAD,
and FAD protein domains of FHP or their combinations. PCR amplifications were performed with a Perkin-Elmer GeneAmp 9600 PCR
system and Pwo polymerase (Boehringer Mannheim).
Oligonucleotides for PCRs were synthesized by Microsynth (Balgach,
Switzerland) and are shown in Table 1.
Translational stop codons (CTA and TTA) were also inserted into the
gene structures encoding new fusion proteins if necessary (Table 1).
The oligonucleotides were designed in a way that the hinge sequences
between the various domains of fusion proteins (globin-reductase)
remained highly conserved and that amino acid sequences relative to the
original sequence of FHP were minimally altered (summarized in Fig.
1). The amino acid sequences of the FHPg
and VHb domains contain 147 and 146 residues, respectively. Thus,
Gly150 within the FHP protein sequence was changed to Thr149 in VHb-FAD
and VHb-FAD-NAD fusion proteins. This change generates a new
restriction site for KpnI. Linkage of the NAD domain
(LHIDVDA) with either the VHb or FHPg domain changed Leu (L) to Phe (F)
or Pro (P), respectively (Fig. 1B). PCR-amplified gene fragments were
purified by using a QIAquick PCR purification kit (Qiagen, Basel,
Switzerland). DNA fragments were separated by agarose gel
electrophoresis and recovered by using a QIAquick gel extraction kit.
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TABLE 1.
Oligonucleotides used for PCR amplifications of
vhb, fhp, and the open reading frames of
fhp encoding FHPg, NAD, and FAD subunits
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FIG. 1.
(A) Protein domains of native Vitreoscilla
VHb and A. eutrophus FHP. Alignment of VHb (residues 1 to
146) (22) and FHP (1 to 403) amino acid sequences reveals
three different modules in FHP: hemoglobin domain (FHPg; positions 1 to
147), FAD-binding domain (153 to 258) and NAD(P)H-binding domain (266 to 403) (8). The linker regions between the FHPg and FAD
(PGGWK) and the FAD and NAD (LHIDVDA) domains are shown. (B) The
protein modules of chimeric proteins and hinge sequences. Gly150 was
changed to Thr149 in pAX4 and pAX9, expressing VHb-FAD-NAD (residues 1 to 402) and VHb-FAD (1 to 257) proteins, respectively, and written in
bold italics. Because VHb is one residue shorter than FHPg (146 versus
147), renumbering of amino acid residues in VHb fusion proteins was
necessary. The LHIDVDA linker sequence was changed to FHIDVDA and
PHIDVDA in pAX12 (VHb-NAD) and pAX14 (FHPg-NAD), respectively.
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A PCR screening method was used to identify
E. coli DH5
transformants carrying the correct gene inserts either in pUC19 or
in
pKQV4. The reaction mixture for one sample contained 0.4 µl
of
dimethyl sulfoxide, 1.0 µl of 10×
Taq buffer, 0.1 µl of
Taq polymerase (5 U/µl; Boehringer Mannheim), 1 µl of
each primer
(final concentration of each primer, 100 pmol/µl),
deoxynucleoside
triphosphates added to a final concentration of 10 mM,
and H
2O
added to give a total volume of 10 µl in a well
of a 96-well PCR
plate (Axon Lab, Baden-Dättwil, Switzerland). A
single ampicillin-resistant
colony was picked aseptically from
overnight-incubated Luria-Bertani
(LB) agar plates (
33)
supplemented with ampicillin (100 µg/ml)
and transferred to a well of
the PCR plate. Standard PCR techniques
were used for DNA amplification
(
32). Reaction mixtures were
analyzed for amplified gene
fragments of the expected molecular
size by agarose gel electrophoresis
(
33), and the correct recombinant
plasmid was used for
further
characterization.
Plasmid DNAs for DNA sequencing were isolated and purified from
overnight-grown bacteria cultures by using a QIAprep Spin
MiniPrep kit
(Qiagen). A Thermo Sequenase fluorescence-labeled
primer cycle
sequencing kit (Amersham International) was used
for DNA sequencing
reactions (
34), and cycle sequencing was
performed as
recommended by the manufacturer. The infrared (IRD-41)-labeled
sequencing primer pair pKK
for (5' CTC AAG GCG CAC TCC CGT
TCT),
plus pKK
rev (5' GAG TTC GGC ATG GGG TCA GGT G) and
universal primer
pair -40 forward plus -40 reverse, for DNA sequencing
of pKQV4
and pUC19 derivatives, respectively, were obtained from
MWG-Biotech
(Ebersberg, Germany). The sequencing reactions were
separated
in a LI-COR 4000 L automated DNA sequencer (LI-COR, Inc.,
Lincoln,
Neb.). IRD-41-labeled DNA fragments were visualized by using a
scanning laser microscope assembly. DNA sequence data were collected
and analyzed by using the LI-COR Base ImagIR
software.
Construction of VHb-reductase, FHP, and FHPg-reductase expression
vectors.
Plasmids pRED2 and pGE276 were used as templates for
vhb and fhp gene amplifications, respectively.
The oligonucleotides were designed so that only one amino acid was
changed within the proposed linker region between the protein domains
(Fig. 1). The gene fragments encoding FHP and the FHPg and FHPg-FAD
domains of FHP were PCR amplified by using oligonucleotides 1 and 3, 1 and 2, and 1 and 10, respectively (Table 1). Purified DNA fragments
were digested with EcoRI and PstI and subcloned
directly into pKQV4 digested with the same enzymes. Correct plasmids
were identified by the PCR screening method, and authenticity of
reading frames of the expression cassettes in pAX1 (FHPg), pAX5 (FHP),
and pAX6 (FHPg-FAD) was verified by DNA sequencing.
Construction of plasmids pAX4 (VHb-FAD-NAD), pAX9 (VHb-FAD), pAX12
(VHb-NAD), and pAX14 (FHPg-NAD) is summarized below (Fig.
1B). In all
cases, two subcloning steps using pUC19 were necessary
for construction
of the expression cassettes. The gene fragment
encoding the FHPg
subunit of FHP was PCR amplified with oligonucleotides
1 and 9 (Table
1) and a purified 6.5-kb
SalI-
XhoI fragment of
pGE276 as a template. The PCR fragments were isolated and digested
with
EcoRI and
HincII. The fragments were subcloned
into pUC19
digested with the same enzymes, and the new plasmid was
named
pAX13. The gene fragment encoding the NAD domain was amplified
with oligonucleotides 3 and 7 (Table
1) and the same template
as
mentioned above. The purified PCR fragments were
HincII-
PstI
digested and subcloned into pAX13 to
generate pAX2. The expression
cassette synthesizing FHPg-NAD was
removed with
EcoRI-
PstI digestion
and subcloned
into
EcoRI-
PstI-digested pKQV4 to generate pAX14.
An identical cloning strategy was used to construct the expression
cassette synthesizing VHb-NAD, using oligonucleotide pairs 4-8
and 3-7 (Table
1) for
vhb and the open reading frame encoding
the
NAD domain of FHP, respectively. The expression vector producing
VHb-NAD was named
pAX12.
Expression cassettes for VHb-FAD and VHb-FAD-NAD production (Fig.
1B)
were constructed by using a slight modification of the
protocol
outlined above. The
vhb gene was amplified with
oligonucleotides
4 and 5 (Table
1). The
vhb PCR fragment was
double digested with
EcoRI and
KpnI and inserted
into pUC19 digested with the same
enzymes, yielding plasmid pAX7. The
open reading frame of the
fhp gene encoding the FAD module
was amplified with oligonucleotides
6 and 10 (Table
1), double digested
with
KpnI and
PstI, and ligated
with pAX7,
resulting in pAX16. The complete cassette for VHb-FAD
expression was
excised from pAX16 by using
EcoRI and
PstI and
ligated with
EcoRI-
PstI digested pKQV4 to form
pAX9. An identical
cloning strategy was applied for the VHb-FAD-NAD
expression cassette.
The
vhb and
fhp gene
fragments encoding FAD-NAD were amplified
with oligonucleotide pairs
4-5 and 3-6, respectively (Table
1).
The final expression plasmid
producing VHb-FAD-NAD was named pAX4.
The amplified expression
cassettes in vectors pAX4 (VHb-FAD-NAD),
pAX9 (VHb-FAD), pAX12
(VHb-NAD), and pAX14 (FHPg-NAD) were subjected
to DNA sequencing, and
the sequences of the reading frames were
verified.
Bioreactor cultivations.
Cultures for both plasmid DNA
isolations and bioreactor inoculations were grown on a shaker at 37°C
and 250 rpm for approximately 14 h in 50-ml shake flasks
containing 10 ml of LB medium (33) supplemented with
ampicillin (100 µg/ml).
Fed-batch cultivations of
E. coli MG1655 carrying various
VHb, VHb-reductase, FHPg, or FHPg-reductase expression vectors were
performed in a defined glucose batch medium supplemented, per
liter,
with 150 mg of Casamino Acids (Difco), 30 mg of yeast extract
(Difco),
and 100 mg of ampicillin (
19), using a Sixfors bioreactor
unit (Infors, Bottmingen, Switzerland) allowing six controlled
cultivations at the same time. To start Sixfors bioreactor fed-batch
cultivations, 3.0 ml of seeding culture was used to inoculate
300 ml of
glucose batch medium, and process parameters were maintained
at 37°C,
pH 7 ± 0.2 (adjusted either with 2 M NaOH or with 2 M
H
3PO
4), 300 rpm, and 120 ml of air per min. The
composition of
feed medium has been described previously
(
19). Expression of
hemoglobins and hemoglobin-reductase
constructions were induced
by IPTG addition (final concentration of 100 µM) when the optical
densities (
A600) of
cultures were approximately 1. Fed-batch mode
was commenced with 1 ml
of feed medium per h when the culture
reached an
A600 of approximately 2, and the feeding rate
was increased
to 2 ml/h when the
A600 was
approximately 4. Thereafter, the feeding
rate was maintained constant
at 2 ml/h until the end of 30 h of
microaerobic fed-batch
cultivations.
Dissolved oxygen concentration and exhaust gases (CO
2 and
O
2) from bioreactors were monitored as described previously
(
17,
19).
Analytical techniques.
The soluble fractions of both
non-globin-producing and globin-expressing E. coli MG1655
cells (for VHb, FHPg, FHPg-FAD-NAD, FHPg-FAD, FHPg-NAD, VHb-FAD-NAD,
VHb-FAD, and VHb-NAD constructions) were prepared by harvesting 100 ml
of bioreactor-cultivated cells followed by centrifugation in a Beckman
J2-21M centrifuge with a JLA 10.500 rotor at 4°C for 10 min and
7,000 × g. Supernatants were discarded, and cell
pellets were resuspended in 10 ml of lysis buffer (100 mM Tris-HCl [pH
7.5], 50 mM NaCl, 1 mM EDTA). Cells were disrupted in a French press
(SLM-Aminco) at 1,200 lb/in2. Cell debris was removed by
centrifugation in a Beckman GS-6R at 4°C for 15 min and
3,200 × g. The supernatants were poured in new tubes,
and clear soluble fractions were recovered by centrifugation for 15 min
at 14,000 rpm at 4°C in an Eppendorf centrifuge. Globin activities
were assayed by CO difference spectroscopy (reduced + CO minus reduced
spectrum) as described previously (13).
Acetate concentrations of bioreactor samples were determined with a
Beckman SYNCHRON CX5CE autoanalyzer. A Boehringer Mannheim
acetate kit
(product no. 148261) was adapted for the Beckman autoanalyzer
system
and used to measure acetate concentrations from the bioreactor
samples.
Ethanol concentrations were measured enzymatically with
a Beckman
autoanalyzer and a Beckman alcohol kit. The measured
concentrations
were normalized to the final
A600 values of the
cultures.
-Lactamase activities expressed by plasmids pKQV4 (control), pAX1
(FHPg), pAX4 (VHb-FAD-NAD), pAX5 (FHP), pAX6 (FHPg-FAD),
pAX9
(VHb-FAD), pAX12 (VHb-NAD), pAX14 (FHPg-NAD), and pPPC1 (VHb)
were
determined with the chromogenic cephalosporin nitrocefin
(Becton
Dickinson) as a substrate (
28). The samples were withdrawn
at the end of hypoxic bioreactor cultivations, cells were disrupted
in
a French press, and the change of absorbance at 482 nm was
recorded at
room temperature (
27). The total soluble protein
of each
sample was determined with a Bio-Rad protein assay kit,
based on the
method of Bradford (
5). Activity of
-lactamase
is
reported in units per milligram of soluble
protein.
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RESULTS |
Construction of hemoglobin (VHb and FHPg) and hemoglobin-reductase
expression vectors.
Most of the known bacterial hemoglobin
proteins, such as FHP and HMP, show two different activities, an
oxygen-binding activity and a reductase activity (8, 43).
These two different biochemical properties are linked in a single
two-domain protein containing N-terminal hemoglobin and C-terminal
reductase modules (Fig. 1A). VHb and the recently identified new globin
of Vitreoscilla stercoraria do not have reductase activity
in the same polypeptide (16, 22, 44). However, it is well
documented that VHb requires reductase activity for physiological
function, and such an unlinked NADH-methemoglobin reductase has been
identified in Vitreoscilla. E. coli also contains an
unidentified reductase system capable of catalyzing the reduction of
VHb in vivo (9, 46). However, maintenance of the redox state
of heme iron of VHb, catalyzed by a heterologous reductase, may be an
activity-limiting factor in a heterologous host and decrease the
beneficial effects of VHb expression such as relieving oxygen stress
under hypoxic conditions. To determine whether fusion of a heterologous
reductase domain to VHb provides improved benefits, the following three
expression vectors were constructed: pAX4 (VHb-FAD-NAD), pAX9
(VHb-FAD), and pAX12 (VHb-NAD). The N-terminal domain of FHP is highly
homologous with VHb (8). Thus, it may also be possible that
FHP is able to expedite growth of E. coli cells under
hypoxic conditions. To study this hypothesis, we also constructed four
new expression vectors, pAX1 (FHPg), pAX5 (FHP), pAX6 (FHPg-FAD), and
pAX14 (FHPg-NAD), as described in Materials and Methods. The VHb
expression plasmid pPPC1 has been described previously (19).
Structures of the novel expression modules with hinge sequences,
relative to the wild-type structures, are summarized in Fig. 1B.
Microaerobic bioreactor cultivations.
VHb-expressing wild-type
E. coli MG1655 cells showed improved growth relative to the
common VHb-positive cloning host E. coli DH5 under
oxygen-limited culture conditions (19). Thus, E. coli MG1655 cells expressing VHb (pPPC1), FHPg (pAX1), or separate globin-reductase fusions (pAX5, pAX6, pAX4, pAX9, pAX12, and pAX14) were cultivated at least twice in a microaerobic Sixfors bioreactor. The dissolved oxygen concentration readings by polarographic electrodes were zero after approximately 6 h of various cultivations and remained there until the end of fed-batch processes. Differences in
growth behavior between various constructions became apparent after
approximately 8 to 9 h of cultivation, where
A600 was approximately 3, and the growth
of non-VHb-expressing control MG1655:pKQV4 ceased rapidly.
Obviously, the supply of oxygen was not sufficient to support effective
growth of nonglobin-expressing E. coli cells beyond this
point (Fig. 2).
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FIG. 2.
OD600 (optical density at 600 nm)
trajectories of E. coli MG1655 expressing proteins showing
FHPg domain activity: pAX1 (FHPg;  ), pAX5 (FHP;  ), pAX6
(FHPg-FAD;  ), and pAX14 (FHPg-NAD;  ). The control plasmid was
pKQV4 ( ). The cells were cultivated under hypoxic conditions in a
Sixfors bioreactor as described in Materials and Methods.
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Growth curves of MG1655:pAX1, MG1655:pAX5, MG1655:pAX6, and
MG1655:pAX14 expressing FHPg or FHP-reductase derivatives of
A. eutrophus are shown in Fig.
2. The growth curves of VHb-expressing
(pPPC1) or VHb-reductase-expressing (pAX4, pAX9, and pAX12)
E. coli MG1655 cells are shown in Fig.
3. Non-globin-expressing MG1655:pKQV4
was
used as an internal control between various cultivations,
and the final
A600 of the control was 5.5 ± 0.5 at the
end of
30 h of cultivation.
E. coli MG1655:pAX1
(FHPg-expressing) cells
reached a final
A600 of
8.3 ± 0.9, which is similar to the final
A600 of 7.9 ± 0.5 for
Vitreoscilla VHb-expressing MG1655:pPPC1
cells (Fig.
2 and
3).
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FIG. 3.
Time course of OD600 (optical densities at
600 nm) of E. coli MG1655 with the various VHb constructs:
pPPC1 (VHb; ), pAX4 (VHb-FAD-NAD; ), pAX9 (VHb-FAD; ), and
pAX14 (VHb-NAD; ). , MG1655 cells carrying the control plasmid,
pKQV4.
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Coexpression of the NAD part of the reductase together with the VHb
(MG1655:pAX12) or FHPg (MG1655:pAX14) domain reduced the
final
A600 values of
E. coli approximately
20% relative to either
MG1655:pPPC1 (VHb) or MG1655:pAX1 (FHPg), but
these constructions
eventually attained a final optical density
slightly higher than
that of the cultures carrying the control plasmid
pKQV4. Final
A600 values for MG1655:pAX12 and
MG1655:pAX14 were 6.4 ± 1.0 and
6.4 ± 0.9, respectively.
MG1655:pAX6 (FHPg-FAD) and MG1655:pAX9
(VHb-FAD) showed slightly
increased final optical densities, 9.0
± 1.0 and 9.9 ± 0.9, respectively, relative to VHb- or FHPg-expressing
cells (Fig.
2 and
3).
E. coli MG1655:pAX5 expressing full-length
A. eutrophus FHP flavohemoprotein (
A600 of
12.0 ± 1.5) reached
approximately 2.2-fold and 50% higher final
cell densities relative
to the control MG1655:pKQV4 (5.5 ± 0.5)
and VHb-expressing MG1655:pPPC1
(7.9 ± 0.5), respectively.
MG1655:pAX4 (VHb-FAD-NAD) cells reached
the highest final optical
densities,
A600 of 13.9 ± 1.4, at the
end
of 30 h of hypoxic fed-batch cultivations (Fig.
3). This value
was
on average approximately 75 and 15% higher than those for
the original
VHb-expressing clone MG1655:pPPC1 (7.9 ± 0.5) and
the
FHP-expressing construct MG1655:pAX5 (12.0 ± 1.5), respectively
(Fig.
2 and
3). These results clearly show that the beneficial
effect
of VHb can be improved substantially by using protein engineering
to
combine directly its advantageous oxygen-binding properties
with a
fused reductase
activity.
All expression vectors were also analyzed for insert maintenance by a
PCR screening method. The results obtained with different
oligonucleotide primer combinations showed that the expression
cassettes were stably maintained during prolonged hypoxic bioreactor
cultivations (data not
shown).
By-product accumulation during hypoxic bioreactor
cultivations.
The excretion of by-products such as acetate and
ethanol allows E. coli cells to discard a surplus of
reduction equivalents, regenerating NAD+ and
NADP+, which are needed to metabolize glucose. Final
concentrations of acetate and ethanol from samples withdrawn from
bioreactors at the end of 30 h cultivation were assayed in a
Beckman autoanalyzer, and values were normalized to 1 unit of
A600. Our results show reduced excretion of
acetate from strains expressing various VHb, FHPg, and
hemoglobin-reductase constructions relative to the control MG1655:pKQV4. For MG1655:pAX4 (VHb-FAD-NAD) and MG1655:pAX5 (FHP), the
smallest specific acetate levels were measured: 4.9 ± 0.3 mM/A600 and 4.6 ± 0.1 mM/A600, respectively. These values were approximately 40% lower than specific acetate accumulation of the
vector control MG1655:pKQV4 cultivation (8.2 ± 0.7 mM/A600). Expression of the hemoglobin domain
alone, either in MG1655:pAX1 (FHPg; 6.4 ± 0.3 mM/A600) or in MG1655:pPPC1 (VHb; 5.9 ± 0.7 mM/A600), resulted in smaller decreases
(approximately 22 and 28%, respectively) in the production of acetate
relative to the control (Table 2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Final acetate concentrations of E. coli MG1655
expressing various hemoglobin constructions cultivated in a
hypoxic bioreactora
|
|
The excretion of ethanol from the samples was also measured at the end
of cultivations. The results indicate that FHPg-expressing
cells
produced 40% less ethanol (1.4 ± 0.4 mM/
A600) relative to
MG1655:pKQV4 (2.3 ± 1.5 mM/
A600). Surprisingly, VHb-expressing
MG1655:pPPC1 cells produced 2.9 times more ethanol (4.0 ± 0.1
mM/
A600) relative to FHPg-producing strain
MG1655:pAX1 under similar
culture conditions. In addition, accumulation
of ethanol was also
reduced in MG1655:pAX6 and MG1655:pAX4,
approximately 25 and 38%,
respectively, relative to the control
MG1655:pKQV4. These results
show that ethanol accumulation is not
always decreased in hemoglobin-expressing
cells relative to the
hemoglobin-negative
control.
Analysis of biological activities of VHb-, FHPg-, and
globin-reductase hemoproteins by CO-binding assay.
Cells were
harvested at the end of hypoxic fed-batch cultivations, and samples for
CO-binding assays were prepared as described in Materials and Methods.
The biochemical activity of the globins in all constructions can be
determined by using a standard technique (13). The maximum
and minimum values of the recorded spectra of the different
constructions are given in Table 3. Due
to lack of a reported extinction coefficient either for FHP or for FHPg and to the novel nature of the heme-reductase fusion proteins, it is
impossible to predict the effects of the reductase tails on the
extinction coefficients. Thus, it is not possible to compare the
specific activities of the different heme domains. Our results are only
qualitative, giving no information about the specific activity of
globins per milligram of soluble protein. However, the results clearly
show that biologically active hemoproteins were produced in all
E. coli strains expressing various recombinant globins
(Table 3). The CO-difference spectrum of MG1655:pKQV4 revealed no
hemoglobin activity, as shown previously (19), indicating that the recorded curves represent the activity of overexpressed heterologous hemoproteins and are not artifacts of E. coli
HMP expression (data not shown).
The hemoglobin domain of
A. eutrophus flavohemoprotein
(FHPg) has maximal absorption at a slightly longer wavelength (422
nm)
relative to the
band of VHb (419 nm) (Table
3). A similar
maximal
absorption value is observed in strains expressing FHPg
protein fused
with either the FAD-NAD or FAD domain. A slight
shift of the absorption
maximum (419 nm) for the FHPg-NAD-expressing
strain was recorded. In
addition, the absorption maximum has changed
in VHb-FAD-NAD-expressing
(422 nm) and VHb-NAD-expressing (416
nm) strains relative to the
VHb-producing strain (419 nm) (Table
3). This variation may result from
a slightly varying three-dimensional
structure of the hemoglobin
domains of the fusion proteins. These
results also verify that FHP
contains a CO-binding
b-type heme
cofactor such as VHb and
also HMP of
E. coli (
30,
41,
43).
Analysis of -lactamase activity at the end of hypoxic
cultivations.
Production of a model recombinant protein,
-lactamase, was also assayed for samples withdrawn from a
microaerobic bioreactor (Table 4). The
results showed that MG1655:pAX1 (FHPg; 109 ± 3 U/mg), MG1655:pAX5
(FHP; 113 ± 10 U/mg), MG1655:pAX9 (VHb-FAD; 106 ± 5 U/mg),
and MG1655:pPPC1 (VHb; 119 ± 13 U/mg) produced 24, 28, 20, and
35% more -lactamase, respectively, relative to MG1655:pKQV4
(88 ± 3 U/mg). The production of -lactamase was 3.1- or
2.3-fold higher in the fastest-growing strain, MG1655:pAX4 (VHb-FAD-NAD; 271 ± 3 U/mg), relative to MG1655:pKQV4 (control) or MG1655:pPPC1 (VHb; 119 ± 13 U/mg), respectively, at the end of
30 h of hypoxic fed-batch cultivations. The production of
-lactamase was reduced in MG1655:pAX6 (FHPg-FAD; 40 ± 1 U/mg),
MG1655:pAX12 (VHb-NAD; 62 ± 15 U/mg), and MG1655:pAX14 (FHPg-NAD;
36 ± 3 U/mg) relative to the control, MG1655:pKQV4 (Table 4).
This observation was surprising because recombinant E. coli
MG1655 strains expressing various hemoprotein gene constructions were
always able to reach higher final optical densities relative to the
non-VHb-expressing control.
-Lactamase results reported here do not take into account the
various problems and limitations encountered with the optimization
of
heterologous protein production in
E. coli (
47).
However,
each type of experiment conducted here included the same
genetic
background (
E. coli MG1655), and plasmid
constructions were derived
from the same parental plasmids with the
pBR322 origin of replication.
Thus, the above results suggest that
VHb-reductase-expressing
cells are able to redirect cellular resources
more efficiently
toward recombinant protein production relative to
FHP-expressing
cells.
| |
DISCUSSION |
Our results show that the beneficial effect of VHb expression on
microaerobic bacterial growth can be improved substantially by
expressing instead a fusion protein (VHb-FAD-NAD) containing vhb and the reductase gene module of fhp. The
positive effect of fused reductase expression was also observed when
FHP was expressed in microaerobic E. coli. The expression of
these two proteins resulted in 2.2-fold (FHP) and 2.5-fold
(VHb-FAD-NAD) increases in final cell densities relative to the
VHb-expressing strain. VHb expression has been shown to modulate
cellular metabolism of E. coli (7, 18, 27,
38-40). VHb influences the energy level by interacting with the
respiratory chain, thus leading to increased proton pumping, higher ATP
production rates, and a less reduced contingent of pyridine nucleotide
cofactors (7, 18, 38, 39). Recently, it has been shown that
HMP expression elicits different physiological effects relative to VHb
in E. coli. HMP is able to protect E. coli cells
against nitrosating agents, NO-related species, and oxidative stress
(24). The detailed physiological functions of FHP of
A. eutrophus in E. coli and other heterologous
hosts remain to be investigated. No significant changes in either
aerobic or anaerobic growth were seen in A. eutrophus
strains lacking the entire flavohemoprotein gene in the genome
(8). However, A. eutrophus fhp mutants did not
accumulate nitrous oxide in significant amounts as observed in
wild-type cells. This finding suggest that FHP may interact in an
unidentified way with gas metabolism under denitrification conditions
in A. eutrophus (8).
The reductase domain (FAD-NAD) of A. eutrophus FHP is part
of the FNR-like protein family and is widespread among prokaryotic and
eukaryotic organisms (1). A well-studied example is HMP of
E. coli. HMP has an N-terminal hemoglobin domain and a
C-terminal FNR-like module (43). This module consists of two
functionally inseparable and evolutionarily conserved domains, the FAD-
and NADP+-binding domains. The FNR-like module of HMP
reduces the heme iron of the hemoglobin domain by transferring
electrons from NAD(P)H to the heme moiety via the protein-associated
FAD group (1). Poole et al. (29) have shown that
HMP is a reductase of broad substrate specificity, and the prosthetic
heme group of the hemoglobin domain is not necessarily involved in
electron transfer in E. coli. Thus, the FAD-binding domain
is able to donate electrons directly to other acceptors such as
cytochrome c (29). There are at least two
different ways for electrons to be channeled from NAD(P)H: either via
FAD to the oxidized heme iron or via FAD to different putative electron
acceptors. It may be possible that FHP functions in a similar way in
A. eutrophus and even in heterologous prokaryotes.
Normally, the binding of O2 to the heme iron is a
reversible reaction, but the binding of oxygen can lead to a redox
reaction in which the ferroheme [Fe(II)] group of the hemoglobin is
oxidized to ferriheme [Fe(III)] (26). The oxidized iron
[Fe(III)] is incapable of binding oxygen, therefore limiting the
biochemical and physiological effects of the hemoglobins. HMP has been
shown to possess flavin reductase activity capable of reducing iron complexes such as ferric citrate with a negligible rate relative to
main ferric/flavin reductase activities in E. coli (11,
29). Previously, VHb and an associated reductase protein, called
NADH-cytochrome o reductase, have been shown to constitute
an electron-transferring path for the oxidation of NADH and to increase
the oxygen uptake several fold in Vitreoscilla
(46). Recently, it was also shown that the coexpression of
NADPH cytochrome P450, an alkane-inducible monooxygenase, with an NADPH
cytochrome P450 oxidoreductase of Candida tropicalis led to
an elevated level of P450-catalyzed monooxygenase activity in S. cerevisiae (35). This experiment shows that the
metabolic activity of a heterologous hemeprotein, such as VHb, may be
limited by reductase activity in a novel host and suggests that the
coexpression of an additional reductase may increase electron flux from
NAD(P)H to the heme of the hemoglobin-like domain. The electron flow
may concomitantly increase the regeneration rate of the ferriheme to
ferroheme of the prosthetic heme of oxygen binding hemoglobin in
E. coli.
All constructions showed VHb and FHPg activity, as judged by the
CO-binding assay. Our results indirectly suggest that the recombinant
reductase system was also expressed in an active form because the
fusion constructions expressing either VHb-FAD, FHPg-FAD, VHb-reductase, or FHP were able to increase cell growth relative to
either VHb- or FHPg-expressing E. coli (Fig. 2 and 3). This assumption is supported by results showing that the native enzyme of
E. coli HMP transfers two H+ ions from NADH to
FAD and consecutively passes the electrons in two steps, via the heme
group to a putative substrate (29). Therefore, it is likely
that either reduction equivalents could not be passed from NADH to the
heme due to lack of the electron transfer-mediating FAD moiety or the
novel three-dimensional structure of fusion proteins is sterically able
to hinder the transfer of reduction equivalents from endogenous
reductase domain to VHb-NAD and FHPg-NAD constructions. As a
consequence, hemoglobin has reduced affinity toward oxygen after
oxidation of the prosthetic heme group of the heme moiety. Thus, the
beneficial effect of heterologous hemoglobin expression is lost in
hypoxic E. coli.
| |
ACKNOWLEDGMENTS |
This research was supported by the ETH.
We thank Heidi Ernst for skillful DNA sequencing of the plasmid constructions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biotechnology, ETH-Zürich, CH-8093 Zürich, Switzerland.
Phone: 41 1 633 3446. Fax: 41 1 633 1051. E-mail:
kallio{at}biotech.biol.ethz.ch.
| |
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Abstract
Introduction
