Journal of Bioscience and Bioengineering
VOL. 113 No. 3, 300 – 306, 2012
www.elsevier.com/locate/jbiosc
Mutations to the active site of 3-ketoacyl-ACP synthase III (FabH) increase
polyhydroxyalkanoate biosynthesis in transgenic Escherichia coli
Alexander P. Mueller and Christopher T. Nomura⁎
State University of New York, College of Environmental Science and Forestry, Department of Chemistry, 1 Forestry Dr., Syracuse, NY 13210, USA
Received 29 July 2011; accepted 26 October 2011
Available online 3 December 2011
Polyhydroxyalkanoate (PHA) production has been enhanced with engineered 3-ketoacyl-ACP synthase III (FabH) enzymes
that accept diverse fatty acyl-ACP substrates and convert them to fatty acyl-CoA substrates for polymerization by PHA
synthase enzymes resulting in the production of diverse polymers. Two mutations in the monomer supplying enzyme FabH,
His244Ala and the Asn274Ala, were investigated to assess the impact of these mutations on PHA monomer production. PHA
production increased more than six-fold with the mutation His244Ala in the FabH enzyme. Engineering of the FabH enzyme
for improved PHA monomer supply led to a more productive system for PHA copolymer production.
© 2011, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Recombinant Escherichia coli; Protein engineering; polyhydroxyalkanoates; Bioplastic; 3-Ketoacyl-ACP synthase III; fabH]
Polyhydroxyalkanoates (PHAs) are a class of polyesters that can be
biologically produced and are completely biodegradable, making them
an environmentally friendly alternative to their petroleum based
counterparts (1–3). These materials have many applications ranging
from use in bulk-commodity plastics to medical applications (1,2,4). The
production and use of PHAs are dwarfed by petroleum based plastics
due to their limited physical properties and higher cost of production.
The physical properties of PHAs are largely dependent upon
monomer composition. PHAs composed of short-chain-length (SCL)
monomers, with repeating units 3 to 5 carbons in length, form highly
crystalline thermoplastics which are quite brittle. PHAs composed of
medium-chain-length (MCL) monomers, with repeating units 6 to 14
carbons in length, form semi-crystalline elastomers which range from
tacky to free-flowing (5). PHA copolymers composed of SCL and MCL
monomers (SCL–MCL) can possess a wide range of physical properties
dependent on the monomer composition (5). Because there is currently
not a significant source of these copolymers, few opportunities exist
to test them and fully explore their potential applications. Due to
the variety of applications that enhanced physical properties of the
SCL–MCL PHAs could allow for, developing a cost-effective and efficient
biological strategy to produce them would be beneficial in extending the
use of PHAs to replace petroleum based plastics.
While pathways for the production of SCL PHAs have been well
defined (3,6–8) MCL PHA production is not as well understood.
Transgenic production of MCL PHA has been accomplished in
Escherichia coli, but most of these pathways require fatty acids or oils
as carbon sources (9–13). Such pathways rely on β-oxidation, and are
not effective at producing PHAs from carbon sources that are initially
⁎ Corresponding author. Tel.: +1 315 470 6854; fax: +1 315 470 6856.
E-mail address: ctnomura@esf.edu (C.T. Nomura).
unrelated to fatty acids, like sugars or CO2. As such, an alternative model
system may lead to lower production cost where SCL–MCL PHAs are
produced from simple carbon sources unrelated to fatty acids, by way of
fatty acid biosynthesis metabolism. The ability to produce SCL–MCL
PHAs from unrelated carbon sources is especially desirable in plants, in
order to convert CO2 directly into polymers photosynthetically in the
chloroplast. There are few studies that have shown potential for
transgenic production of MCL PHAs from unrelated carbon sources
(14–17), and even fewer have produced SCL–MCL copolymers (18–21).
Thus, the potential for development in this area is largely unexplored.
One pathway that has successfully generated SCL–MCL PHAs from
sugars in recombinant E. coli utilizes an engineered 3-ketoacyl-ACP
synthase III (FabH) as a MCL-monomer-supplying enzyme (Fig. 1).
The pathway makes use of intermediates from fatty acid biosynthesis,
which are present in all organisms, giving the pathway potential for
introduction to a wide range of organisms, including plants. FabH is a
member of the β-ketoacyl synthase family of enzymes. Its native
function is the condensation of malonyl-ACP and acyl-CoA units in
fatty acid biosynthesis, but it has been demonstrated to have a
transacylase activity capable of converting β-ketoacyl-ACP units,
intermediates from fatty acid biosynthesis, to β-ketoacyl-CoA units
which can be converted into monomer supplying units recognizable
by PHA synthase enzymes (19,22–24). Overexpression of fabH genes
encoding proteins with mutations to amino acid 87 of the binding
pocket led to low level MCL PHA monomer supply in transgenic E. coli
(19). Mutations to the active site, amino acids histidine 244 (H244)
and asparagine 274 (N274), have been reported to increase the
transacylase activity of the enzyme (22). This transacylase activity is
the critical activity in the monomer supplying function of the enzyme,
but the effect of these mutations on PHA monomer supplying activity
has not been tested.
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2011.10.022
VOL. 113, 2012
ENGINEERED FabH FOR PHA PRODUCTION IN E. COLI
301
FIG. 1. Metabolic pathway for PHA production from glucose in recombinant E. coli adapted from Nomura et al. (19). 3-ketoacyl-ACP intermediates from fatty acid biosynthesis are
converted to 3-ketoacyl-CoA by the engineered FabH (shown in bold) to produce medium-chain-length monomers. The native FabH continues the normal function condensing
acetyl-CoA and malonyl-ACP into acetoacetyl-ACP, which can be converted to acetoacetyl-CoA by the engineered FabH and processed to produce short-chain-length monomers. The
monomers are assembled into a polymer by the PHA synthase (PhaC).
In this work we improved the PHA monomer supplying efficiency of
the E. coli FabH enzyme for PHA production in transgenic E. coli through
introduction of specific mutations to the active site of the enzyme.
MATERIALS AND METHODS
Bacterial strains, plasmids, and cultivation conditions
Competent cell
preparation, transformation, and plasmid DNA isolation were preformed according to
common protocols (25) using E. coli JM109 (Promega, USA). Plasmids were isolated
from E. coli cells by alkaline lysis with sodium dodecylsulfate. Transgenic production of
PHA was performed in E. coli K-12 MG1655. Bacterial strains were grown in LB medium
supplemented with glucose as described below. To maintain and select for plasmids
within recombinant E. coli, 100 μg L− 1 of ampicillin was used. Plasmids and strains used
in this study are listed in Table 1.
Construction and analysis of plasmids
All protocols were carried out according
manufacturer's instructions as outlined below. Primers were purchased from Invitrogen
(USA) and all enzymes were purchased from Promega (USA). The pTrc99A plasmids
containing the wild type and F87 mutant fabH genes were obtained from a previous study
by Nomura et al. (19). Site-directed mutagenesis for introduction of active site mutations
was performed with the QuickChange kit (Stratagene, USA). Briefly, complimentary pairs
of oligonucleotide primers were designed which contained the modified codon of interest
and annealed to the same sequence on opposite strands of the plasmid (Table 2). These
primers were used in a polymerase chain reaction (PCR) with Pfu polymerase and Dam
methylated pTrcFabH plasmids as the template to generate copies of the plasmid with the
desired mutation. The PCR products were then treated with DpnI to digest the methylated
template. The DpnI treated PCR product was then used to transform E. coli, and
302
MUELLER AND NOMURA
J. BIOSCI. BIOENG.,
TABLE 1. Bacterial strains and plasmids used in this study.
Strain or plasmid
Relevant characteristics
Source or reference
E. coli JM109
E. coli K-12 MG1655
pGEMC1AB–STQK
pTrc99A
recA1 endA1 gyrA96 thi-1 hsdR17 (rK− mK+) supE44 relA1 l-lac [F′proAB lacIq ZΔM15]
F− λ−ilvG-rfb-50 rph-1
pGEM derivative; PhaC (ST/QK), phbAB, Ampr, colE1 ori
Expression vector; Apr PtrcrrnB TtT2ori (pBR322) lacIq
pBS-PhaCSTQK
pTrcFabH
pTrcFabH(F87C)
pTrcFabH(F87T)
pTrcFabH(F87S)
pTrcFabH PhaCSTQK
pTrcFabH(H244A) PhaCSTQK
pTrcFabH(N274A) PhaCSTQK
pTrcFabH(H244A,N274A) PhaCSTQK
pTrcFabH(F87C,H244A) PhaCSTQK
pTrcFabH(F87I,H244A) PhaCSTQK
pTrcFabH(F87T,H244A) PhaCSTQK
pTrcFabH(F87S,N274A) PhaCSTQK
pBS derivative; phaC (ST/QK), Ampr, colE1 ori
pTrc99A derivative; wild-type fabH,
pTrc99A derivative; fabH(F87C)
pTrc99A derivative; fabH(F87T)
pTrc99A derivative; fabH(F87S)
pTrc99A derivative; wild-type fabH, phaCPs ST/QK
pTrc99A derivative; fabH(H244A) phaCPs ST/QK
pTrc99A derivative; fabH(N274A) phaCPs ST/QK
pTrc99A derivative; fabH(H244A,N274A) phaCPs ST/QK
pTrc99A derivative; fabH(F87C,H244A) phaCPs ST/QK
pTrc99A derivative; fabH(F87I,H244A) phaCPs ST/QK
pTrc99A derivative; fabH(F87T,H244A) phaCPs ST/QK
pTrc99A derivative; fabH(F87S,N274A) phaCPs ST/QK
Promega
29
26
Amersham Pharmacia
(USA)
This study
19
19
19
19
This study
This study
This study
This study
This study
This study
This study
This study
Ps, Pseudomonas sp. 61–3.
transformants were selected on ampicillin. The mutations were verified by sequence
analysis by Genewiz (USA). The nucleotide sequence encoding the engineered PHA
synthase, phaCPs ST/QK, was amplified from the pGEMC1AB–STQK plasmid (26) using
custom oligonucleotide primers (Table 2). The resulting PCR product was then inserted
into the XhoI site of pBS using the In-Fusion kit (Clontech, USA) and sequenced to verify
fidelity of PCR. The synthase was then cut from the verified pBS-STQK plasmid with XhoI
and PstI and ligated into the SalI and PstI sites of the pTrcFabH constructs using T4 DNA
ligase (Fig. 2).
PHA production from glucose in recombinant E. coli
Confirmed pTrcFabH
plasmids containing appropriate fabH genes and engineered PHA synthase genes were
transformed into E. coli K-12 MG1655. Transformants were selected and the presence
of the proper plasmid was verified by extraction and enzymatic digest. For assessing
PHA production, all samples were run in triplicate. Single colonies were cultured
overnight in 50 mL of LB medium and 1 mL was used to inoculate 500-mL baffled
culture flasks containing 100 mL of LB medium. Cultures were grown at 30°C and
shaken at 250 rpm for 4 h, when the optical density of cultures reached between 0.5
and 0.8 at 600 nm, at which time expression of the fabH and synthase was induced by
the addition of 1 mmol L− 1 isopropyl-β-D-thiogalactopyranoside (IPTG). The cultures
were grown for an additional 3 h, at which time 3 mL of 50% glucose was added. An
additional 1 mL of 50% glucose was added again at 24 h and 36 h. After 48 h of total
growing time, the cells were harvested by centrifugation.
Determination of PHA production by gas chromatography
Cell pellets were
rinsed in NANOpure water by resuspension and centrifugation. The rinsed cells were
then resuspended in 3 mL of NANOpure water, and the resulting cell suspension was
frozen and dried by lyophilization. The weight of the dried cells was recorded. The PHA
content and composition of samples were determined by methanolysis and gas
chromatography (GC) as previously described (19) with modifications in the amount of
cells used and column for separation of methyl esters. Briefly, 40 mg of dried cells was
mixed with 2 mL of chloroform and 2 mL of 15% (v/v) sulfuric acid in methanol. The
mixture was then heated to 110°C for 140 min. Samples were then cooled, and 1 mL of
water was added and vortexed 1 min to separate the methanol and sulfuric acid from
the chloroform containing the methyl esters of the PHA monomers. The chloroform
layer was then filtered (PTFE membrane 0.22 μm). The filtered organic layer (0.5 mL)
was mixed with 0.5 mL 0.1% (wt/v) caprilic acid in chloroform. This mixture was then
analyzed by GC with a flame ionization detector (FID) by split injection of 1 μL onto a
30-m Rtx®-5 (5% diphenyl-95% dimethyl polysiloxane) column with a 0.25 mm ID
(Restek, USA). The injector port was held at 280°C and the temperature of the oven was
held at 100° for the first 3 min. Then the oven temperature was increased at a rate of
20°C min− 1 to a final temperature of 310°C which was held for 10 min. Products were
determined by FID, and their peak areas and retention times were compared to
standards of 3-hydroxybutyrate, 3-hydroxyhexanoate, and 3-hydroxyoctanoate for
identification and quantification.
RESULTS
Growth characteristics of recombinant E. coli expressing
engineered FabH monomer supplying genes with the PHA synthase
gene phaC STQK A number of mutations were introduced into FabH
enzymes (Table 3) in order to improve their abilities to supply
substrates for SCL and MCL PHA production in recombinant E. coli. The
effect of overproducing these mutant FabH enzymes in E. coli on cell
growth was unknown. To verify that there were no deleterious effects
on cell growth, total biomass production in each culture was measured.
Cell dry weights of all cultures after 48 h were between 1.2 and
2.0 g L− 1 (Fig. 3). Cultures with plasmids containing the dual active
site mutant FabH enzyme, harboring both the His-244-Ala (H244A)
and Asn-274-Ala (N274A) mutations, grew to the highest density at
2.0 g L− 1 among all mutant strains. However the growth of the strains
harboring the FabH H244A and N274 mutations was not significantly
higher, by t-test, than that of the culture overproducing the wild-type
FabH. Single active site mutations had significantly lower total growth
at 1.6 g L− 1 (N274A) and 1.7 g L− 1 (H244A) respectively, by t-test
(p b 0.001). Cultures expressing FabH enzymes with H244A mutation
as well as various binding pocket mutations at position 87 yielded still
lower total cell growth, between 1.2 and 1.6 g L− 1.
Effect of FabH modifications on PHA production in recombinant
E. coli The mutant FabH constructs were expressed in E. coli with a
PHA synthase to determine the effects of the mutations on PHA
production. The two mutations to the active site affected polymer
productivity differently (Fig. 3). Expression of the FabH harboring the
N274A mutation and the PhaC STQK enzyme had little effect on
polymer content relative to expression of the wild type FabH and the
PhaC STQK enzyme, with strains producing only slightly more polymer
than the wild type. Strains expressing the FabH N274A and wild type
FabH both produced polymers composed solely of SCL monomers, 3hydroxybutyrate. The culture expressing FabH harboring the H244A
mutation and the PhaC STQK enzyme had a greater than six-fold
TABLE 2. PCR primers.
Primers
His244Ala-s
His244Ala-a
Asn274Ala-s
Asn274Ala-a
PhaC-s
PhaC-a
a
Target gene
Sequence a
fabH
fabH
fabH
fabH
phaC
phaC
5′-GTT GAC CTG ACC GAC CAA GGC GCA GTC CGA TTG GAC GC-3′
5′-GC GTC CAA TCG GAC TGC GCC TTG GTC GGT CAG GTC AAC-3′
5′-CG CTG GAT CGC CAC GGT GCA ACC TCT GCG GCC T-3′
5′-A GGC CGC AGA GGT TGC ACC GTG GCG ATC CAG CG-3′
5′-CGAATAGTGACTCGAGTCTAGAAATAATTTTGTTTAACTTT-3′
5′-TACCGTCGACCTCGACGTCAGTAATTGTGTAGTCCTTTC-3′
Bold sequences indicate amino acid mutations introduced into the primers.
VOL. 113, 2012
ENGINEERED FabH FOR PHA PRODUCTION IN E. COLI
303
FIG. 2. Plasmid construction scheme. Active site mutations were generated on the pTrcFabH constructs through site-directed mutagenesis. The plasmid shown is the H244A mutation
to the wild type FabH construct. The phaC STQK was cut from pBSphaCSTQK with XhoI and PstI and ligated into the SalI and PstI sites of the mutated pTrc FabH constructs to generate
the pTrcFabHphaCSTQK constructs.
increase in polymer, and the polymer produced by the H244A mutant
and the PhaC STQK enzyme contained more than 2 mole-percent MCL
monomers: 1.6% 3-hydroxyhexanoate and 0.7% 3-hydroxyoctanoate.
Based on the proximity of this amino acid to the active site, this change
in substrate specificity is due to slight expansion of the binding pocket
of the enzyme, allowing for incorporation of the larger subunits. The
double mutant, containing the H244A and N274A mutations and the
PhaC STQK enzyme produced slightly less PHA per cell mass than the
wild type.
PHA production was relatively consistent among cultures expressing a FabH enzyme with various mutations to the binding pocket,
position 87, in combination with the H244A mutation and the PhaC
STQK enzyme. A previous study had identified a number of mutations
TABLE 3. Mutations made to the 3-ketoacyl-ACP-synthase III (FabH) enzyme, and the
abbreviations used to represent them.
Abbrev.
Mutation
WT
H244A
N274A
F87C
F87I
F87S
F87T
Wild type (control)
Amino acid 244 histidine to alanine
Amino acid 274 asparagine to alanine
Amino acid 87 phenylalanine to cysteine
Amino acid 87 phenylalanine to isoleucine
Amino acid 87 phenylalanine to serine
Amino acid 87 phenylalanine to threonine
to F87 that allowed the FabH enzyme to produce 3-hydrdoxyacyl-CoA
substrates for incorporation into SCL–MCL PHA polymers (19). We
selected four mutations (F87C, F87I, F87S, and F87T) that were
characterized from this previous study and combined them with the
H244A mutation in this study in order to determine if the mutations
would have a synergistic effect on substrate specificity and overall
activity of the enzyme to produce SCL–MCL PHA polymers. Although
these mutations are far apart in the primary structure of FabH, when
the dimer forms, they are in close proximity to one another within the
binding and active site of the FabH enzyme based on its crystal
structure (22). All recombinant strains harboring the FabH F87/H244A
mutations and the PhaC STQK enzyme produced less PHA per cell
mass than the H244A mutant and the PhaC STQK enzyme, but more
than the wild type and the PhaC STQK enzyme. However, the
monomer composition of the PHA polymers varied dependent on
the specific mutations in FabH (Fig. 3). All strains expressing FabH
with F87/H244A mutations and the PhaC STQK enzyme produced PHA
copolymers with a greater mole-percent of MCL monomers than the
wild type FabH and the PhaC STQK enzyme or the FabH H244A mutant
and the PhaC STQK enzyme, with FabH F87C/H244A and the PhaC
STQK enzyme producing a PHA copolymer with the highest mol
percent of MCL monomer at 6.6%. The mean total PHA produced by the
samples ranged from 2.8 mg L− 1 in the H244A, N274A dual mutant to
17.5 mg L− 1 in the H244A mutant.
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MUELLER AND NOMURA
FIG. 3. Dry cell mass of E. coli K-12 MG1655 cultures over expressing the various mutant
FabH enzymes after 48 h of growth, values represent the mean of three samples with error
bars representing one standard deviation (A). PHA production content of the cultures after
48 h of growth (B). Values represent the mean of three samples with error bars
representing one standard deviation. Monomer composition of produced polymers: 3hydroxybutyrate (HB), 3-hydroxyhexanoate (HHX), and 3-hydroxyoctanoate (HO) (C).
The mutant type abbreviations are defined in Table 3.
DISCUSSION
In this study, we have shown that engineering the FabH enzyme
from E. coli through mutations to the active site greatly enhances its
PHA monomer supplying efficiency in vivo (Fig. 3). In previous work
with the FabH enzyme some of the tested mutations to the binding
pocket enhanced the enzyme's ability to supply MCL monomers, but
all of the tested mutations led to a drop in total PHA production per
J. BIOSCI. BIOENG.,
cell mass relative to the wild type enzyme (19). Here we demonstrate
that while production of PHA enriched in MCL monomers was
retained, the previous observed reduction in PHA levels in the FabH
F87 mutants was overcome by adding the H244A mutation to these
binding pocket mutants. Based on PHA production per cell mass, the
PHA monomer supplying activity of all the F87/H244A mutants
generated in this study surpassed that of recombinant E. coli strains
that were expressing the wild type FabH enzyme.
Although there are many barriers to the photosynthetic production
of SCL–MCL PHA polymers, the improved enzymes developed in this
study could be incorporated into other hosts and even improve the
success of PHA production in plants (27). Production of PHAs in plants
could significantly reduce the costs of PHAs. Because most bacterial
production methods utilize an agriculturally derived carbon source,
direct production in plants would effectively reduce the number of steps
between atmospheric carbon and the biobased plastic products (27,28).
PHA copolymer production requires the production of both SCL and
MCL monomers. The FabH enzymes described in this study are unique in
their ability to supply both of these monomer types for PHA copolymer
production. The changes in PHA monomer-supplying efficiency in the
active-site mutants closely match the changes in transacylase activity,
where the liberation of free CoA from acetyl-CoA was measured as
reported in previous work (22). In that study, the H244A mutant led to a
634% increase in transacylase activity over wild type (22) and the same
mutation led to a 679% increase in polymer production in the current
study. The N274A mutant led to a 118% increase in transacylase activity
over wild type in the previous study (22) and 118% increase in polymer
production in the current study. The original amino acids at these two
positions were reported to stabilize the oxyanion generated in the
decarboxylative condensation reaction (Fig. 4) since the mutation of
either of these sites led to a drop in condensation reaction to less than
5% of the wild type. The increased rate of transacylation can be attributed
to the loss of the condensation reaction as a competing reaction.
Considering the differences in transacylase activity between the active
site mutants, the two positions play unequal roles in oxyanion
stabilization. The high transacylase activity of the H244A mutant implies
that the role of that position is primarily this stabilization, and the
substrate still binds to the enzyme as efficiently as previously. The
relatively lower transacylase activity of the N274A mutant and the
H244A/N274A double mutant implies that the amino acid at position 274
plays another important role in the activity of the enzyme, whether it be
binding of the substrate or maintenance of the shape of the active site.
Improving MCL monomer supply from de novo fatty acid
biosynthesis is an important step toward producing SCL–MCL PHAs
to replace some of the petroleum-based plastics currently in use.
Efficient MCL monomer supply will improve production of SCL–MCL
PHAs with enhanced material properties opening up new potential
uses for these biobased, biodegradable polymers. A pathway for
producing the monomers from de novo fatty acid biosynthesis will
allow for use of cheaper carbon sources when producing PHA in
bacterial strains and would allow for the transfer of the pathway for
photosynthetic production of PHA polymers.
ACKNOWLEDGMENTS
This work was supported by NSFEAPSI 1015089 awarded to A.P.
Mueller and NSF DMR 0907085 awarded to C.T. Nomura. The authors
wish to thank S. Taguchi and K. Matsumoto (Hokkaido University) and
FOR 694 (SUNY-ESF) for their input.
FIG. 4. Active site and proposed mechanism of the wild-type FabH enzyme adapted from Davies et al. (22) on the following page. Asn274 and His244 are understood to play key roles
in stabilizing the shown oxyanion generated in the native condensation reaction through hydrogen bonding. The substitution of an alanine for histidine 244 reduces this stabilization,
inhibiting the native condensation reaction, leading to an increase in transacylase activity. (A) Native decarboxylase and condensation reactions of FabH. (B) Conversion of the acylACP to acyl-CoA form in the native FabH enzyme. The presence of His244 limits the substrate binding pocket size. (C) His244Ala mutation prevents stabilization of the oxyanion thus
preventing the condensation reaction. (D) His244Ala mutation allows larger substrates (C6) to be subjected to acyl-ACP to acyl-CoA transacylation.
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A
B
C
D
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