Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases

Article abstract of DOI:10.1002/anie.201812623

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final-product profiles are still obscure. In this study, the FabA-type dehydratase domain (DH_FabA) in the C-subunit and the polyketide synthase-type dehydratase domain (DH_PKS) in the B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C₄- to C₈-acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DH_PKS and DH_FabA, depending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.

Full text of DOI:10.1002/anie.201812623

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Control mechanism for cis double bond formation by
polyunsaturated fatty acid synthases
Authors: Shohei Hayashi, Yasuharu Satoh, Yasushi Ogasawara,
Chitose Maruyama, Yoshimitsu Hamano, Tetsuro Ujihara, and
Tohru Dairi
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201812623
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10.1002/anie.201812623
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Control mechanism for cis double bond formation by
polyunsaturated fatty acid synthases
Shohei Hayashi[b], Yasuharu Satoh[a], Yasushi Ogasawara[a], Chitose Maruyama[c], Yoshimitsu
Hamano[c], Tetsuro Ujihara[d], and Tohru Dairi*[a]
elongation steps to synthesize PUFAs from oleic acid (C18:1 ω9),
as represented by the abovementioned practical EPA and ARA
production[1b,1c]. In the latter pathway, PUFA synthases composed
of huge enzyme complexes with multiple catalytic domains
synthesize PUFAs from acetyl units as exemplified by microalgal
Abstract: Polyunsaturated fatty acids (PUFAs) such as
docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and
arachidonic acid (ARA) are essential fatty acids for humans. Some
microorganisms biosynthesize these PUFAs through PUFA
synthases composed of four subunits with multiple catalytic domains.
These PUFA synthases each create a specific PUFA without
undesirable byproducts even though the multiple catalytic domains in
each huge subunit are very similar. However, the detailed biosynthetic
pathways and mechanisms for controlling final product profiles are still
obscure. In this study, the FabA-type dehydratase domain (DHFabA) in
the “C”-subunit and the polyketide synthase-type dehydratase domain
(DHPKS) in the “B”-subunit of ARA synthase were revealed to be
essential for ARA biosynthesis by in vivo gene exchange assays.
Furthermore, in vitro analysis with truncated recombinant enzymes
and C4 to C8-acyl ACP substrates showed that ARA and EPA
DHA production[1a,2]
.
These PUFA synthases possess
acyltransferase (AT), acyl carrier protein (ACP), malonyl CoA
transacylase (MAT), ketoacyl synthase (KS), ketoacyl reductase
(KR), two types of dehydratase—polyketide synthase (PKS)-type
dehydratase (DHPKS
)
and FabA-type DH (DHFabA)[3], enoyl
reductase (ER), and chain length factor (CLF) domains, and all of
these domains are perhaps involved in the elongation cycle for
PUFA biosynthesis in a similar manner to fatty acid synthases
(FASs) and PKS[4].
Among marine prokaryotic microorganisms, biosynthetic
genes for DHA, EPA, and ARA have been identified in Moritella
marina, Photobacterium profundum, and Aureispira marina,
respectively[5]. These PUFA synthases are composed of four
subunits and each create a specific PUFA even though the
multiple catalytic domains in each subunit are very similar (Fig.
1B), suggesting that a key domain with specific catalytic activity is
responsible for the determination of carbon chain length (C20 or
C22) and the first unsaturated position (ω3 or ω6). Although
functional analyses of some domains have been carried out[6], the
detailed biosynthetic machinery remains unclear. Here, we
revealed that two types of DH domains, DHPKS and DHFabA, were
key domains for controlling the first unsaturated carbon position
of the final products of bacterial EPA and ARA synthases.
synthases utilized two types of DH domains, DHPKS and DHFabA
,
depending on the carbon chain length to introduce saturation or cis
double bonds to growing acyl chains.
Polyunsaturated fatty acids (PUFAs, Fig. 1A), such as
docosahexaenoic acid (DHA; C22:6 ω3), eicosapentaenoic acid
(EPA; C20:5 ω3), and arachidonic acid (ARA; C20:4 ω6), are
essential fatty acids for humans and we ingest them from dietary
sources such as fish and fish oils. Recently, alternative and
sustainable sources of PUFAs are required and commercial
fermentative processes using microalgae, yeasts, and fungi have
been developed to produce DHA, EPA, and ARA, respectively[1].
PUFAs are biosynthesized by two pathways, the aerobic
desaturase/elongase pathway and the anaerobic PUFA synthase
pathway. In the former pathway, specific oxygen-dependent
desaturases and elongases catalyze individual desaturation and
[a]
Dr. Y. Satoh, Dr. Y. Ogasawara, Prof. Dr. T. Dairi*.
Graduate School of Engineering
Hokkaido University,
N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
E-mail: dairi@eng.hokudai.ac.jp
Mr. S. Hayashi
Graduate School of Chemical Sciences and Engineering
Hokkaido University,
N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
Dr. C. Maruyama, Prof. Dr. Y. Hamano.
Department of Bioscience
[b]
[c]
[d]
Fukui Prefectural University
Fukui, 910-1195, Japan.
Dr. T. Ujihara.
Kyowa Hakko Bio Co. Ltd.
1-6-1, Ohtemachi, Chiyoda-ku, Tokyo, 100-8185, Japan.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201812623
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synthase genes (ara) of A. marina each comprise four genes (A
to D). These genes were cloned into different and compatible
expression vectors and introduced into E. coli BLR(DE3)ΔfadE for
evaluation of PUFA profiles. Because ARA productivity was
significantly lower than that of EPA in the heterologous expression
system[6d], the PUFA profiles were evaluated by replacing each
epa gene with the corresponding ara gene. When epa-D was
replaced with ara-D, the PUFA profile was almost the same as
that of the parents. By replacing epa-C with ara-C, however, both
EPA and ARA production were observed, indicating that Ara-C is
responsible for ARA production (Fig. 2A). Moreover, the
transformants expressing epa-AD with ara-BC mostly produced
ARA (Fig. 2A), showing that Ara-B also impacts specificity.
We next examined which domain in Ara-C is essential for
ARA production. We constructed ara-C-AT0, in which the catalytic
Ser residue in the AT-domain[7] was mutated to Ala, and co-
expressed it with epa-ABD (Fig. S1). The PUFA profile of the
transformants was the same as that with ara-C (Fig. 2B), showing
that the AT domain in Ara-C was unrelated to ARA production and
was complemented by the AT domain in Epa-B. We therefore
constructed chimeric genes in which the DHFabA domains in Epa-
C and Ara-C were reciprocally swapped to yield epa-C-ara-
DHFabA-chimera1 and ara-C-epa-DHFabA-chimera2 (Fig. S2).
When epa-C-ara-DHFabA-chimera1 was co-expressed with epa-
ABD, almost the same amounts of ARA and EPA were produced
(Fig. 2C), suggesting that the DHFabA domains in Ara-C are
Figure 1. (A) Chemical structures of docosahexaenoic acid (DHA; C22:6 ω3),
docosapentaenoic acid (DPAω3; C22:5 ω3), eicosapentaenoic acid (EPA;
C20:5 ω3), and arachidonic acid (ARA; C20:4 ω6). (B) Domain organizations of
bacterial PUFA synthases. KS: β-ketoacyl synthase, MAT: malonyl CoA
transacylase, ACP: acyl carrier protein, KR: β-ketoacyl reductase, DHPKS
:
polyketide synthase-type dehydratase, AT: acyltransferase, CLF: chain length
factor, DHFabA: FabA-type dehydratase, ER: enoyl reductase.
To find clues to how the enzymes control the formation of the
first cis double bond, we employed in vivo gene exchange assays.
The EPA synthase genes (epa) of P. profundum and ARA
essential for ARA production. However, ara-C-epa-DHFabA
-
chimera2 predominantly conferred ARA productivity when co-
Figure 2. GC-MS analysis traced at m/z 79[6d] of products produced in the gene replacement assay. (A) Gene replacement of epa genes with ara genes (from 3rd
to bottom). (B) Co-expression of ara-C (wild type) (middle) or ara-C-AT0 (bottom) with epa-ABD. (C) Co-expression of ara-C-epa-DHFabA-chimera2 with ara-B and
epa-AD (middle) and epa-C-ara-DHFabA-chimera1 with epa-ABD (bottom). (D) Co-expression of ara-B-KR0 (middle) or ara-B-DH0 (bottom) with epa-AD and ara-C.
(A) to (D) Authentic standards of methyl ester derivatives of ARA, EPA (0.3 mM, (A), 1.2 mM (B), 0.3 mM (C), 3 mM (D)), eicosatetraenoic acid (ETA),
docosapentaenoic acid (DPAω3), and DHA (top) were injected. Asterisks in (C) indicated minor uncharacterized PUFA products.
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10.1002/anie.201812623
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expressed with epa-AD and ara-B (Fig. 2C), suggesting that the
KS/CLF domains in Ara-C are also important. We then tried
constructing dozens of chimeric genes fused at different points
but none of the constructs showed activity.
To investigate the essential domain in Ara-B for ARA
production, we constructed mutated enzymes. Tyr in the KR
domain and His in the DHPKS domain, which are catalytically
essential amino acid residues[8], were mutated to Phe to construct
ARA-B-KR0 and ARA-B-DH0 (Figs. S3 and S4). The transformant
expressing ara-B-KR0 with epa-AD and ara-C produced ARA (Fig.
2D), plausibly because the function of Ara-B-KR0 was
complemented by the KR domain in Epa-A, while ARA production
was drastically decreased by expressing ara-B-DH0 with epa-AD
and ara-C (Fig. 2D). These results showed that the DHPKS domain
in Ara-B is essential for ARA production. Taking these results
together, the KS/CLF and DHFabA domains in Ara-C and the DHPKS
domain in Ara-B perhaps control ARA biosynthesis.
We then carried out in vitro analysis with truncated
recombinant enzymes. Because there have been no reports on
the geometrical analysis of PUFA-KR products formed from 3-
oxoacyl-ACP, we therefore performed this type of analysis.
Because chiral analysis of 3-hydroxyacyl-ACP products was
unsuccessful, we utilized N-acetylcysteamine (SNAC) thioester
derivatives, mimics of acyl-ACPs usually used as substrates for
in vitro experiments. For the enzymes, we tried to express a
truncated enzyme containing only the KR domain but no
recombinant enzyme was obtained. Then, we tried expressing a
truncated enzyme containing both the KR and neighboring DH
domains (Epa-A-KR-DHPKS (Epa-KR-DHPKS)) and obtained the
enzymes as a soluble form. When 3-oxobutyryl-SNAC (1-SNAC)
was incubated with Epa-KR-DHPKS and NADPH, 3-
hydroxybutyryl-SNAC (2-SNAC) whose m/z and retention time of
chiral LC-MS analysis were consistent with those of the ketoacyl
reductase EcFabG of E. coli was clearly detected (Figs. S5 and
S6), suggesting the formation of (3R)-hydroxybutyryl-SNAC[9].
Furthermore, we also confirmed that the Epa-KR-DHPKS products
from 3-oxohexanoyl-SNAC (3-SNAC) and 3-oxooctanoyl-SNAC
(4-SNAC) were (3R)-hydroxyhexanoyl-SNAC (5-SNAC) and
(3R)-hydroxyoctanoyl-SNAC (6-SNAC), respectively (Figs. S5
and S6).
Figure 3. HPLC analysis of in vitro hydration reaction mixtures. (A) Proposed
biosynthetic pathway of ω3 and ω6 PUFAs from C6- to C8-ACP intermediates.
(B) HPLC traces (210 nm) of in vitro hydration reactions of crotonyl-ACP (9-
ACP) with Epa enzymes (left) or Ara enzymes (right). Without enzyme (top),
with KR-DHPKS (middle), or with DHFabA (bottom). (C) HPLC traces (210 nm) of
in vitro hydration reactions of 2-trans hexenoyl-ACP (7-ACP) with Epa enzymes
(left) or Ara enzymes (right). Without enzyme (top), with KR-DHPKS (middle), or
with DHFabA (bottom).
only the DHFabA domains, were expressed as soluble forms (Fig.
S7). For the substrates, we prepared crotonyl-ACP (9-ACP), 2-
trans hexenoyl-ACP (7-ACP), and 2-trans octenoyl-ACP (10-
ACP) (Fig. S8) because 3-hydroxyacyl-ACP was more
thermodynamically stable than 2-trans acyl-ACP in FAS[3] and
hydration activities were measured instead of the forward
We next examined how the enzymes introduce the first cis
double bond into the products. A branching point between EPA
and ARA biosynthesis would be brought about by specific
reactions during the carbon elongation from a C6- to a C8-ACP
intermediate. A saturation reaction of 2-trans hexenoyl-ACP (7-
ACP) occurs in ARA biosynthesis while a 2,3-isomerization
reaction from 2-trans to 3-cis hexenoyl-ACP (7-, 8-ACP) takes
place in EPA biosynthesis (Fig. 3A). To investigate this branching
point, we carried out in vitro experiments using acyl-ACP
substrates and truncated recombinant enzymes. As mentioned
above, DHPKS and DHFabA were crucial for EPA and ARA
biosynthesis, so we prepared truncated DH enzymes. We tried to
express a truncated enzyme containing only the DHPKS domain
but no recombinant enzyme was obtained. Therefore, Epa-KR-
DHPKS and Ara-B-KR-DHPKS (Ara-KR-DHPKS) were prepared as
the DHPKS domain. For the DHFabA domain, Epa-C-DHFabA (Epa-
DHFabA) and Ara-C-DHFabA (Ara-DHFabA), both of which posssess
dehydration reactions. When crotonyl-ACP (9-ACP),
a C4
substrate, was used, Epa-KR-DHPKS exhibited higher hydration
activity than Epa-DHFabA (Fig. 3B). The same tendency was also
observed with Ara-KR-DHPKS and Ara-DHFabA (Figs. 3B and S9).
However, in the case of a C6 substrate, 2-trans hexenoyl-ACP (7-
ACP), Epa-KR-DHPKS and Ara-DHFabA showed very weak
activities while EPA-DHFabA and ARA-KR-DHPKS showed high
hydration activities (Figs. 3C and S9). These results suggested
that the two different DH domains strictly recognized different
intermediates to create EPA or ARA. Indeed, Dha-KR-DHPKS and
Dha-DHFabA (Fig. S7) showed the same substrate specificities as
Epa enzymes (Fig. S10). We also tried examining this hypothesis
with a C8 substrate and Ara-DHFabA. However, both ARA-KR-
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DHPKS and Ara-DHFabA showed no hydration activity when 2-trans
octenoyl-ACP (10-ACP) was used as the substrate (Fig. S11).
We therefore prepared 3-hydroxyoctanoyl-ACP (6-ACP),
the intrinsic substrate, for the forward dehydration reaction (Fig.
S12). As shown in Figs. 4A and S13, Ara-DHFabA catalyzed
dehydration of 3-hydroxyoctanoyl-ACP (6-ACP). As geometry
determination of the ACP-product is very difficult because of its
high molecular weight, we used SNAC derivatives as substrates.
When 3-hydroxyoctanoyl-SNAC (6-SNAC) was incubated with
Ara-DHFabA, a dehydrated product was again detected and
confirmed to be 2-trans octenoyl-SNAC (10-SNAC, Fig. 4B).
Taken together, these results suggested that the DHPKS domain
of EPA synthase recognized the C4 substrate while that of ARA
synthase recognized both the C4 and C6 substrates for full
reduction in the early biosynthetic stage.
Figure 5. UPLC analysis of in vitro isomerization reaction mixtures. (A) UPLC
traces (260 nm) of the 2,3-isomerization reaction of 3-cis hexenoyl-SNAC (8-
SNAC) with Epa-DHFabA. Standard (top) and without enzyme (bottom). (B) UPLC
traces (260 nm) of the 2,2-isomerization reaction of 2-cis octenoyl-SNAC (11-
SNAC) with Ara-DHFabA (2nd). Standard (top and bottom) and without enzyme
(3rd).
In this study, we showed that PUFA synthases cleverly
utilize two types of DH domains, DHPKS and DHFabA, depending on
the carbon chain length to introduce saturation or cis double
bonds to growing acyl chains. In ARA biosynthesis, dehydration
by DHPKS and subsequent enoyl reduction by the ER domain
would occur to form hexanoyl-ACP from (3R)-hydroxyhexanoyl-
ACP (5-ACP). Then, condensation of hexanoyl-ACP with
malonyl-ACP, reduction by the KR domain, and dehydration and
2,2-isomerization of the C8 intermediate by DHFabA would occur.
By contrast, in EPA biosynthesis, dehydration and 2,3-
isomerization of a C6 intermediate would be catalyzed by DHFabA
to form 3-cis hexenoyl-ACP (8-ACP, Fig. 6).
Figure 4. HPLC/UPLC analysis of in vitro dehydration reaction mixtures. (A)
HPLC traces (210 nm) of in vitro dehydration reactions of 3-hydroxyoctanoyl-
ACP (6-ACP) with Ara-KR-DHPKS (middle), Ara-DHFabA (bottom), or without
enzyme (top). (B) UPLC traces (260 nm) of in vitro reaction products with Ara-
DHFabA and 3-hydroxyoctanoyl-SNAC (6-SNAC). Standards (top and 2nd) and
without enzyme (bottom).
Because the DHFabA domain acted on substrates to which a
cis double bond was introduced and the DHFabA domains show
high similarity to the dehydratase FabA, which is responsible for
the introduction of cis double bonds during unsaturated fatty acid
biosynthesis in type II FAS[3], we next examined whether trans to
cis isomerization occurs during the dehydration reaction.
However, detection of isomerization activity with trans-substrates
was reported to be difficult[3,10]. We therefore examined whether
the DHFabA domains catalyzed the reverse 2,3- and 2,2-
isomerization reactions with cis-acyl-SNAC substrates. When 3-
cis hexenoyl-SNAC (8-SNAC) was incubated with Epa-DHFabA, 2-
trans hexenoyl-SNAC (7-SNAC) was clearly detected (Fig. 5A).
Moreover, Ara-DHFabA also catalyzed the conversion of 2-cis
octenoyl-SNAC (11-SNAC) to 2-trans octenoyl-SNAC (10-SNAC)
(Fig. 5B). These results suggested that DHFabA domains catalyze
trans to cis isomerization, and 2,3- and 2,2-isomerizations,
besides dehydration reactions. Although the reaction
mechanisms of 2,3- and 2,2-isomerization were unclear, 2,3-
isomerization might occur by the same reaction mechanism as
that of FabA in type II FAS. FabA is a bifunctional enzyme
catalyzing dehydration and successive isomerization[3,11]. First,
FabA abstracts a C2 proton concomitant with the C3 hydroxyl
group elimination from the same face and then isomerizes to
generate C3–C4 double bond in cis configuration by abstracting
the pro-R proton from C4 of the substrate.
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Figure 6. Proposed biosynthetic pathway from C2 to C8-ACP intermediates of ω3 and ω6 PUFAs.
1913; c) T. Ujihara, M. Nagano, H. Wada, S. Mitsuhashi, FEBS Lett.
2014, 588, 4032–4036.
Acknowledgements
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This study was supported in part by Grants-in-Aid for Research
on Innovative Areas from MEXT, Japan (JSPS KAKENHI Grant
Number 16H06452 to T.D.) and Grants-in-Aid for Scientific
Research from JSPS (15H03110 and 18H03937) to T.D. We
thank
Robbie
Lewis,
MSc,
from
Edanz
Group
(www.edanzediting.com/ac) for editing a draft of this manuscript.
Keywords: biosynthesis • cis double bond • dehydratase •
polyketide • polyunsaturated fatty acid synthase
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Entry for the Table of Contents
COMMUNICATION
Eicosapentaenoic acid synthase and
arachidonic acid synthase, both of
which are composed of huge enzyme
complexes with multiple catalytic
domains, utilized two types of
dehydratase domains depending on
the carbon chain length to introduce
saturation or cis double bonds to
growing acyl chains.
Shohei Hayashi, Yasuharu Satoh,
Yasushi Ogasawara, Chitose
Maruyama, Yoshimitsu Hamano,
Tetsuro Ujihara, and Tohru Dairi*
Page No. – Page No.
Control mechanism for cis double
bond formation by polyunsaturated
fatty acid synthases
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