(Received for publication, November 27, 1996, and in revised form, January 23, 1997)
From the Instituto de Bioquímica Vegetal y
Fotosíntesis, Universidad de Sevilla-CSIC, Americo Vespucio
s/n, 41092 Sevilla, Spain and the Botanisches Institut,
Biosynthesis Group, Postfach 111932, D-60054 Frankfurt, Germany
We have isolated, based on the knowledge of the
complete genomic sequence of the cyanobacterium
Synechocystis sp. PCC 6803, an open reading frame (slr0088)
similar to known bacterial carotene desaturases and have analyzed the
function of the encoded protein. Surprisingly, this protein has no
detectable desaturase activity with phytoene, hydroxyneurosporene, or
-carotene as substrates, but is rather a
-carotene ketolase that
acts asymmetrically introducing a keto group on only one of the two
-ionone rings of
-carotene to generate echinenone. This is in
contrast to the so far characterized
-carotene ketolases that act
symmetrically, producing the di-keto carotenoid canthaxanthin from
-carotene without significant accumulation of echinenone. We have
designated this new gene crtO. The function of the
crtO gene product has been demonstrated by 1) the
biosynthesis of echinenone when the crtO gene is expressed
in an Escherichia coli strain able to accumulate
-carotene, 2) the in vitro biosynthesis of echinenone
from
-carotene with cell free extracts from E. coli
cells that express the crtO gene, and 3) the absence of
echinenone in a Synechocystis strain in which the
crtO gene has been insertionally inactivated. The primary
structure of the Synechocystis asymmetric ketolase bears no
similarity with the known
-carotene ketolases. crtO is
not required for normal growth under standard or high light conditions,
neither is the photosynthetic activity of the crtO-deficient strain affected.
Carotenoids are pigments that are synthesized by all photosynthetic organisms as essential components of the photosynthetic apparatus (1). They are also synthesized in heterotrophic growing bacteria and fungi. They participate in light collection in photosynthetic organisms and play a protective role against oxidation damage induced by strong oxidants produced in the photosynthetic membranes upon illumination (2).
In plants and cyanobacteria, carotenoids are synthesized by a similar
pathway, from the C40 precursor phytoene (Fig.
1). Phytoene is converted to -carotene by two
sequential desaturations catalyzed by the enzyme phytoene desaturase.
-Carotene is further oxidized by
-carotene desaturase to give
lycopene, which is converted to
-carotene by lycopene cyclase
(reviewed in Refs. 3-5).
Phytoene desaturase from cyanobacteria and plants, which introduce only
two double bonds to produce -carotene, have no homology with the
bacterial type phytoene desaturases, which introduce three or four
double bonds, producing neurosporene or lycopene, respectively (6).
Therefore a second enzyme (
-carotene desaturase) is required in
cyanobacteria and plants for the production of lycopene from
-carotene.
-Carotene desaturase from plants is homologous to the
plant and cyanobacterial type phytoene desaturases (7). Surprisingly,
the only cyanobacterial
-carotene desaturase identified, from
Anabaena sp. PCC 7120, is homologous to the bacterial type
phytoene desaturases (8). It has been suggested that plant and
bacterial desaturases have independent evolutionary origins and that in
cyanobacteria the bacterial type desaturase has been restricted to the
last two dehydrogenation steps, while the plant type desaturase took
over the function for the first two desaturation steps of phytoene. In
Synechocystis no
-carotene desaturase has been identified
so far.
The carotenoid pattern of cyanobacteria is very diverse (4), but
cyanobacteria are not able to synthesize -carotene and its
derivatives as well as epoxy carotenoids. In Synechocystis the major carotenoids accumulated are
-carotene, myxoxanthophyll, zeaxanthin, and echinenone (9, 10). The biosynthetic pathway of those
carotenoids is outlined in Fig. 1. None of the enzymes involved in the
biosynthesis of myxoxanthophyll, zeaxanthin, and echinenone from
-carotene has been characterized, nor have their genes been cloned
in cyanobacteria. The biosynthesis of zeaxanthin requires an
hydroxylase that has been already characterized in some bacteria (11,
12). The biosynthesis of myxoxanthophyll requires several unidentified
enzymes, and some of them might be equivalent to the ones used in
Rhodobacter for the synthesis of spheroidenone (13).
Synechocystis is one of the very few species that
accumulates echinenone in substantial amounts (9). The biosynthesis of
echinenone requires a ketolase that attacks only one of the
-rings
of
-carotene. Genes coding for
-carotene ketolases were cloned
and sequenced from two different bacteria (14) and the green alga
Haematococcus (15, 16). However, all these related enzymes
catalyze the simultaneous introduction of a keto group into position 4 of every ionone ring at each end of the molecule, yielding the
symmetrically di-keto carotenoid canthaxanthin. Echinenone, with only
one keto group, is found as a minor intermediate of the reaction (17,
18).
In the Synechocystis genomic sequence (19) an
ORF1 (slr0088) has been found with
significant homology to bacterial type carotene desaturases. Because it
was possible that this protein was the Synechocystis
-carotene desaturase, we decided to undertake the cloning of the
corresponding gene and the characterization of its function.
Surprisingly slr0088 does not show any desaturase activity, but is
rather a
-carotene ketolase that reacts asymmetrically on its
substrate. We have demonstrated its function by functional complementation in Escherichia coli, by its in
vitro activity, and by inactivation of the corresponding gene in
Synechocystis.
This new -carotene ketolase from Synechocystis is
unrelated in structure to the symmetrically acting ketolases
characterized in algae and bacteria and catalyzes a different reaction.
Therefore we have named its gene crtO to differentiate it
from the symmetrically acting ketolase genes (crtW).
All manipulations were performed by standard methods (20) or as recommended by the manufacturers. Synechocystis cells were grown in BG11 medium (21) and DNA extracted as described (22). Southern blot was performed by standard procedures (20).
Cloning ofORF slr0088 was cloned by polymerase chain reaction based on
the available Synechocystis genomic sequence (19). The
forward primer (5-AACAGAATCACCACCGATGTTGTC-3
)
contains an EcoRI site (underlined) and overlaps the
beginning of the coding sequence. The reverse primer
(5
-AACAGATTACCAAAAACGACGTTG-3
) contains a
BamHI site (underlined) and overlaps the 3
-end of the gene. A polymerase chain reaction product of the expected size was purified and treated with EcoRI and BamHI and cloned in
the polylinker of pTrc99A (Pharmacia Biotech Inc.) to generate plasmid
pTRCRT-O. In this plasmid expression of the crtO gene is
controlled by the inducible pTrc promoter of the vector. The amino end
of the expected recombinant protein has two additional amino acids
(MITTD... versus MITTD...).
To inactivate the crtO gene, a 1.3-kilobase pair HincII fragment containing a kanamycin resistance gene (npt) from Tn5 (23) was used to replace an internal ClaI fragment of the crtO gene. A plasmid with the npt gene inserted in the opposite orientation to the crtO ORF was used to transform Synechocystis sp. PCC 6803 wild type strain and cells were plated on kanamycin-containing plates. Transformants were grown on the same medium for several segregation rounds. Segregation was checked by Southern blot using as a probe an internal BstXI fragment from the crtO gene.
Complementation in E. coliPlasmid pTRCRT-O was introduced
in E. coli JM101 containing different plasmids depending on
the function to be tested (see Table I). Plasmid pACCRT-EB (24)
contains the genes crtE and crtB from
Erwinia. Cells that carry this plasmid accumulate phytoene. Plasmid pACCRT-EBP (25) contains the genes crtE and
crtB from Erwinia and the crtP gene
from Synechococcus, and in plasmid pACCRT-EBIRc the crtP gene is replaced by crtI from
Rhodobacter capsulatus (26). Cells that carry these plasmids
accumulate -carotene and neurosporene, respectively. Plasmid
pACCAR16
crtX (14) contains the genes crtE,
crtB, crtI, and crtY from
Erwinia. Cells that carry this plasmid accumulate
-carotene. These four plasmids are all derivatives of vector
pACYC184 carrying an origin of replication compatible with the origin
of pTRCRT-O. Another vector used for complementation was pRKCRT-C that
is derived from pRK404 and carries the crtC gene from
R. capsulatus coding for a neurosporene hydratase. The
transformants were cultivated at 28 °C in LB medium containing ampicillin (100 µg/ml), chloramphenicol (50 µg/ml), and/or
tetracycline (50 µg/ml) according to the plasmids present. Cells were
grown for 48 h in the presence of 2 mM
isopropyl-
-D-thiogalactopyranoside before harvesting for
carotenoid analysis.
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Carotenoids were extracted from freeze-dried E. coli or Synechocystis 6803 cells with methanol containing 6% KOH by heating for 15 min at 60 °C. After partitioning into 10% ether in petrol, the upper phase was collected, the solvent wad evaporated, and the residual carotenoids were resuspended in acetone. Separation and quantification of carotenoids were done by HPLC with a Nucleosil-C18 3µ column and acetonitrile/methanol/2-propanol (85:10:5, v/v) as eluent at a flow rate of 1 ml/min. Spectra were recorded on-line at the elution peaks with a Kontron 440 photodiode array detector. For identification of carotenoids, authentic standards isolated and identified previously (9) were used.
In VitroFreshly harvested
cells of JM101 carrying pCAR16crtX, which synthesize
-carotene,
and of JM101 carrying pTRCRT-O, in which the ketolase gene is
expressed, were resuspended in 50 mM Tris-HCl, pH 7.5, and
disrupted in a French pressure cell at 95 MPa. Both homogenates were
treated with DNase (5 µg/ml) for 15 min on ice. One served as a
source of substrate and the other as a source of enzyme. The assay
consisted of 250 µl of each extract and 2 mM NADPH.
Incubation was for 15 h at 30 °C. The equivalent to 600 ng of
-carotene was provided by the pCAR16
crtX extract. The reaction
was terminated by addition of 2.5 ml of methanol. After heating to
60 °C, the carotenoids were extracted and separated by HPLC as
described above.
Synechocystis 6803 wild type and
crtO strains were cultured at 30 °C in BG11
and bubbled with a continuous stream of 1.5% (v/v) CO2 in
air under constant illumination (50 µE m
2
s
1). For the crtO
strain, the
medium was supplemented with kanamycin to a final concentration of 25 µg/ml.
When the cultures reached a density of 0.2 absorbance unit at 580 nm,
they were divided in two halves, one was maintained at 50 µE
m2 s
1 and the other was illuminated at 250 µE m
2 s
1. Oxygen evolution was
periodically determined at 30 °C upon irradiation with saturating
white light by using a Clark-type oxygen electrode. Chlorophyll was
determined as described previously (27).
Our interest in the biosynthetic pathway of carotenoids in
cyanobacteria led us to identify the genes coding for the enzymes phytoene desaturase (crtP) and phytoene synthase from
Synechocystis (28, 29) and -carotene desaturase
(crtQ) from Anabaena sp. PCC 7120 (25). In the
Synechocystis genome data base an ORF (slr0088) with
significant homology to bacterial type desaturase (crtI)
genes has been identified. As the Anabaena crtQ gene is homologous to crtI rather than to the plant or
cyanobacterial crtP genes (8), we decided to explore the
possibility that slr0088 was the Synechocystis
-carotene
desaturase.
The open reading
frame identified as slr0088 in the Synechocystis genome was
cloned by polymerase chain reaction and introduced in an expression
vector for E. coli to generate plasmid pTRCRT-O. E. coli cells carrying plasmid pTRCRT-O synthesized a protein of the
expected size (59 kDa) (not shown) upon induction with isopropyl--D-thiogalactopyranoside. To test the function
of the expressed protein, plasmid pTRCRT-O was introduced in E. coli in combination with several plasmids that allow the
accumulation of different carotenoids (Table I). In
E. coli cells that accumulate phytoene (containing
pACCRT-EB), 1-hydroxyneurosporene (containing pACCRT-EBIRc
and pRKCRT-C), and
-carotene (containing pACCRT-EBP), no desaturase
activity could be detected when the crtO gene product was
expressed. These results suggest that the crtO gene product is not a carotene desaturase, as are the enzymes encoded by
crtI and crtD, nor is it related to the
-carotene desaturase gene from Anabaena, because no
detectable desaturation products from phytoene,
-carotene, or
1-hydroxyneurosporene accumulated in E. coli cells that
express crtO. Alternatively crtO could be a carotene desaturase in Synechocystis that cannot be
functionally expressed in E. coli, even though the protein
is synthesized or that requires additional cofactors not present in
E. coli. However, when pTRCRT-O was introduced in E. coli cells that accumulate
-carotene due to the presence of
pACCRT
16crtX, two additional carotenoids were observed, echinenone
and canthaxanthin in lower amounts (Fig. 2). This result
indicates that crtO is a
-carotene ketolase. Previously
identified
-carotene ketolases produce the accumulation of
canthaxanthin with little or none of echinenone. However,
crtO produces the accumulation of much higher amounts of
echinenone than it does of canthaxanthin. This correlates with the
situation in Synechocystis that accumulates echinenone
rather than canthaxanthin.
In Vitro Ketolase Activity of the crtO Gene Product
We have
confirmed the in vivo complementation results by showing
that the crtO gene product can synthesize echinenone from -carotene in vitro. Cell homogenates from E. coli cells that express crtO can convert
-carotene
to echinenone at a conversion rate of 12% in the presence of NADPH
(Table II). Some formation of the di-keto derivative
canthaxanthin (2% conversion) was also observed. This indicates that
the specificity for
-carotene of the crude enzyme is not absolute.
The reaction products echinenone and canthaxanthin were positively
identified not only by co-chromatography with authentic standards but
also by their absorbance spectra (Fig. 3). NADPH was
required for activity.
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Inactivation of the crtO Gene
To test the function of
crtO in Synechocystis, we have inactivated the
crtO gene by insertion of a kanamycin resistance casette (Fig. 4A). The inactivated copy of
crtO could be completely segregated as tested by Southern
blot (Fig. 4B), and cells lacking a functional crtO gene seem to be viable. The carotenoid composition of
the crtO-deficient strain (crtO)
was compared with that of wild type Synechocystis (Fig.
5). Myxoxanthophyll, zeaxanthin, echinenone, and
-carotene were the main carotenoids identified in the wild type
strain. In the mutant strain echinenone was absent, while there were
wild type levels of the other carotenoids. No canthaxanthin was
detected either in the wild type or in the
crtO
strain, even when a different HPLC system
optimized for the separation of canthaxanthin from zeaxanthin (18) was
used. This result indicates that crtO is required for
echinenone biosynthesis in Synechocystis and, together with
the previous results, clearly demonstrates that crtO is a
-carotene ketolase that acts asymmetrically to produce echinenone as
the main product rather that of canthaxanthin.
Characterization of the crtO
The
previously described results show that we have been able to completely
segregate a strain lacking crtO, and that this strain is
deficient in echinenone biosynthesis. To study what could be the
functional role of echinenone, we have analyzed the behavior of the
crtO strain when incubated under either low
(50 µE m
2 s
1) or high (250 µE
m
2 s
1) light intensity. Both wild type and
crtO
strains have similar growth rates at low
and at high light intensity. The photosynthetic rate (as measured by
O2 evolution) of the crtO
strain
is also similar to that of wild type at either low or high light
intensity. Therefore incubation at high light intensity does not reveal
any significant differences between both strains regarding the measured
parameters.
The cyanobacterium Synechocystis sp. PCC 6803 is the
organism of choice for genetical studies. It is naturally
transformable, and genes can be easily inactivated by targeting and
homologous recombination. Furthermore, the complete sequence of the
Synechocystis genome is available (30). Several genes of the
carotenoid biosynthesis pathway have been identified in
Synechocystis. The availability of the complete genome
sequence gives us the opportunity to search for the so far unidentified
genes of this pathway in a rational way. Among the open reading frames
present in the Synechocystis genome, there is one with
significant homology to bacterial type phytoene desaturases (slr0088).
The only cyanobacterial -carotene desaturase characterized so far is
homologous to bacterial phytoene desaturases rather than to plant type
phytoene desaturases, therefore it was reasonable to expect slr0088 to
be the Synechocystis
-carotene desaturase. The results
presented in this work clearly indicate that slr0088 is a
-carotene
ketolase. This fact illustrates the risk of assuming functions for
unknown ORFs based solely on weak homologies and indicates that
functional studies are required to confirm the role of the expected
protein product.
The Synechocystis mutant strain lacking crtO does
not accumulate echinenone, but has normal levels of -carotene.
Therefore crtO is not required for the desaturation steps
necessary to produce
-carotene, excluding that crtO is
the functional
-carotene desaturase in Synechocystis. It
should be noted that crtO is closely linked and in the same
orientation as several other genes, possibly organized as an operon.
Therefore inactivation of crtO could affect the expression
of these other genes that could account for the phenotype observed.
However, the function of those other genes is hypothetically unrelated
to carotenoid biosynthesis (as deduced from sequence homologies), and
some of them are expected to be essential. In any case a polar effect
on genes placed downstream of crtO could not account for the
in vitro activity of the crtO gene product and
the complementation observed in E. coli.
As the plot in Fig. 6 clearly shows, crtO is
homologous to bacterial type phytoene desaturases as exemplified by the
Erwinia herbicola enzyme. The mechanism of carotene
ketolases is not known in detail, but the symmetrically acting enzyme
is most likely a dioxygenase.2 Although the
in vitro formation of echinenone by the
crtO-derived ketolase was carried out in cell homogenates, a
dependence of the reaction on NADPH was observed. This may be an
indication for a different reaction mechanism responsible for the
introduction of the keto group, involving a monooxygenase-type of
hydroxylation followed by dehydrogenation. crtO contains a
nucleotide binding site at the amino end similar to the one in phytoene
desaturases. This is most likely the site for the binding of the
cofactor NADPH.
The three symmetrically acting -carotene ketolases characterized so
far, two from bacteria and one from a green algae, are homologous among
themselves. They can be aligned, and four conserved regions have been
identified (15). They do not contain a nucleotide binding site at their
amino end. We have tried to identify in crtO regions of
similarity with any of the four conserved domains in ketolases but
there are no significant similarities (Fig. 6). Therefore
crtO is a completely different enzyme, phylogenetically related to carotene desaturases rather than to ketolases. In addition crtO has also a different mechanism of substrate
recognition, acting asymmetrically on
-carotene to introduce a keto
group on only one of the
-ionone rings. In other words,
crtO can use as substrate mainly
-carotene but echinenone
very poorly. The previously identified
-carotene ketolases do not
produce the accumulation of significant amounts of echinenone.
When crtO is expressed in E. coli, about 10% of
the ketocarotenoid produced is canthaxanthin. Therefore crtO
has not a strict specificity for -carotene but can, at a low rate,
introduce a keto group in the second ionone ring of echinenone to
generate canthaxanthin. However, this effect is due to the high level
expression of the ketolase and is restricted to the heterologous
environment in E. coli, because no significant canthaxanthin
accumulation is seen in Synechocystis.
The availability of a Synechocystis mutant strain deficient in the biosynthesis of echinenone will allow the study of the function of this carotenoid. It has been suggested that echinenone is located close to the reaction centers in the thylakoid membrane in a related cyanobacterium (31). However, our results indicate that the absence of echinenone has no effect on the growth rate or photosynthetic oxygen evolution at either low or high light. The specific role of echinenone in Synechocystis will require further investigation.
Carotene ketolases and their genes are of significant biotechnological
interest for the production of carotenoids of commercial interest (32).
The new -carotene ketolase described here has a different substrate
specificity of the previously known ketolases and therefore provides
additional flexibility in the design of biotechnological procedures for
the production of carotenoids of potential applied interest. The
Synechocystis
-carotene ketolase could be useful for
engineering of the biosynthesis of not only echinenone, but also other
asymmetric carotenoids such as 3-hydroxyechinenone, 3
-hydroxyechinenone, and adonixanthin when expressed in combination with a carotenoid hydroxylase gene (crtZ).