Noncyanobacterial bacteria and fungi use a single CrtI-type phytoene desaturase to carry out the four dehydrogenation steps producing lycopene.. We show here that the first three genes of
Trang 1production of lycopene in Myxococcus xanthus
Antonio A Iniesta1,2, Marı´a Cervantes1and Francisco J Murillo1
1 Departamento de Gene´tica y Microbiologı´a, Facultad de Biologı´a, Universidad de Murcia, Spain
2 Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, CA, USA
Carotenoids constitute one of the most widely
distri-buted and structurally diverse classes of natural
pig-ments, with important functions in photosynthesis,
nutrition, and protection against photooxidative
dam-age Carotenoids are ubiquitously found in bacteria,
fungi, algae, and plants Even though the end-products
of carotenoid biosynthesis are extremely diverse, a
gen-eral common pathway leading to the formation of
lycopene (red carotene), and cyclic b-carotene (yellow)
is observed in many organisms However, the nature
of the involved enzymes varies among different
organ-isms [1] Precursors for the synthesis of carotenoids are
derived from the general isoprenoid biosynthetic
path-way (along with a variety of other important natural
substances) [2], and start with the precursor farnesyl
diphosphate (Fig 1) The condensation of two geranyl-geranyl diphosphate (GGPP) molecules produces phy-toene, mostly in the cis conformation Generally, phytoene is dehydrogenated in four desaturation events, producing phytofluene, f-carotene, neurospo-rene, and lycopene, respectively, in that order (Fig 1)
On the basis of sequence homology, there are two unrelated groups of phytoene desaturases, CrtI-like and Pds-like Noncyanobacterial bacteria and fungi use a single CrtI-type phytoene desaturase to carry out the four dehydrogenation steps producing lycopene The Pds-type phytoene desaturase is found in plants, algae, and cyanobacteria, where it is known as CrtP Both Pds and CrtP converts phytoene into f-carotene
in two steps The two remaining desaturation events
Keywords
carotenes; CrtI; dehydrogenation;
isomerization; phytoene
Correspondence
A A Iniesta, Beckman Center B355, 279
Campus Drive, Stanford, CA 94305, USA
Fax: +1 650 725 7739
Tel: +1 650 723 5685
E-mail: ainiesta@stanford.edu
F J Murillo, Facultad de Biologı´a,
Universidad de Murcia, Campus de
Espinardo, Murcia 30071, Spain
Fax: +34 957 355 039
Tel: +34 957 355 024
E-mail: francisco.murillo@juntadeandalucia.es
(Received 10 May 2007, revised 26 June
2007, accepted 28 June 2007)
doi:10.1111/j.1742-4658.2007.05960.x
In Myxococcus xanthus, all known carotenogenic genes are grouped together in the gene cluster carB–carA, except for one, crtIb (previously named carC) We show here that the first three genes of the carB operon, crtE, crtIa, and crtB, encode a geranygeranyl synthase, a phytoene desatur-ase, and a phytoene synthdesatur-ase, respectively We demonstrate also that CrtIa possesses cis-to-trans isomerase activity, and is able to dehydrogenate phytoene, producing phytofluene and f-carotene Unlike the majority of CrtI-type phytoene desaturases, CrtIa is unable to perform the four dehy-drogenation events involved in converting phytoene to lycopene CrtIb, on the other hand, is incapable of dehydrogenating phytoene and lacks cis-to-trans isomerase activity However, the presence of both CrtIa and CrtIb allows the completion of the four desaturation steps that convert phytoene
to lycopene Therefore, we report a unique mechanism where two distinct CrtI-type desaturases cooperate to carry out the four desaturation steps required for lycopene formation In addition, we show that there is a difference in substrate recognition between the two desaturases; CrtIa dehydrogenates carotenes in the cis conformation, whereas CrtIb dehydro-genates carotenes in the trans conformation
Abbreviations
CTT, casitone ⁄ Tris; GGPP, geranylgeranyl diphosphate.
Trang 2are carried out by other desaturases, Zds in plants and
algae, and CrtQ in cyanobacteria, which are related to
the Pds-type desaturases In organisms with a
CrtI-type desaturase, the cis-to-trans isomerization
convert-ing cis-phytoene into trans-phytoene is also performed
by CrtI However, the Pds-type desaturases lack
iso-merase activity, and a CrtI-like enzyme (CrtISO in
plants and CrtH in cyanobacteria) is used for
cis-to-transisomerization, converting cis-lycopene into
trans-lycopene [1] A few exceptions to these generalizations about the dehydrogenation or the isomerization pro-cess have been reported [3–7]
Myxococcus xanthus is a Gram-negative bacterium that produces carotenoids in response to blue light [8,9] and the presence of copper [10] M xanthus accu-mulates mainly esterified carotenoids such as ester of myxobacton (final carotenoids), and all-trans-phytoene (Table 1) [11,12] In M xanthus, all known and pre-dicted carotenoid biosynthesis genes are grouped together in the carB–carA gene cluster [13], except for carC (hereafter renamed crtIb) (Fig 2) [14] The first six ORFs of carB–carA are located in the carB operon, and the rest are at the carA locus We characterize here the first three genes from the M xanthus carB operon, and show that their products, CrtE, CrtIa, and CrtB, possess GGPP synthase, phytoene desatur-ase and phytoene synthdesatur-ase activity, respectively In addition, we show that M xanthus uses two
desaturas-es, CrtIa and CrtIb, to complete the four desaturation processes required to transform phytoene into lyco-pene This is the first report of such unusual and unique collaboration between two CrtI-like
desaturas-es, providing additional evidence for the wide plasticity
of carotenoid biosynthesis Finally, we also show here that CrtIa possesses cis-to-trans isomerase activity, and recognizes substrates in the cis conformation, whereas CrtIb has similar desaturase activity but recognizes substrates in the trans conformation
Results
Phytoene isomerization
In M xanthus, a mutant with a transposon insertion
in the coding region of crtIb accumulates all-trans-phy-toene (93%) and phytofluene (7%) [11] Therefore, CrtIb is required for phytoene dehydrogenation steps producing lycopene, but is dispensable for the 15-cis-phytoene to all-trans 15-cis-phytoene isomerization This sug-gests the existence of a second enzyme to carry out the phytoene isomerization and, possibly, the first phyto-ene dehydrogenation step leading to phytofluphyto-ene The product encoded by crtIa showed high similarity to the CrtI-type phytoene dehydrogenase from fungi and noncyanobacterial bacteria [13], including the previ-ously described CrtIb of M xanthus [14] An M xan-thus mutant with a transposon insertion in crtIa is unable to produce carotenoids, indicating an early role
in carotenogenesis The crtIa insertion could have a polar effect on the expression of downstream genes [12] To clarify the possible function of crtIa in the carotenoid synthesis pathway, we generated an
Fig 1 Schematic of the initial carotenoid biosynthesis pathway in
M xanthus The addition of an isopentenyl diphosphate unit (IPP)
to farnesyl diphosphate generates a GGPP molecule The
conden-sation of two GGPPs results in the synthesis of 15-cis-phytoene.
After its isomerization to the all-trans conformation, the phytoene
undergoes four dehydrogenation steps, producing phytofluene,
f-carotene, neurosporene, and lycopene, respectively Dashed
circles represent the site where the desaturation events take place.
Trang 3in-frame deletion of crtIa in M xanthus strain MR151.
This strain contains a mutation, carR3, which renders
the expression of the carB operon independent of
external stimulation [10,15] Colonies of the
MR151-derived strain with the crtIa deletion (MR841)
com-pletely lose the red color typical of colonies from the
parental strain, suggesting that the absence of CrtIa
blocks the synthesis of colored carotenoids The
possi-ble accumulation of carotenoid precursors by MR841
was analyzed by carotene extraction and
chromatogra-phy on an alumina column Only the presence of chromatogra-
phy-toene was detected (Fig 3A and Table 1) The pattern
of absorbance of this carotene is similar to that shown
by 15-cis-phytoene and different from that shown by
all-trans-phytoene (Fig 3B) Therefore, CrtIa is
required, at least, for the isomerization of
15-cis-phyto-ene to all-trans-phyto15-cis-phyto-ene
Two CrtI-type enzymes cooperate in phytoene
dehydrogenization to lycopene
In M xanthus, a transposon insertion in crtE or in
crtB of the carB operon prevents the accumulation of
Table 1 Carotenoid content of several strains of M xanthus and E coli with plasmids bearing different carotenogenic genes Mx,
M xanthus; Ec, E coli; ND, not detected.
Host Strain
Genotype (Mx)
Plasmids (Ec)
Carotenoid content (lgÆg)1of protein)a
Phytoene Phytofluene f-carotene Neurosporene Lycopene
Final carotenoids, esterified
a The average of three or more independent determinations is given b In the presence of light.
Fig 2 carB–carA gene cluster and crtIb The carB operon is transcribed by the light-inducible promoter P B and contains the first six genes of the carB–carA cluster The carA operon includes the last five genes of the cluster, and its transcription is driven by a light-independent promoter, PA.
Fig 3 The absence of CrtIa blocks the cis-to-trans isomerization
of phytoene (A) Absorption spectra in hexane of carotenoids extracted from strain MR841 (DcrtIa and carR3), showing only the presence of cis-phytoene (B) Absorption spectra in hexane of all-trans-phytoene produced by M xanthus (continuous line) and of 15-cis-phytoene produced by the fungus P blakesleeanus (dashed line), taken from Martinez-Laborda et al [11] All-trans-phytoene presents three well-defined peaks (276 nm, 286 nm, and 297 nm), unlike the spectrum of 15-cis-phytoene, which shows a maximum
at 286 nm and two inflections at 276 nm and 297 nm [46].
Trang 4carotenoids or their C40 precursors [11,12] The amino
acid sequences of CrtE and CrtB showed significant
similarities, respectively, to the GGPP and phytoene
synthase from bacteria, fungi, and plants [13] To
confirm the predicted enzyme activities of CrtE and
CrtB, the corresponding M xanthus genes were both
expressed in Escherichia coli, which lacks carotenogenic
genes E coli was transformed with a plasmid
(pFD6) harboring crtE, crtIa and crtB from the carB
operon, to generate strain FD6 In pFD6, the
expres-sion of the crt genes was under the control of the
arti-ficial constitutive promoter Part-1-2 Strain FD6 was
grown in LB medium to stationary phase, and
carote-noids were purified from the cell extract and analyzed
The absorption spectra of the extract showed the
pres-ence of all-trans-phytoene, phytofluene and f-carotene,
at decreasing concentrations (Table 1) Thus, CrtE,
CrtB and CrtIa are sufficient to carry out the synthesis
of phytoene, its isomerization, and the first two
phyto-ene dehydrogenation steps up to f-carotphyto-ene
produc-tion Similar results were obtained when crtIa was
coexpressed in E coli with the crtE and crtB genes
from Rhodobacter (data not shown) This confirms that
CrtE and CrtB have GGPP and phytoene synthase
functions, respectively, and that CrtIa, besides its
isomerase activity, is responsible for the double
dehy-drogenation of phytoene up to f-carotene CrtIa,
how-ever, seems unable to drive the rest of the desaturation
events that produce neurosporene and lycopene
As mentioned above, crtIb is somehow required for
the dehydrogenation steps converting phytoene to
lyco-pene However, the expression of crtIb in the E coli
strain producing cis-phytoene, using the crtE and crtB
genes from Rhodobacter, did not transform the initial
cis-phytoene at all (data not shown) In order to
deter-mine the specific function of CrtIb, we analyzed the
carotenoids accumulated by an M xanthus crtIb
dele-tion mutant, which also carries the carR3 mutadele-tion
(MR728) [16] MR728 was shown to accumulate
all-trans-phytoene, phytofluene, and f-carotene, in
decreas-ing concentrations (Table 1) This pattern of carotene
accumulation, notably similar to that resulting from the
heterologous expression of crtIa in E coli (strain FD6
in Table 1), indicates that CrtIb is acting at one or two
of the last dehydrogenation steps in lycopene
produc-tion, after the CrtIa isomerase and desaturase activities
The low ratio of f-carotene to phytofluene found in
both the M xanthus crtIb deletion mutant and the
E colistrain expressing crtIa indicates a low efficiency
of desaturation by CrtIa in the absence of CrtIb A
high relative accumulation of all-trans-phytoene is not
unusual, as it is also seen in an extract from an M
xan-thus wild-type strain, which, however, produces only
traces of partially desaturated phytoene products (Table 1) Altogether, the accumulated evidence sug-gests novel cooperation between two CrtI-type desatu-rases in the dehydrogenation of phytoene to lycopene
In order to determine whether both CrtIa and CrtIb are necessary and sufficient for the complete dehydro-genation to lycopene, we generated E coli strain FD9 This strain contains plasmid pFD9, which bears the
M xanthus genes crtE, crtIa, crtB and crtIb under the control of the Part-1-2 promoter Colonies from strain FD9 developed a very strong red color on LB agar plates Carotenoid analysis showed that FD9 accumu-lates high amounts of lycopene almost exclusively (Table 1) This is also seen in an M xanthus mutant defective in lycopene cyclization, where lycopene is more abundant than other desaturated precursors [12] Therefore, in M xanthus, CrtIa and CrtIb are both required to complete the four phytoene dehydrogeniza-tion steps necessary for lycopene formadehydrogeniza-tion Moreover, the exclusive accumulation of lycopene suggests some kind of cooperation between both CrtI-type dehydro-genases for the efficient processing of the partially dehydrogenated intermediate substrates
Different isomeric substrates for each dehydrogenase
The requirement in M xanthus for two CrtI-type pro-teins to carry out the four dehydrogenation steps involved in converting phytoene to lycopene is atypical
in the biogenesis of carotenoids The precise dehydro-genase activity of CrtIa and CrtIb could be due to dif-ferential substrate recognition, based on the substrate desaturation state, on its isomer conformation, or both To discriminate between these possibilities, we expressed crtIa or crtIb in E coli containing plasmid pACCRT-EBP This plasmid harbors the crtE and crtB genes from Erwinia uredovora and the gene crtP from the cyanobacterium Synechococcus PCC7942 Carotene analysis of extract from E coli with pACCRT-EBP (FD100) identified cis-f-carotene as a major carotene, and trace amounts of all-trans-f-caro-tene and cis-phytoene [5] The expression of crtIa in this strain resulted in the accumulation of the four de-hydrogenated phytoene derivatives phytofluene, f-caro-tene, neurosporene, and lycopene (FD101 in Table 1)
On the other hand, the same strain expressing crtIb produced f-carotene, neurosporene and lycopene, all in low amounts, with no phytofluene being detected (FD102 in Table 1) These data seem to indicate that CrtIa is able to carry out two consecutive dehydro-genation events on carotenes in the cis conformation: the conversion of cis-phytoene into phytofluene and
Trang 5f-carotene, and that of cis-f-carotene into
neurospo-rene and lycopene However, CrtIb appears to only
transform the small amounts of trans-f-carotene into
neurosporene and lycopene
To further confirm the hypothesis of the substrate
isomer specificity of CrtIa and CrtIb, we expressed
crtIa or crtIb in an E coli strain producing
trans-neu-rosporene [5] This strain (MR2301) contains plasmid
pACCRT-EBI bearing the crtE and crtB genes from
Er uredovora and crtI from Rhodobacter capsulatus,
which encodes a dehydrogenase that is capable of
transforming cis-phytoene into trans-neurosporene but
is unable to catalyze efficiently the fourth desaturation
step to produce lycopene The expression of crtIa
in the trans-neurosporene-producing strain did not
change the dehydrogenation state of the accumulated
carotenes (FD103 in Table 1) However, the expression
of crtIb caused the almost complete transformation of
trans-neurosporene into lycopene (FD104 in Table 1)
All of these results suggest a scenario where CrtIa and
CrtIb specific substrate recognition properties depend
on the cis–trans conformation of the substrate, rather
than on its desaturation state
Discussion
A variety of isoprenoid compounds, such as
choles-terol, dolichol, ubiquinone, coenzyme Q, isoprenoid
quinines, sugar carrier lipids, and carotenoids, are
syn-thesized by polyprenyl synthases in eukaryotic and
prokaryotic organisms Two distinct types of
evolu-tionarily conserved prenyltransferases, CrtE and CrtB,
catalyze the early reactions of carotenoid biosynthesis
from farnesyl diphosphate to phytoene [2] As
pre-dicted from sequence alignments [13], we report here
that the crtE and crtB genes from the M xanthus carB
operon encode enzymes with GGPP and phytoene
syn-thase activity, respectively After phytoene synthesis,
this carotene undergoes several desaturation events
(Fig 4) In M xanthus, an enzyme similar in sequence
to the CrtI-type phytoene dehydrogenases, previously
called CarC [11,14] and referred to here as CrtIb, was
shown to be involved in carotenoid biosynthesis The
crtIb gene is not linked to the carotenogenic carB
operon, which contains a gene predicted to encode a
second phytoene dehydrogenase [13], referred here as
CrtIa Interestingly, CrtIa is unable to catalyze the
four desaturations necessary for lycopene production
in the absence of CrtIb, and instead it leads to the
accumulation of the intermediates phytofluene and
f-carotene in decreasing amounts On the other hand,
CrtIb is itself incapable of introducing any double
bonds into phytoene We have demonstrated that a
unique collaboration between CrtIa and CrtIb is used
to successfully introduce four double bonds into phy-toene To our knowledge, this is the first case reported where two CrtI-type desaturases function together to generate lycopene
Although changes of CrtI-type enzymes producing loss or gain of dehydrogenation activities are not frequent, some cases have been reported In the bacteria
R capsulatus and R sphaeroides, CrtI introduces three double bonds into phytoene, producing neurosporene, but lacks the capacity to introduce the fourth double bond needed to produce lycopene [17,18] A fifth dehydrogenation step is carried out by a CrtI-type desaturase from the fungus Neurospora crassa, produc-ing 3,4-dihydrolycopene [19] Other variations in the lycopene biosynthesis pahtway have been reported
in some cyanobacteria species The cyanobacteria Anabaena PCC7120 converts the f-carotene produced
by CrtP into lycopene using a CrtI-type desaturase instead of the typical cyanobacterial CrtQ [5] Like
M xanthusCrtIb, the Anabaena CrtI-type desaturase is unable to introduce any double bonds into phytoene In the cyanobacterium Gloebacter, a single CrtI-type desat-urase is responsible for the four dehydrogenation steps required for lycopene formation, and homologs of crtP and crtQ are not found in its genome [6,7] It has been proposed that in the course of evolution, cyanobacteria acquired a gene encoding an unrelated CrtI-type desat-urase, which was duplicated, resulting in crtP and crtQ These two genes would be the ancestors of pds and zds from algae and plants [1] The lack of CrtP and CrtQ in Gloebacter may be related to its evolutionary distance from other groups of cyanobacteria [20] This organism
Fig 4 General representation of the pathway for synthesis of trans-lycopene from cis-phytoene and the enzymes involved in dif-ferent organisms, including M xanthus.
Trang 6is thought to retain traces of the ancestral properties of
cyanobacteria [4]
The formation of b-carotene is one of the most
com-mon steps in the synthesis of carotenoids It requires the
cyclization of lycopene to ionone end-groups Lycopene
in the cis conformation cannot be cyclized, due to its
steric arrangement, and therefore it must be synthesized
in the all-trans configuration, or be converted to that
form [1,21] In organisms that use Pds⁄ CrtP-type and
Zds⁄ CrtQ-type desaturases, where the dehydrogenation
steps are performed on carotenes in the cis
confor-mation, the final isomerization from cis-lycopene to
all-trans-lycopene is performed by a CrtI-type enzyme
called CrtISO in algae and plants, and CrtH in
cyano-bacteria [21–24] (Fig 4) cis-to-trans isomerization can
be also enhanced by light [5,25,26] However, in all
organisms that use a CrtI-type desturase, the
cis-to-trans isomerization is associated with the desaturation
processes producing trans-phytoene [6,27,28] (Fig 4)
In the case of Anabaena, the cis-to-trans isomerization
is carried out on f-carotene, instead of on phytoene, by
its CrtI-type f-carotene dehydrogenase [5] It is not clear
why two different biosynthetic pathways for lycopene
exist in nature The discovery of the biosynthetic
enzymes CrtP, CrtQ and CrtH in the green sulfur
bacte-rium Clorobium tepidum, an obligate photoautotroph,
suggests that these enzymes originated from a common
ancestor of modern-day green sulfur bacteria and
cyanobacteria [3] The fact that organisms with
CrtP⁄ CrtQ ⁄ CrtH also contain type I photosynthetic
reaction centers, and that cis carotenoids appear to
perform important functions in these reaction centers,
suggests a link between photosynthesis and the presence
of cis carotenoids [3,29] We show here that in
M xanthus, the cis-to-trans isomerization is catalyzed
by CrtIa CrtIa recognizes phytoene and also f-carotene
in the cis conformation In addition, CrtIa is unable
to dehydrogenate trans-neurosporene, indicating its
preference for carotenes in the cis conformation
However, CrtIb seems unable to recognize substrates
in the cis conformation, but can transform
trans-neurosporene into lycopene Therefore, we propose
that, in M xanthus, the whole biosynthetic process
from cis-phytoene to trans-lycopene is carried out by
the cooperation of two CrtI-type desaturases, CrtIa and
CrtIb They may form a protein complex, where CrtIa
recognizes cis-phytoene, isomerizes it to trans-phytoene,
and dehydrogenates it twice to produce
trans-f-caro-tene This last carotene would be then transferred
directly from CrtIa to CrtIb, and this desaturase would
introduce two new double bonds, forming
trans-lycopene The high water insolubility of carotenoids,
and the improvement of the desaturase efficiency of
CrtIa in the presence of CrtIb, suggest a mechanism for the direct transfer of substrates from CrtIa to CrtIb
In the absence of CrtIb, CrtIa would be blocked when bound to f-carotene The idea of a linear assembly chain for carotenoid synthesis was proposed years ago,
on the basis of work with the fungus Phycomyces blakesleeanus[30,31]
Why M xanthus uses two CrtI-type desaturases for the dehydrogenation of phytoene to lycopene is cer-tainly an unanswered question One possibility is that having two unlinked desaturase genes provides more regulatory options The crtIa gene is inserted in the carB operon, which is driven by a light-activated pro-moter [15] The expression of crtIb is also activated by light, but through a tight mechanism that operates only when the cells have reached the stationary phase
or are starved of a carbon source [14] This may have advantages if carotenoids are synthesized only when needed, in stationary phase but not before, leaving the isoprenoid components for metabolic uses in the growth phase In the presence of light, the carotenoid biosynthetic machinery would be present but blocked, waiting for the last enzymatic element, CtrIb, which reaches a very high level soon after the cell’s entrance into the stationary phase [14] This scenario would be possible if, in the course of the evolution, CrtIa lost its capacity to perform the two final desaturation steps of the four catalyzed by a typical CrtI-type enzyme, and these activities were taken over by a second desaturase, CrtIb An obvious idea is that the crtIb gene arose by duplication of the original, single M xanthus crtI gene However, the CrtIb protein is more closely related (46% identity) to the f-carotene desaturase from Anabaena PCC7120 [5] Therefore, a possible horizon-tal gene transfer event from a cyanobacterium to myxobacteria cannot be ruled out
Experimental procedures
Bacterial strains, media, and transducing phages
E coliDH5a [32] was used for cloning and carotenoid pro-duction, and E coli MC1061 [33] for transducing plasmids into M xanthus with the coliphage P1clr100Cm (hereafter called P1) [34] M xanthus strains, derived from MR151 [35], were grown in casitone⁄ Tris (CTT) rich medium [36], and E coli was grown in LB rich medium [37] When nec-essary, 40 lgÆmL)1kanamycin was added to CTT medium
Plasmid and strain construction
Standard protocols were followed for DNA manipulation [37] PCR-derived clones were generated using Pfu DNA
Trang 7polymerase, and sequenced to verify the absence of
PCR-generated mutations
To generate an M xanthus crtIa deletion mutant, we
digested plasmid pMAR161 with MluI, and the biggest
fragment was autoligated, resulting in plasmid pMAR162
Plasmid pMAR161 is a pUC9 vector [38] that contains a
4.3 kb fragment including the first three genes of the carB
operon (Fig 2) and a part of the fourth gene Plasmid
pMAR162 is similar to pMAR161 but with deletion of a
fragment encoding 160 amino acids of the coding region of
crtIa The M xanthus DNA fragment from pMAR162 was
cloned into plasmid pDAH160 [39], which carries a
kana-mycin resistance gene and the incompatibility region of P1
for transferring the plasmid from E coli to M xanthus
by P1-specialized transduction The resulting plasmid,
pMAR164, was transduced into M xanthus MR151, where
it integrated by homologous recombination to generate a
kanamycin-resistant merodiploid We grew this merodiploid
in CTT without kanamycin to allow a second
recombina-tion event that causes the loss of the kanamycin resistance
marker, generating kanamycin-sensitive colonies, either with
a wild-type crtIa or with the crtIa deletion The presence of
this deletion was confirmed by Southern blot analysis using
as a template a 4.3 kb RcaI-digested fragment from
pMAR161, and M xanthus genomic DNA digested with
NcoI The strain with the crtIa deletion was named
MR841
To make plasmid pFD3, a DNA fragment bearing crtE,
crtIa, and crtB, which also includes the ribosomal-binding
site upstream of crtE, was PCR-amplified using pMAR161
as template and the oligonucleotides ORF1-3 (5¢-GGT
TCTTCGGAGGAAAGACATATGGCACTCACGCTTCC
C-3¢) and ORF3-2 (5¢-CCGAAGCTCCGTCTAGATTCC
CTCGCCACGC-3¢) as primers The fragment was digested
by NdeI and XbaI, and cloned into the expression vector
pUC19 [40] An artificial constitutive-expression promoter,
Part-1-2, was inserted just before crtE in plasmid pFD3,
generating plasmid pFD6 and strain FD6 This promoter
was generated by hybridization of two complementary
oli-gonucleotides, Part-1 (5¢-AGCTTGACAGGCCGGAATAT
TTCCCTATAATGCGCTGCA-3¢) and Part-2 (5¢-GCG
contain the E coli RNA polymerase r70consensus binding
site, TTGACA, in position) 35 and TATAAT in position
) 10 [41], and was cloned in vectors digested with HindIII
and PstI The sequence between the) 35 and ) 10 positions
was based on the highly expressed promoter 1 of Es coli
rrnA, which encodes ribosomal RNA [42]
A DNA fragment containing the crtIb coding region plus
12 and 26 additional bp upstream and downstream,
respec-tively, was PCR-amplified using pMAR202 as a template
[14], and CRTI-1a (5¢-GTGGGATTCCGTTCATCTAGAT
ACCGGAGGGCCTTGGC-3¢) and CRTI-2 (5¢-GAGCGC
GCCACTGGATCCCGCGGCGCTCACC-3¢) as primers
The fragment obtained was cloned, after digestion with
XbaI and BamHI, into vector pUC19, resulting in plasmid pFD1 To generate plasmid pFD9 (present in E coli strain FD9), plasmid pFD1 was digested with XbaI and BamHI, and the crtIb fragment was cloned into pFD6
To generate plasmid pMAR183, a DNA fragment was amplified by PCR using pMR161 as a template, and ORF2-1 (5¢-ACCGCGCCGCCTGCAGATCCCATGAGT GCATCG-3¢) and ORF2-2 (5¢-ACCAGCGCCTTGTCGA CAGGCGGGC-3¢) as primers This fragment was digested with PstI and SalI to generate a product containing the crtIa coding region plus 2 and 14 bp upstream and downstream, respectively; this was then cloned into vector pUC9
To generate plasmid pFD39, the same crtIb fragment used for pFD9 was PCR-amplified using pMAR202 as a template, and CRTI-3 (5¢-GTGGGATTCCGTTCATCGA TATACCGGAGGGCC-3¢) and CRTI-2 as primers After digestion by ClaI and BamHI, this fragment was cloned into vector pACYC184 [43], to create plasmid pFD24 An artificial constitutive-expression promoter, Par-5-6, was inserted just before crtIb in plasmid pFD24, generating plasmid pFD26 The Part-5-6 promoter was generated
by hybridization of two complementary oligonucleotides, Part-5 (5¢-CTAGATTGACAGGCCGGAATATTTCCCTA TAATGCGCAT-3¢) and Part-6 (5¢-CGATGCGCATTAT
con-tain, like the Part-1-2 promoter, the E coli RNA polymer-ase r70 consensus binding site, and was cloned in vectors digested with XbaI and BamHI Plasmid pFD26 was digested by XbaI and BamHI, and the Part-5-6-crtIb frag-ment was cloned into vector pBJ114 [44], resulting in plas-mid pFD39
Carotenoid extraction and analysis
E coli was grown in LB medium to stationary phase, and
1 mL of this culture was inoculated into 100 mL of LB medium and incubated at 37C for 12 h FD100, FD101 and FD102 E coli strains were supplemented with isopro-pyl thio-b-d-galactoside (0.5 mm) to increase expression of crtP, and incubated at 28C for 48 h [5] In the case of
M xanthus, 100 mL of CTT was inoculated with 1 mL of culture in stationary phase, and incubated at 33C until this culture reached stationary phase Additional experi-mental procedures were identical for all cultures A 1.5 mL aliquot was taken for protein assay [15], and the remaining volume was centrifuged at 15 700 g using an Eppendorf 5415D 24-place rotor for 1.5–2.0 ml tubes (Eppendorf AG, Hamburg, Germany) to pellet the cells The pellet was stored at) 20 C until analysis
The pellet was resuspended in 20 mL of methanol by vigorous shaking for 15 min, and then centrifuged at
27 200 g using a Beckman J2-21 centrifuge and JA-20 rotor (GMI Inc., Albertville, MN, USA) and re-extracted until the sample was totally colorless An equal volume of
Trang 8light petroleum (40–60C) was added to the methanol
extract The two-phase sample was vigorously shaken for
5 min, and the upper, light petroleum phase was removed
Three further extractions with light petroleum were
per-formed, and the fractions were pooled and concentrated
under vacuum Carotenes were resuspended in 1–10 mL of
hexane, and identified by their absorption spectra and
quantified by their extinction coefficient [45] In some
cases, the concentrated extract was resuspended in 1 mL
of light petroleum and chromatographed on a Brockman
grade III deactivated alumina column [45], which was
developed using light petroleum with increasing quantities
of acetone When chromatographed in activated alumina,
the cis-phytoene accumulated by M xanthus strain
MR841 (Fig 3A) behaves like 15-cis-phytoene extracted
from the fungus P blakesleeanus, as described in detail in
Martinez–Laborda et al [11] The separated carotenes
were collected, concentrated and resuspended in hexane
for their analysis All manipulations of carotenoids were
carried out in the dark at 4C The same carotenes were
always detected in various independent analyses of the
same strain, although some quantitative differences were
observed, particularly in the heterologous expression
experiments
Acknowledgements
We thank Jose´ A Madrid for technical assistance, and
Dr Gerhard Sandmann and Dr Agustı´n Vioque for
providing plasmids and strains This work was
supported by the Spanish Ministerio de Educacio´n
y Cultura (grant PB96-1096 and fellowship to
M Cervantes), Ministerio de Ciencia y Tecnologı´a
(grant BMC2000-1006), and Fundacio´n Se´neca
(fellow-ship to M Cervantes)
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