Biotechnology and Bioprocess Engineering 2007, 12: 312-317
1 Department of Microbiology, Pukyong National University, Busan 608-737, Korea
2 Department of Biomaterial Control, Dong-Eui University (BK 21 program), Busan 614-714, Korea
3 Korea BIO-IT Foundry Center, Pusan National University, Busan 609-735, Korea
4 Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Korea
^Äëíê~Åí= Carotenoids are important natural pigments produced by many microorganisms and plants. We have previously reported
the isolation of a new marine bacterium, m~ê~ÅçÅÅìë=Ü~ÉìåÇ~Éåëáë, which produces carotenoids, mainly in the form of
astaxanthin. The astaxanthin biosynthesis gene cluster, consisting of six carotenogenic genes, was cloned and character-
ized from this organism. Individual genes of the carotenoid biosynthesis gene cluster were functionally expressed in bëJ
ÅÜÉêáÅÜá~=Åçäá and each gene product was purified to homogeneity. Their molecular characteristics, including enzymatic ac-
tivities, were previously reported. Here, we report cloning the genes for crtE, crtEB, crtEBI, crtEBIY, crtEBIYZ, and crtEBI-
YZW of the mK=Ü~ÉìåÇ~Éåëáë carotenoid biosynthesis genes in bK=Åçäá and verifying the production of the corresponding
pathway intermediates. The carotenoids that accumulated in the transformed cells carrying these gene combinations were
analyzed by chromatographic and spectroscopic methods. © KSBB
hÉóïçêÇëW=~ëí~ñ~åíÜáå, Å~êçíÉåçáÇ, ÉñéêÉëëáçå, Paracoccus haeundaensis, éáÖãÉåí=
Carotenoids are natural lipid-soluble pigments, produced
primarily within bacteria, algae and plants, that have recently
attracted increased attention because of their beneficial ef-
fects on human health, including their function as antioxi-
dants [1,2], which are involved in cancer prevention [3-5],
and enhancing immune responses [6-8]. The area of carote-
noid research is therefore extremely important from both
fundamental and applied perspectives, and extensive studies
have been conducted on the general aspects of the chemical
structures, physical and biochemical properties, biosynthetic
and molecular genetics, and biotechnological applications of
Many carotenoid biosynthesis genes have been cloned and
characterized from various organisms and the functions of
the gene products have been determined [12-19]. In recent
years, the individual carotenoid biosynthesis genes have
most frequently been used to study carotenoid biosynthesis
[9,20-22], and the carotenoid biosynthetic pathways have
Tel: +82-51-620-6366 Fax: +82-51-611-6358
been elucidated through analyses of the pigments accumu-
lated in E. coli cells carrying various combinations of the
carotenoid biosynthetic genes [17,23] or through in vitro
assays of the gene products synthesized in E. coli [24-27]. E.
coli is one of the most popular host organisms for the high-
level production of recombinant proteins [28,29]. The func-
tions of many carotenoid biosynthesis genes from a variety
of carotenogenic organisms such as bacteria  as well as
higher plants [31-33] have been studied using transformed E.
coli. These genes are easily expressed in E. coli cells and
their recombinant proteins utilize suitable substrates for ca-
rotenoid biosynthesis within the cells.
The functions of the individual gene products involved in
the astaxanthin biosynthesis pathways have been extensively
studied. Biosynthesis of carotenoids includes the formation
of geranyl geranyl pyrophosphate (GGPP) from farnesyl
pyrophosphate (FPP) by a GGPP synthase (CrtE; encoded
by a crtE gene), the formation of phytoene from GGPP by a
phytoene synthase (CrtB; crtB gene), the formation of lyco-
pene from phytoene by a phytoene dehydrogenase (CrtI; crtI
gene), and the formation of β-carotene by a lycopene β-
cyclase (CrtY; crtY gene). In the final stages, β-carotene can
Biotechnol. Bioprocess Eng. PNP=
q~ÄäÉ=NK Oligonucleotides used in this study
Name Nucleotide sequence Remarks
CrtW-1 5′-CCATATGAGCGCACATGCCCTG-3′ Primer for Åêít, Forward (kÇÉI site)
CrtZ-1 5′-GCATATGACCAATTTCCTGATC -3′ Primer for Åêíw, Forward (kÇÉI site)
CrtY-1 5′-GCATATGACCATGACGTGCTG-3′ Primer for Åêív, Forward (kÇÉI site)
CrtI-1 5′-TCATATGAACGCCCATTCGC-3′ Primer for Åêíf, Forward (kÇÉI site)
CrtB-1 5′-GCATATGAGCGATCTGGTCCTG-3′ Primer for Åêí_, Forward (kÇÉI site)
CrtB-3 5′- CTCGAGCCTAGACGTGATGCGG-3′ Primer for Åêí_, Reverse (uÜçI site)
CrtE-1 5′-CCATATGAGACGAGACGTCAA-3′ Primer for Åêíb, Forward (kÇÉI site)
CrtE-3 5′-GCCGCCTCGAGTCTAGGCGC-3′ Primer for Åêíb, Reverse (uÜçI site)
T7-1 5′-CTCGAGTAATACGACTCACTATA-3′ Primer for Åêíb, Forward (uÜçI site)
cáÖK=NK Schematic of the carotenoid biosynthesis pathways from
farnesyl pyrophosphate (FPP) to astaxanthin.
be converted to astaxanthin using only two enzymes: a β-
carotene ketolase (CrtW; crtW gene) and a β-carotene hy-
droxylase (CrtZ; crtZ gene). The astaxanthin biosynthesis
pathway is summarized in Fig. 1.
We previously reported the isolation of a new marine bac-
terium, Paracoccus haeundaensis, which produces carote-
noids, mainly in the form of astaxanthin  and described
the cloning and sequence analysis of genes encoding the
astaxanthin biosynthetic enzymes from this organism .
All six genes of the astaxanthin biosynthesis gene cluster of
P. haeundaensis (crtW, crtZ, crtY, crtI, crtB, and crtE, which
contain 726, 486, 1158, 1503, 912, and 879 base pairs, re-
spectively) are necessary for astaxanthin biosynthesis, and
those genes were cloned and characterized . Individual
carotenoid biosynthesis genes of P. haeundaensis were func-
tionally expressed in E. coli and each gene product was puri-
fied to homogeneity. Their molecular characteristics, includ-
ing enzymatic activities, were reported . Detailed studies
of the involvement of the astaxanthin biosynthetic enzymes
on carotenoid biosynthesis, however, have not yet been con-
ducted. In order to elucidate the mechanism responsible for
controlling the astaxanthin biosynthetic pathway and the
intracellular carotenoid concentration, it would therefore be
necessary to conduct a comparative analysis of the structure,
expression, and function of the astaxanthin biosynthesis
genes through various combinations of these genes.
To create the expression plasmid for the genes containing
both the crtB and crtE genes, each gene was amplified by
PCR using a pair of gene-specific oligonucleotides (Table 1)
with the plasmid pCR-XL-TOPO-Crt-full as a template,
which carries the full-length astaxanthin biosynthesis gene
cluster . The amplified fragment was subcloned into the
vector pGEM-T (Promega, Madison, WI, USA). The sub-
cloned plasmid was digested with NdeI and XhoI restriction
enzymes, and the excised fragment was then ligated into the
expression plasmid pET44-a(+) vector. The resulting plas-
mids carrying an individual gene of the carotenoid biosyn-
thesis enzymes were designated pET-44a(+)-CrtB and pET-
44a(+)-CrtE (Fig. 2), respectively. Next, PCR amplification
was performed using a pair of oligonucleotides with T7-1
and CrtE-3 primers (Table 1) using pET-44(a)-CrtE (Fig. 2)
as a template, and ligated into the pGEM-T-easy vector. The
resulting plasmid, pGEM-T-easy-CrtE, was digested with
XhoI enzyme and ligated into the pET-44(a)-CrtB plasmid
previously digested with XhoI enzyme and treated with calf
intestinal alkaline phosphatase (CIAP) to prevent self-
ligation. The resulting plasmid, pET-44(a)-CrtEB (Fig. 2),
was transformed into E. coli BL21(DE3) Codon Plus. Also,
the plasmids, pET-44(a)-CrtEBI, pET-44(a)-CrtEBIY, pET-
44(a)-CrtEBIYZ, and pET-44(a)-CrtEBIYZW (Fig. 2), were
constructed by the methods described as above. The result-
ing plasmids were transformed into E. coli BL21(DE3)
Codon Plus. The cells harboring the various carotenoid
genes were cultured in LB medium (containing 50 μg/mL
ampicillin) and induced by adding final concentration of
0.05 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at a
cáÖK=OK= Construction of the expression plasmids carrying various combinations of the m~ê~ÅçÅÅìë=Ü~ÉìåÇ~Éåëáë carotenoid biosynthesis
genes. A: pET-44(a)-CrtE, B: pET-44(a)-CrtEB, C: pET-44(a)-CrtEBI, D: pET-44(a)-CrtEBIY, E: pET-44(a)-CrtEBIYZ, F: pET-
cell density corresponding to OD600 = 0.5. Induced cells
were incubated in a rotary shaking incubator at 37oC and
150 rpm for 12 h.
To test the functions of the astaxanthin biosynthesis en-
zymes, we performed chromatographic and spectroscopic
analyses of pigments produced from the transformed E. coli
with the combinations of the carotenoid biosynthesis genes.
The pigments produced in the E. coli transformants carrying
various combinational constructs of the carotenoid biosyn-
thesis genes of P. haeundaensis were analyzed with chroma-
tographic and spectroscopic methods. Ten grams of the ly-
ophilized cells of E. coli BL21(DE3) Codon Plus carrying a
plasmid were resuspended in 10 mL acetone and incubated
overnight at 4oC. The acetone was then evaporated and the
pellet was dissolved in 10 mL n-hexane-ethanol (1:1, v/v).
The extract was diluted to one-half with distilled water, and
the two phases were divided using a separating funnel. The
organic phase (n-hexane phase) was washed with 30%
aqueous ethanol until colorless and near neutral pH. After
separation, the organic phase was blown to dryness under a
stream of nitrogen, and the residue was stored in a refrigera-
The carotenoid extract was dissolved in 2-propanol and
subjected to high-performance liquid chromatography (HPLC).
Chromatography was performed using an Agilent 1100
HPLC system (Agilent Technologies, Palo Alto, CA, USA)
equipped with a temperature-controlled autosampler and a
diode array detector and the column was a YMC carotenoid
C30 column (5 micron, steel, 250 mm long × 4.6 mm i.d.;
Waters Corp., Milford, MA, USA). The guard column was a
Pelliguard LC-18 cartridge (20 mm; SUPELCO, Bellefonte,
PA, USA). The mobile phase was a methanol/methyl tert-
butyl ether (A/B) gradient having the following parameters
(all percentages expressed as v/v): start, 80% A/20% B; 10
min, 65% A/35% B; 20 min, 10% A/90% B. A flow rate of
1.0 mL/min was used. The injection volume and column
temperature were 10 μL and 15oC, respectively. Carotenoids
were detected by absorbance at 470 nm (except for a phy-
toene, which was detected at 286 nm). Astaxanthin, β-
carotene, and lycopene were purchased from Sigma (St.
Louis, MO, USA), and phytoene and zeaxanthin were pur-
chased from Carl Roth GmbH (Karlsruhe, Germany) as au-
To calculate the amount of the accumulated carotenoids
from the transformed cells, the following equation was used
Total carotenoid (g) = (1)
The specific absorbance coefficient, (= specific ex-
tinction coefficient, ), where representing the absorb-
ance of 1% (w/v) solution (1 g/100 mL) in a 1-cm path cu-
vette at the appropriate wavelength, was applied for the de-
termination of the concentrations of carotenoids.
Biotechnol. Bioprocess Eng. PNR=
cáÖK=PK= HPLC analysis of the products synthesized in bëÅÜÉêáÅÜá~=Åçäá cells carrying (A) pET-44(a)-CrtEBI, (B) pET-44(a)-CrtEBIY, (C)
pET-44(a)-CrtEBIYZ, and (D) pET-44(a)-CrtEBIYZW plasmids.
The maximal wavelength and absorbance coefficient
( ) of a phytoene are 286 nm and 1,250, respectively. A
colorless main peak of the extracts prepared from the cells
transformed with pET-44(a)-CrtEB was absorbed at 286 nm,
eluted at a retention time of 25.4 min from the HPLC, and
determined to be phytoene by comparing it to the standard
(data not shown). From the calculation using the above Eq.
(1), the concentration of the phytoene produced was 1.4
mg/g DCW (dry cell weight).
The main peak of the HPLC analysis from the cell ex-
tracts transformed with pET-44(a)-CrtEBI was found to be
lycopene by comparing it to the standard. This peak was
eluted at a retention time of 21.7 min and absorbed at maxi-
mal wavelength of 470 nm (Fig. 3A). The absorbance coef-
ficient ( ) of a lycopene is 3,450. The amount of lyco-
pene calculated with the above Eq. (1) was 1.1 mg/g DCW.
The elution peaks of the HPLC analysis from the cell ex-
tracts transformed with pET-44(a)-CrtEBIY were found to
be two pigments corresponding to β-carotene and lycopene
when these peaks were compared with standards. These
peaks were eluted at a retention time of 17.4 min for β-
carotene and 21.7 min for lycopene, respectively (Fig. 3B).
The maximal wavelength and absorbance coefficient ( )
of a β-carotene are 450 nm and 2,592, respectively. The
amounts of β-carotene and lycopene using the above for-
mula (1) were calculated to be 0.8 and 0.1 mg/g DCW, re-
The HPLC elution profile of the cell extracts transformed
with pET-CrtEBIYZ was shown in Fig. 3C. The peaks were
turned out to be three pigments corresponding to zeaxanthin,
β-carotene, and lycopene by comparing them to the standard
pigments. These peaks were eluted at retention times of 8.2
min for zeaxanthin, 17.4 min for β-carotene, and 21.7 min
for lycopene, respectively. The maximal wavelength and
absorbance coefficient ( ) of a zeaxanthin are 451 nm
and 2,348, respectively. The amounts of zeaxanthin, β-
carotene, and lycopene calculated using the above formula
(1) were 0.7 mg/g DCW, 0.2 mg/g DCW, and 50 μg/g DCW,
The result of the HPLC analysis from the cells trans-
formed with pET-44(a)-CrtEBIYZW was shown in Fig. 3D.
The main peaks were turned out to be three pigments corre-
sponding to astaxanthin, zeaxanthin, and β-carotene, when
these peaks were compared with standard pigments. These
peaks were eluted at retention time at 6.9 min for astaxanthin,
at 8.2 min for zeaxanthin, and at 17.4 min for β-carotene,
respectively. The maximal wavelength of astaxanthin is at
470 nm and the absorbance coefficient (E) of astaxanthin is
2,500. The amounts of astaxanthin, zeaxanthin, and β-
carotene using the above formula (1) were calculated to 0.4
mg/g DCW, 0.2 mg/g DCW, and 80 μg/g DCW, respec-
In this study, we have conducted a comparative analysis
of the structure, expression, and function of the astaxanthin
biosynthesis genes of P. haeundaensis through various com-
binations of these genes. We have further studied their ex-
pression, organization, and characteristics of the carotenoid
biosynthesis enzymes using chromatographic and spectro-
scopic analyses. The observations and genetic manipulations
of the astaxanthin biosynthesis enzymes from P. haeundaen-
sis make this species a very useful model in which to study
the mechanism of astaxanthin biosynthesis. In addition, the
results of this study can be used to enhance the production of
astaxanthin through the manipulation of carotenoid biosyn-
thesis genes in P. haeundaensis, an important application
since astaxanthin is a pigment of high economic value.
These data will provide a wider base of knowledge on the
primary structure of the astaxanthin biosynthesis gene clus-
ter at the molecular level as well as further the biotechno-
logical applications of carotenoids.
^ÅâåçïäÉÇÖÉãÉåíë This research was supported by a
grant (M2007-05) from the Marine Bioprocess Research
Center of the Marine Bio 21 Center funded by the Ministry
of Maritime Affairs and Fisheries, Republic of Korea.
Received December 7, 2006; accepted March 13, 2007
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