APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1998, p. 2676–2680 Vol. 64, No.

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0099-2240/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Increased Carotenoid Production by the Food Yeast Candida utilis

through Metabolic Engineering of the Isoprenoid Pathway
HIROSHI SHIMADA,* KEIJI KONDO, PAUL D. FRASER,† YUTAKA MIURA, TOSHIKO SAITO,
AND NORIHIKO MISAWA

Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., Kanazawa-ku, Yokohama-shi,
Kanagawa 236, Japan
Received 15 December 1997/Accepted 16 April 1998

The yeast Candida utilis does not possess an endogenous biochemical pathway for the synthesis of carote-
noids. The central isoprenoid pathway concerned with the synthesis of prenyl lipids is present in C. utilis and

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active in the biosynthesis of ergosterol. In our previous study, we showed that the introduction of exogenous
carotenoid genes, crtE, crtB, and crtI, responsible for the formation of lycopene from the precursor farnesyl
pyrophosphate, results in the C. utilis strain that yields lycopene at 1.1 mg per g (dry weight) of cells (Y. Miura,
K. Kondo, T. Saito, H. Shimada, P. D. Fraser, and N. Misawa, Appl. Environ. Microbiol. 64:1226–1229, 1998).
Through metabolic engineering of the isoprenoid pathway, a sevenfold increase in the yield of lycopene has
been achieved. The influential steps in the pathway that were manipulated were 3-hydroxy methylglutaryl
coenzyme A (HMG-CoA) reductase, encoded by the HMG gene, and squalene synthase, encoded by the ERG9
gene. Strains overexpressing the C. utilis HMG-CoA reductase yielded lycopene at 2.1 mg/g (dry weight) of cells.
Expression of the HMG-CoA catalytic domain alone gave 4.3 mg/g (dry weight) of cells; disruption of the ERG9
gene had no significant effect, but a combination of ERG9 gene disruption and the overexpression of the HMG
catalytic domain yielded lycopene at 7.8 mg/g (dry weight) of cells. The findings of this study illustrate how
modifications in related biochemical pathways can be utilized to enhance the production of commercially
desirable compounds such as carotenoids.

The aim of metabolic engineering is defined as the purpose-
ful modification of metabolic networks in living cells to pro-
duce desirable chemicals with superior yields and productivity
by using recombinant DNA technologies (1, 18). Its main field
should be investigations undertaken to produce chemicals of
commercial interest efficiently and abundantly by using the
appropriate microorganisms (8). It has traditionally been pos-
tulated that microbes naturally synthesizing desirable chemi-
cals should be used as hosts. However, the use of suitable
microorganisms which have the ability to produce the precur-
sors for the desired chemicals with superior yields and at high
levels of productivity is also feasible (14). This notion signifi-
cantly extends the range of microbes utilizable as productive
hosts. In order to achieve these objectives, three main research
approaches are usually employed: (i) introducing exogenous
genes, which convert the final precursor of a host organism to
a desirable chemical, at a viable yield; (ii) enhancing the met-
abolic flux through a pathway to increase the synthesis of the
final precursor (this may, for example, be achieved by ampli-
fying rate-limiting reactions or eliminating mechanisms of
feedback inhibition); and (iii) increasing precursors by mini-
mizing metabolic flow to biosynthetically related products.
Lycopene is a red carotenoid pigment present in the tomato,
watermelon, and red grapefruit. This pigment has recently
attracted great attention, due to its beneficial effect on health.
For example, lycopene has been shown to have preventive
effects against certain cancers, e.g., prostate cancer (4). Lyco-
pene is also said to be the most effective antioxidant (12).
* Corresponding author. Present address: Department of Biological
Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of
Technology, Nagatsuta, Midoriku, Yokohama 226, Japan. Phone: 81-
45-924-5737. Fax: 81-45-924-5805. E-mail: hshimada@bio.titech.ac.jp.
† Present address: Biochemistry Department, Royal Holloway Uni-
versity of London, Egham, Surrey TW20 OEX, United Kingdom.
The objective of the present study was to produce, through
a metabolic engineering approach, a microorganism giving a
high yield of carotenoid. The yeast Candida utilis is an indus-
trially important microorganism approved by the U.S. Food
and Drug Administration as a safe substance. Through its
large-scale production, C. utilis has become a promising source
of single-cell protein as well as a host for the production of
several chemicals, such as glutathione and RNA (3, 7). C. utilis
does not synthesize carotenoid pigment but does accumulate
large quantities of ergosterol (10 to 15 mg/g [dry weight] of
cells). Like carotenoids, ergosterol is an isoprenoid, and it is
biosynthetically related to them by a common prenyl lipid
precursor, farnesyl diphosphate (FPP). Thus, although C. utilis
does not form carotenoids, it does possess their potential pre-
cursors. By introducing the three carotenogenic genes (crtE,
crtB, and crtI) required for lycopene synthesis from FPP under
the control of C. utilis promoters, a C. utilis strain that pro-
duces 1.1 mg of lycopene per g (dry weight) of cells has been
generated; the lycopene produced in C. utilis is a pure product
(13) (Fig. 1). In the present study, we have applied concepts (ii)
and (iii) of metabolic engineering described above to the yeast
strain in order to redirect carbon flux away from ergosterol
formation for potential utilization by the carotenoid pathway.
The resultant strains producing high-yields of lycopene and
possessing commercial potential are described and discussed.
MATERIALS AND METHODS
Strains and growth conditions. C. utilis ATCC 9950 was used in this study.
Cells were cultured at 30°C in YPD medium (2% glucose, 1% Bacto Yeast
Extract, 2% Bacto Peptone). Escherichia coli DH5a was used as a host for
plasmid construction (17).
Molecular cloning techniques. The construction of the C. utilis genomic DNA
library has been previously described by Kondo et al. (10). Colony hybridization
and Southern hybridization were performed as described by Sambrook et al.
(17). In order to obtain fragments containing either the HMG or ERG9 gene,
PCRs were performed with C. utilis genomic DNA as a template. The oligo-
nucleotides HMG1-59 (59-GGTGAYGCHATGGGTATGAACAT-39) and

2676
VOL. 64, 1998
FIG. 1. Metabolic pathway of endogenous ergosterol biosynthesis and exog-
enous lycopene biosynthesis. The solid arrows show the one-step conversions of
the biosynthesis, and the dashed arrows show the several steps. crtE, crtB, and crtI
are the endogenous lycopene synthesis genes. GGPP, geranylgeranyl diphos-
phate.
HMG1-39 (59-GTACCVACCTCGATNWSTGGCAT-39) were used for ampli-
fying the HMG gene. The nucleotide sequence of primer HMG1-59 was deduced
from the amino acid sequence between 1807 and 1814 of the HMG1 protein of
Saccharomyces cerevisiae, and the nucleotide sequence of primer HMG1-39 was
deduced from the amino acid sequence between 1951 and 1958 of the HMG1
protein of S. cerevisiae. PCR was carried out with a LA PCR kit, version 2
(Takara Shuzo Co., Ltd., Kusatsu, Japan). The amplified DNA fragments were
sequenced and analyzed by the protocol provided with the ABI PRISM cycle
sequencing kits. The PCR products were used as probes for colony hybridization
and Southern hybridization. The clone obtained did not encode the 39 region of
the HMG gene. To obtain the 39 region of the HMG gene, PCR was performed
with the following primers: HMG-3-1 (59-GGGGAATTCAAGAAAGCCTTCA
ACTCTACTTCCAGATTT-39) and pBR322-39 (59-TGATGCCGGCCACGAT
GCGTCCGGCGTAGA-39). Primer HMG-3-1 contained the nucleotide se-
quence between 12023 and 12061 of the C. utilis HMG gene, and primer
pBR322-39 contained the nucleotide sequence of pBR322. The C. utilis genomic
library which was constructed in pBR322 (10) was used as a template.
In order to obtain a probe for cloning the ERG9 gene, PCR was performed
with C. utilis genomic DNA as a template. The oligonucleotides ERG9-59 (59-
TTYSTNCARAAGACNAACATCAT-39) and ERG9-39 (59-ATVACYTGTGG
GATDGCACARAA-39) were used as PCR primers. The nucleotide sequence of
primer ERG9-59 was deduced from the amino acid sequence between 1217 and
1224 of the ERG9 protein of S. cerevisiae, and the nucleotide sequence of
primer ERG9-39 was deduced from the amino acid sequence between 1295 and
1302 of the S. cerevisiae ERG9 protein. The amplified DNA fragments were
analyzed, and the DNA fragments were used as a probe for cloning the ERG9
gene.
Construction of an HMG overexpression vector and an ERG9 disruption
vector. Plasmid pGhrH, used for overexpression of the HMG gene, was con-
structed as follows. The oligonucleotides F-HMG-59 (59-GGGGCTAGCATGT
TCTACCACGGTGCAAGCGCAAACCAA-39) and F-HMG-39 (59-GGGAGA
TCTTGCTTATTTCTTAGCAGCGGCTCTGTTGTG-39) were used as PCR
primers for amplifying the HMG gene in C. utilis. Primer F-HMG-59 corre-
sponded to the sequence between 11 and 130 of the HMG gene, and primer
F-HMG-39 corresponded to the sequence between 12779 and 12805 of the
HMG gene. C. utilis genomic DNA was used as the template for PCR. The PCR
products were digested by NheI and BglII and ligated between the XbaI and
BamHI sites of pGAPPT10, which contains the GAP promoter and terminator
(17). The plasmid was designated pGh. The 1.2-kb EcoRI fragment of plasmid
pCLRE2 containing C. utilis ribosomal DNA (rDNA) (10) was ligated into the
EcoRI site of pGh, and the resulting plasmid was designated pGhr. The 3.2-kb
NotI fragment containing an expression cassette of the hygromycin B (HYG)
phosphotransferase gene (HPT) was isolated from pGKHPT1 and inserted at the
NotI site of pGhr to construct the plasmid pGhrH. pGKHPT1 was constructed
as follows. The HPT gene was obtained by PCR with the 59 primer 59-GGTCT
AGATATGAAAAAGCCTGAAC-39 and the 39 primer 59-GGAGATCTATTC
CTTTGCCCTCGGA-39. Plasmid pBIB-HYG (2) was used as a template for
PCR. The PCR products were digested with XbaI and BglII and then ligated
between the XbaI and BamHI sites, which contain PGK promoter and terminator
fragments.
Plasmid pChGRH, used for the expression of a truncated form of the HMG
IMPROVED CAROTENOID PRODUCTION BY C. UTILIS 2677
gene, was constructed as follows. The oligonucleotides T-HMG-59 (59-GGGGCT
AGCATGATCCATACCACAAGATTGGAGGATGCAATC-39) and T-HMG-39
(59-GGGAGATCTTGCTTATTTCTTAGCAGCGGCTCTGTTGTG-39) were
used as primers for PCR. Primer T-HMG-59 corresponded to the sequence
between 11339 and 11368 of the HMG gene, and primer T-HMG-39 corre-
sponded to the sequence between 12779 and 12805 of the HMG gene. Genomic
DNA from C. utilis was used as the template for PCR. The PCR products were
digested with NheI and BglII and ligated between the XbaI and BglII sites of the
plasmid pGAPPT10 to construct plasmid pChG. The 1.2-kb EcoRI fragment of
rDNA and the 3.2-kb NotI fragment of the HPT gene expression cassette were
ligated into the EcoRI site of pChG to construct pChGRH. Plasmid pGARH,
which we used as a control, was constructed by inserting the EcoRI fragment of
rDNA and the NotI fragment of the HPT gene at the EcoRI and NotI sites of
pGAPPT10.
Plasmid p8EBN8 z G4, used for disruption of the ERG9 gene, was constructed
as follows. Plasmid p8EB9 was constructed by subcloning a 2.0-kb EcoT221-
BamHI fragment containing the full-length ERG9 gene between the PstI and
BamHI sites of pUC18. Plasmid p8EBN8 was constructed by inserting a NotI
linker into the BglII site at 1722 of the ERG9 gene of p8EB9 after treating the
BglII site with T4 DNA polymerase. Plasmid p8EBN8 z G4 was constructed by
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ligation of the 2.5-kb NotI fragment containing a G418 (Geneticin) resistance
gene expression cassette of pGKAPH3 into the NotI site of p8EBN8.
Transformation of C. utilis. Yeast transformations were carried out by elec-
troporation as described by Kondo et al. (10) with a slight modification: the
incubation of the cells in YPD medium after the pulse was extended to 12 h
instead of 6 h. Plasmids were used for transformation after they were linearized
by restriction enzyme digestion. The plasmids phmrDH, pChGRH, and pGhrH
were digested with BglII, and plasmid p8EBN8 z G4 was digested with BamHI
and SphI. The transformed cells were grown in YPD medium containing appro-
priate antibiotics (200 mg of G418/ml, 40 mg of cycloheximide [CYH]/ml, and 800
mg of HYG/ml).
Measurements of lycopene and ergosterol contents. Lycopene and ergosterol
were extracted from yeast cells as described previously (19). The extracted
lycopene and ergosterol were subjected to high-performance liquid chromatog-
raphy as follows: the column (3.9 by 300 nm; Nova-pak HR 6 m C18; Waters) was
developed with acetonitrile–methanol–2-propanol (90:6:4) at a flow rate of 1
ml/min, and on-line spectra were recorded with a Waters photodiode array
detector 996. The precise amounts of ergosterol and lycopene in the samples
were determined by comparison to known amounts of standard compounds
chromatographed under identical conditions.
Nucleotide sequence accession numbers. The nucleotide sequence accession
numbers for the HMG-CoA reductase gene (HMG) and the squalene synthase
gene (ERG9) are ABO12603 and ABO12604, respectively.
RESULTS
Cloning of the HMG and ERG9 genes. Genes encoding
HMG-CoA reductase (HMG) have been cloned and se-
quenced from various organisms (6). Comparison of the
known amino acid sequences indicated that HMG proteins
share highly conserved amino acid sequences at their carboxyl
termini. From the conserved regions, the mixed oligonucleo-
tide primers (HMG-1-59 and HMG1-39; see Materials and
Methods) were synthesized and used as primers for PCR. Se-
quence analysis of the amplified fragments indicated strong
similarities (72.4% identity in the deduced amino acid se-
quence) to the known HMG1 gene in S. cerevisiae. The DNA
fragment was used as a probe for Southern hybridization and
colony hybridization. The HMG gene in C. utilis consists of a
2,805-bp open reading frame (ORF) and encodes a polypep-
tide of 935 amino acids; the deduced amino acid sequence had
52.2% identity with that of HMG-CoA reductase encoded by
the HMG1 gene of S. cerevisiae (data not shown). Hydropho-
bicity analysis of the amino acid sequence of C. utilis HMG
protein indicated that it contained seven putative membrane-
spanning regions in the amino-terminal region between resi-
dues 1 and 476 (data not shown). It has been shown that the
HMG1 protein of S. cerevisiae contains seven membrane-span-
ning domains at the amino terminus, and the stability of the
protein is controlled by protease attack upon these domains
(6). The carboxyl-terminal region of the HMG1 protein is
considered to be the catalytic domain. The region between
residues 477 and 935 of the C. utilis HMG protein exhibited
75.5% identity with the HMG1 protein of S. cerevisiae.
2678 SHIMADA ET AL.
FIG. 2. Structures of plasmids used for HMG gene overexpression. Plasmids
pGhrH and pChGRH have the full-length HMG gene and the truncated HMG
gene, respectively. GAP-P, promoter of the gene encoding glyceraldehyde-3-
phosphate dehydrogenase; GAP-T, terminator of the gene encoding glyceralde-
hyde-3-phosphate dehydrogenase; HYGr, HYG phosphotransferase gene.
An approach similar to that used to clone the HMG gene
was used to isolate the C. utilis squalene synthase gene
(ERG9). From the conserved regions of the isolated ERG9
gene (9, 16), the mixed oligonucleotide primers were synthe-
sized and used as primers for PCR (see Materials and Meth-
ods). Sequence analysis of the amplified fragments indicated
that the sequence of the DNA fragment had high similarity to
the ERG9 genes isolated in S. cerevisiae (72.9% identity in the
deduced amino acid sequences). The DNA fragment was used
as a probe for Southern hybridization or colony hybridization.
An ERG9 gene of C. utilis was cloned, and its sequence was
determined. The ERG9 gene consisted of a 1,320-bp ORF and
a 63-bp intron. The ERG9 gene encoded a polypeptide of 440
amino acids that had 60.0% identity with that of the squalene
synthase of S. cerevisiae (data not shown). Southern hybridiza-
tion showed that the genomic DNA digested with BamHI and
HindIII yielded a single band hybridized to the ERG9 gene
probe DNA (data not shown). However, when the genomic
DNA was digested with EcoRI, two bands were observed in the
analysis. The sequence analysis of the ERG9 gene indicated
that there is no EcoRI site in the ORF or intron. These results
suggested that C. utilis has two copies of the ERG9 gene, as C.
utilis is diploid. The ERG9 gene copies were encoded by 5.0-
and 17.0-kb EcoRI fragments, respectively, in the C. utilis ge-
nome.
Increase of lycopene content by overexpression of the HMG
gene. Production of lycopene in C. utilis was achieved by ex-
TABLE 1. Lycopene and ergosterol contents of strains carrying various HMG and ERG9 constructs
Strain Descriptiona
0988 Wild type
12-2
C-1 Carrying no HMG
FHL Carrying full-length HMG
THL Carrying truncated HMG
LDL Disrupted upper ERG9
UDL Disrupted lower ERG9
ehL-1 Carrying truncated HMG and disrupted lower ERG9
ehL-2 Same as ehL-1
a
All strains carry the lycopene synthesis genes, crtE, crtB, and crtI, except the wild-type strain, 0988.
b
Average of two independent clones (except strains ehL-1 and ehL-2). Standard deviations are all ,0.2 mg per g (dry weight) of cells.
c
Average of three independent assays. Standard deviations are all ,0.1 mg per g (dry weight) of cells.
d
NA, not assayed.
e
Average of three independent assays.
APPL. ENVIRON. MICROBIOL.
pressing the lycopene biosynthesis genes, crtE, crtB, and crtI.
The lycopene content of the resulting strain (strain 12-2) was
1.1 mg per g (dry weight) of cells (14). Previous studies have
indicated that HMG-CoA reductase is a key enzyme involved
in ergosterol biosynthesis in yeast (5, 6) (Fig. 1). Therefore, the
plasmids phmrDH, containing the full-length HMG gene, and
pChGRH, containing a portion of the gene (between 11339
and 12805) encoding the catalytic domain of the HMG pro-
tein, were constructed (Fig. 2). Plasmid pGAPRH, containing
no HMG gene, was used as a control plasmid. These plasmids
were used for transformation of the lycopene-producing strain
12-2 after the plasmids were cut with BglII. The transformants
were cultured in YPD medium containing CYH and HYG for
2 days at 30°C (see Fig. 4). In the stationary phase, the lyco-
pene content of C-1, a strain carrying the control plasmid
pGAPRH, was almost the same as that of the control strain,
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12-2 (Table 1). The lycopene content of FHL strains, carrying
the full-length HMG gene, was 2.1 mg per g (dry weight) of
cells, while in THL strains, carrying the truncated HMG gene,
the lycopene content was 4.3 mg per g (dry weight) of cells.
Thus, compared to the control strain (12-2), two- and fourfold
increases in lycopene content were achieved. The ergosterol
contents of C-1, FHL, and THL were almost the same as that
of the control strain, 12-2 (Table 1). These results indicated
that the overexpression of the HMG gene under the control of
the glyceraldehyde-3-phosphate reductase gene (GAP) pro-
moter was effective in increasing the accumulation of lycopene
in C. utilis and that the expression of the catalytic domain of
the HMG protein alone was more effective than that of the
full-length protein.
Effect of ERG9 disruption on lycopene content. Farnesyl
pyrophosphate is the precursor diverted from the endogenous
C. utilis isoprenoid pathway into the engineered pathway for
lycopene formation. It is also the substrate for squalene syn-
thase, the formation of squalene being the first step committed
in ergosterol biosynthesis (Fig. 1). In order to increase the
carbon flux towards lycopene biosynthesis, disruption of the
ERG9 gene encoding squalene synthase was conducted. Plas-
mid p8EBN8 z G4 was used to disrupt the ERG9 gene; the
gene was divided by an insertion of the expression cassette of
the G418r gene (Fig. 3a). Strain 12-2 was transformed with
p8EBN8 z G4, and Southern analysis of the transformants in-
dicated that each of the two copies of the ERG9 gene was
disrupted by homologous recombination (Fig. 3b). The strains
in which disruption of the ERG9 gene had occurred within the
5.0- and 17.0-kb EcoRI fragments were designated “Lower
Lycopene contentb (mg/g Ergosterol contentc (mg/g
[dry weight] of cells) [dry weight] of cells)
NAd 13.2
1.1 6.4
1.5 6.8
2.1 6.4
4.3 6.5
1.0 8.5
1.4 5.5
7.8e 6.7
6.5e 6.5
VOL. 64, 1998
FIG. 3. Genomic Southern hybridization of strains with disrupted ERG9. (a)
Schematic representation of the disruption of ERG9 by the plasmid p8EBN8 z
G4. (b) Genomic Southern hybridization. Lane 1, wild type; lane 2, Upper
Disruption strain; lane 3, Lower Disruption strain. The upper and lower arrows
indicate the 17.0- and 5.0-kb bands, respectively. All genomic DNAs were di-
gested by EcoRI.
Disruption” and “Upper Disruption,” respectively. The trans-
formants were cultured in YPD medium containing CYH and
G418 for 2 days at 30°C (Fig. 4). The growth rates of strains
with the disrupted ERG9 genes were virtually the same as that
of the control strain, 12-2 (data not shown). In the stationary
phase, the lycopene contents of UDL (Upper Disruption and
lycopene synthesis) strains were slightly increased (1.4 mg per
g [dry weight] of cells) compared to that of the control strain,
12-2 (1.1 mg per g [dry weight] of cells). However, the lycopene
contents of LDL (Lower Disruption and lycopene synthesis)
strains were almost the same (1.0 mg per g [dry weight] of cells)
FIG. 4. Strains of C. utilis harboring various vectors. (A) Wild-type strain of C. utilis; (B) control strain carrying only lycopene synthesis genes (crtE, crtB, and crtI);
(C) strain carrying lycopene synthesis genes and plasmid pChGRH (the truncated HMG expression vector); (D) strain carrying lycopene synthesis genes, plasmid
pChGRH, and disrupted ERG9.
IMPROVED CAROTENOID PRODUCTION BY C. UTILIS 2679
as that of the control strain. The ergosterol contents of UDL
strains were almost identical (5.5 mg per g [dry weight] of cells)
to that of the control strain (6.4 mg per g [dry weight] of cells),
but the ergosterol contents of LDL strains were increased (8.5
mg per g [dry weight] of cells).
Effect of ERG9 gene disruption and HMG gene overexpres-
sion on lycopene content. In order to assess the effect of the
lower ERG9 gene disruption and the HMG gene overexpres-
sion in combination, plasmid pChGRH, containing the trun-
cated form of the HMG gene, was introduced into the strain
(Lower Disruption) in which the 5.0-kb EcoRI fragment of the
ERG9 gene was disrupted. The transformants obtained were
further transformed with plasmid pCLR1EBI, containing the
lycopene synthesis genes, crtE, crtB, and crtI (14). Two clones,
ehL-1 and ehL-2, were obtained, and the transformants were
cultured in YPD medium containing CYH, HYG, and G418
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for 3 days at 30°C (Fig. 4). In the stationary phase, the lycopene
contents of ehL-1 and ehL-2 were 7.8 and 6.5 mg per g (dry
weight) of cells, respectively (Table 1). The ergosterol contents
of strains ehL-1 and ehL-2 were almost the same as those of
strains FHL and THL. Thus, a fourfold increase in lycopene
was achieved by the combination of ERG9 gene disruption and
HMG gene overexpression compared to that achieved by HMG
gene overexpression alone, and compared to the lycopene con-
tent of strain 12-2, a sevenfold increase was achieved.
DISCUSSION
C. utilis HMG-CoA reductase is similar to other HMG-CoA
reductases, with the amino terminus containing seven diversi-
fied membrane-spanning domains and a highly conserved do-
main at the carboxyl terminus. It is this extremely conserved
domain that has been shown to be responsible for catalytic
activity in most organisms (6). Formation of HMG-CoA re-
ductase is a highly regulated enzymatic step in animal and
2680 SHIMADA ET AL.
fungal isoprenoid biosynthesis, i.e., regulation occurs at tran-
scriptional, posttranscriptional, and posttranslational levels (5,
6). It can be postulated that due to these regulatory mecha-
nisms an additional copy of the HMG-CoA reductase in the
cell would also be regulated and its action would be controlled
in such a manner that the overall flux through the pathway
would not be significantly altered. However, in C. utilis carrying
the HMG gene overexpression vector, the exogenous HMG
gene transcriptional regulation may not function because the
exogenous HMG gene is transcribed by the GAP promoter.
The posttranslational regulation of the truncated HMG gene
may also not be functional because the membrane-spanning
region which is concerned with stability in vivo was deleted.
The increase in the lycopene contents of the strains carrying
the full-length HMG gene and those strains carrying the trun-
cated HMG gene reflect the possible absence of transcrip-
tional, posttranscriptional, and posttranslational regulation.
In order to increase the carbon flux into lycopene biosyn-
thesis, we attempted to decrease squalene synthase activity by
disruption of the ERG9 gene encoding squalene synthase.
Strains with disrupted ERG9 genes (Upper Disruption and
Lower Disruption) were obtained by the introduction of the
ERG9 disruption vector, p8EBN8 z G4, into the strains carrying
the lycopene synthesis genes. Neither ERG9 gene disruption
alone changed the lycopene contents. Presumably, the second
endogenous C. utilis ERG9 gene, which was not disrupted, can
compensate for the loss of the first gene product. To a certain
extent this is understandable, as the total loss of squalene
formation, and thus of ergosterol, would be lethal.
In the sterol biosynthesis pathway, there are regulation steps
other than HMG-CoA reductase. For example, HMG-CoA
synthase is regulated by the end product in the cholesterol
biosynthesis pathway (5). It is possible that HMG-CoA syn-
thase might be regulated in the strains carrying the HMG gene
overexpression vector. In the lycopene production strain with
HMG gene overexpression, ergosterol as well as lycopene con-
tent may be increased. The elevation of the ergosterol content
might reduce HMG-CoA synthase activity by feedback regu-
lation. This could provide an explanation of why expression of
the ERG9 gene and the HMG gene in combination was so
effective compared to HMG gene expression alone.
This study reports the first successful application of meta-
bolic engineering to the production of carotenoid pigments.
Although lycopene synthesis genes have been used in this
study, the formation of b-carotene and astaxanthin in C. utilis
has also been achieved (14). The application of metabolic
APPL. ENVIRON. MICROBIOL.
modification to these strains will hopefully increase their con-
tents of these commercially desirable carotenoids.
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