Biotechnol Lett (2009) 31:10111017

DOI 10.1007/s10529-009-9970-z

ORIGINAL RESEARCH PAPER

Improvement of arachidonic acid and eicosapentaenoic

acid production by increasing the copy number
of the genes encoding fatty acid desaturase
and elongase into Pichia pastoris
Yun-Tao Li Mao-Teng Li Chu-Hua Fu
Peng-Peng Zhou Jian-Min Liu Long-Jiang Yu

Received: 5 December 2008 / Revised: 23 February 2009 / Accepted: 23 February 2009 / Published online: 22 March 2009
Springer Science+Business Media B.V. 2009

Abstract Genes encoding D6 desaturase, D6 fatty

acid elongase, and D5 desaturase from the alga,
Phaeodactylum tricornutum, were co-expressed in
Pichia pastoris to produce arachidonic acid (ARA;
20:4 D5, 8, 11, 14) and eicosapentaenoic acid (EPA;
20:5 D5, 8, 11, 14, 17). A panel of Pichia clones
carrying progressively increasing copies of the heterologous gene expression cassette was created using
an in vitro multimerization approach. ARA and EPA
accumulated up to 0.3 and 0.1% of total fatty acids,
respectively, in the recombinant P. pastoris carrying
with double copies of these three heterologous genes,
as compared to 0.1 and 0.05%, respectively, in the
recombinant P. pastoris with single copy.
Keywords Arachidonic acid

Eicosapentaenoic acid

Yun-Tao Li and Mao-Teng Li contributed equally to this work.

Y.-T. Li
M.-T. Li
C.-H. Fu
P.-P. Zhou

J.-M. Liu
L.-J. Yu (&)
Institute of Resource Biology and Biotechnology, College
of Life Science and Technology, Huazhong University
of Science and Technology, Wuhan 430074, China
e-mail: yulongjiang@mail.hust.edu.cn
Y.-T. Li
M.-T. Li
C.-H. Fu
P.-P. Zhou

J.-M. Liu
L.-J. Yu
Key Laboratory of Molecular Biophysics, Ministry
of Education, Wuhan 430074, China

Fatty acid desaturases and elongase

Pichia pastoris
Polyunsaturated fatty acids

Introduction
Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA; 20:4 D5, 8, 11, 14) and eicosapentaenoic acid (EPA; 20:5 D5, 8, 11, 14, 17), play
important roles as structural components of membrane phospholipids and as precursors of several
biologically active eicosanoids (Spector 1999; Jump
2002). They also have important physiological and
medical functions, such as promoting development of
the brain and retina, regulating of blood pressure and
immune response (Benatti et al. 2004), and antiinflammatory (Das 2002). Regular consumption, and
an accordingly sustainable source of these compounds, is highly desirable. At present, the main
source of n3-long chain PUFAs for human consumption is oily ocean fish (Hoffmann et al. 2008). Because
of the continuous decrease of marine resources by
overfishing and the environmental impact of fish
farming (Pauly et al. 2002; Hites et al. 2004), neither
farmed nor caught fish can be considered a sustainable
source of the long chain PUFAs quantities required for
a healthy nutrition of the growing human population
(Abbadi et al. 2004). To obtain a suitable alternative
source of these desired PUFAs, the genetic modification of the fatty acid biosynthetic pathway in fungi
and algae would be a preferable alternative.

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Pichia pastoris can synthesize linoleic acid (LA;

18:2 D9, 12) and a-linolenic acid (ALA; 18:3 D9, 12, 15).
LA and ALA are substrates for D6-desaturases. In this
study, we reconstituted the pathways for EPA and
ARA biosynthesis in P. pastoris by co-expression of
the genes encoding D6 desaturase, D5 desaturase
(Domergue et al. 2002), and D6 fatty acid elongase
(GenBank accession no. AY746355) from Phaeodactylum tricornutum. In addition, we investigated the
influence of gene copy numbers on the content of ARA
and EPA in P. pastoris.

Materials and methods

E. coli and yeast strains
E. coli DH5a (New England Biolabs) was used to
construct the recombinant plasmids. Pichia pastoris
GS115 (his4) (Invitrogen) was used as a host strain to
express heterologous genes.

Biotechnol Lett (2009) 31:10111017

unique BamHI site (Fig. 1b). The pAO-29D6E6D5

was constructed used the same procedure.
Construction of P. pastoris GS115 recombinant
strains
Pichia pastoris strain GS115 was transformed with
StuI-linearized recombinant plasmid by electroporation with Multiporator (Eppendorf). Cells were spread
on minimal dextrose (MD, 1.34% YNB, 4 9 10-5%
biotin, 2% dextrose, 1.5% agar) plate and incubated at
20C for 72 h until colonies formed. His? transformants were replica plated on minimal methanol (MM,
1.34% YNB, 4 9 10-5% biotin, 1.5% agar) and MD
medium for Mut? and Muts screening. The selected
integrants were confirmed by PCR. The His?Mut?
transformants were grown in a minimal glycerol
medium (MGY, 1.34% YNB, 4 9 10-5% biotin, 1%
glycerol) to an OD600 of 0.5, then transferred to the
MM containing 0.5% methanol and grown to saturation at 20C for 3 days.
Identification of transgenic yeast

Vector construction
The coding regions of D5, D6-desaturase, and
D6-elongase genes were amplified by RT-PCR using
a template of total RNA extracted from Phaeodactylum tricornutum. The primers used for the amplification were introduced with EcoRI sites. The PCR
products were cloned into pMD18-T Simple (TaKaRa)
and sequenced. The ORFs of the D5, D6-desaturase
and D6-elongase were released from the cloning
vector by digestion with EcoRI and then ligated into
the EcoRI site of the P. pastoris expression vector
pAO815 (Fig. 1a). After proving the correct insertion
orientation of every ORF in pAO815 (pAOD6,
pAOE6, pAOD5), these plasmids were used as a
source of expression cassettes (50 AOX1aFD6
30 AOX1TT, 50 AOX1aFE630 AOX1TT, 50 AOX1a
FD530 AOX1TT) for further cloning.
Restriction of pAOD6, pAOE6, pAOD5 with BglII
and BamHI resulted in the release of the D6, E6, D5
expression cassettes (Fig. 1b). To achieve pAOD6E6,
pAOD6 was linearized at the unique BamHI site.
Insertion of the E6 expression cassette in correct
orientation involved a BglII/BamHI cohesive end
ligation. Similarly, pAO-19D6E6D5 was constructed
by ligating D5 expression cassette to pAOD6E6 at the

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Genomic DNA of a number of transformants with

HIS?Mut? phenotype was isolated with the method
as previously reported (Harju et al. 2004). The DNA
was used as template for PCR amplifications with
primers corresponding to 50 -region of the AOX1 gene
and 30 -region of target genes in order to verify that
heterologous gene was integrated into the P. pastoris
genome.
Southern blot hybridization was conducted to
confirm the gene copy number. Genomic DNA from
PCR-positive transformant was digested to completion
with BamHI and BglII separated by electrophoresis
and then transferred onto a Hybond? membrane
(Amersham). The filter was hybridized with a fulllength cDNA fragment probe of D6, D5 desaturase, or
E6 elongase gene labeled using the DecaLabel DNA
Labeling Kit (Fermentas).
A direct semi-quantitative RT-PCR method was
used to measure the expression of D6, D5 desaturase
and E6 elongase. Total RNAs were isolated from the
induced expression clones using TRIZOL (Invitrogen) and first-strand cDNAs were synthesized from
total RNA using the First Strand cDNA Synthesis Kit
(Fermentas). The three heterologous genes were
respectively co-amplified with the internal control

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1013

Fig. 1 Construction flow

chart of Pichia expression
vectors. a Integrative
pAO815 cloning vector
with the methanol promoter
(AOX1); b Diagram of the
expression cassettes
contained in the expression
vectors

(b-actin) using 28 cycles. The semi-quantitative

evaluation of RT-PCR bands identified in 1% agarose
gels was carried out using Smartview software.

Results
Construction of Pichia expression vectors
and molecular analysis of transgenic yeast

Fatty acid extraction and analysis

Induced cultures were harvested by centrifugation.
The total lipid contents of the dry yeast cells were
analyzed by Buchi Extraction Unit B-811 Standard.
Total fatty acids were extracted and transmethylated
as previously described (Tonon et al. 2003). Qualitative analysis of fatty acid methyl esters (FAMEs)
was by GC-MS using an Agilent 7890A-5975C GCMS Network system. The mass spectrum of new peak
was compared with that of standard for identification
of fatty acid.

Restriction analysis with BglII and BamHI of the

resultant clones identified pAO815 derivatives
containing D6, E6, D5, D6E6, 19D6E6D5, and
29D6E6D5 expression cassette(s), and the result
showed that pAO-19D6E6D5 and pAO-29D6E6D5
were successfully constructed.
Pichia transformants with HIS?Mut? phenotype
were verified by PCR analysis, then, positive Pichia
clones were analyzed by Southern hybridization to
determine the number of D6E6D5 expression cassettes integrated into their genome. The results of

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Biotechnol Lett (2009) 31:10111017

Fig. 2 Southern blots of Pichia constructs from strains containing 19 and 29 copies of D6E6D5. Lane M: an optimized mix
of plasmids, containing 19D6E6D5 and 29D6E6D5; lanes
1, 3, and 5: DNA from the transgenic P. pastoris carrying
19D6E6D5; lanes 2, 4, and 6: DNA from transgenic P. pastoris
carrying 29D6E6D5

Southern hybridization showed that the genome of

Pichia transformed by pAO-19D6E6D5 was successfully integrated with a single copy of the D6E6D5
expression cassette, and the genome of Pichia transformed by pAO-29D6E6D5 was successfully integrated with a double copy of the D6E6D5 expression
cassette (Fig. 2). The size of the hybridization band of
Pichia transformed by pAO-29D6E6D5 was 2 times
of that of Pichia transformed by pAO-19D6E6D5
(Fig. 2), because the digestion of the resultant constructs with BglII and BamHI generates D6E6D5
containing fragments which size correlates with the
number of D6E6D5 tandem repeats.
The results of semi-quantitative RT-PCR revealed
that the expression levels of D6, E6 and D5 were
about 2 times in recombinant P. pastoris carrying the
29D6E6D5 than in recombinant P. pastoris carrying
the 19D6E6D5 (Fig. 3). These results suggest that
increasing in the copy number of expression cassettes
had enhanced the expression levels of heterologous
mRNAs.

Fatty acid analysis

The total lipid content of P. pastoris was 6.8 mg/g dry
cells. The total lipid contents of transgenic P. pastoris
transformed with pAO815, pAO19D6E6D5, and
pAO29D6E6D5 were 7, 7.3, and 7.2 mg/g dry cells,
respectively. The GC analysis results of the FAMEs
from the induced P. pastoris cells, which genome
integrated with 19D6E6D5 and 29D6E6D5, showed

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Fig. 3 Result of semi-quantitative RT-PCR. A. RT-PCR

results of variety of mRNAs for D6, E6, and D5 in transgenic
P. pastoris. Lane 1, 3, 5: mRNA from transgenic P. pastoris
carrying 19D6E6D5; lane 2, 4, 6: mRNA from transgenic
P. pastoris carrying 29D6E6D5. B. Comparison of the
expression of D6, E6, and D5 in transgenic P. pastoris carrying
19D6E6D5 and 29D6E6D5

that several new peaks were detected (Fig. 4).

The results of GCMS analysis of their methyl
esters showed that new peaks represented c-linolenic
acid (GLA; 18:3 D6, 9, 12), dihomo-c-linolenic acid
(DGLA; 20:3 D8, 11, 14), ARA and stearidonic acid
(SDA; 18:4 D6, 9, 12, 15), eicosatetraenoic acid (ETA;
20:4 D8, 11, 14, 17), EPA (Fig. 4). These productions
were not detected in P. pastoris and P. pastoris
transformed with an empty vector. These results
indicate that ARA and EPA biosynthetic pathways
were successfully reconstituted in P. pastoris.
The fatty acids of the recombinant P. pastoris are
shown in Table 1. With the increasing copy number
of expression cassettes, the contents of ARA and
EPA increased. ARA and EPA, respectively, accumulated up to 0.1 and 0.05% of total fatty acids in
recombinant P. pastoris carrying the 19D6E6D5,
and, respectively, accounted for 0.3 and 0.1% of total
fatty acids in recombinant P. pastoris carrying the
29D6E6D5; the yield of ARA was thus raised threefold and the yield of EPA was doubled. At the same
time, the content of DGLA and ETA (See Table 1) in

Biotechnol Lett (2009) 31:10111017

1015

and 28.6%) in recombinant P. pastoris carrying the

19D6E6D5.

Discussion

Fig. 4 Results of GC-MS analysis of fatty acids in transgenic

P. pastoris. A, B, C, and D represent the fatty acids in the
P. pastoris, P. pastoris transformed with empty vector,
pAO19D6E6D5 and pAO29D6E6D5, respectively

recombinant P. pastoris carrying the 29D6E6D5 (2.4

and 0.2% of total fatty acids) was higher than that in
recombinant P. pastoris carrying the 19D6E6D5 (1.4
and 0.1% of total fatty acids). Particularly, the
content of LA and ALA decreased along with the
increase of the heterologous genes. The conversion
efficiency of each step in the n-6 and n-3 pathways
was calculated as products/(substrate ? products).
These results are summarized in Table 2 and show
that the desaturation (11.133.9%) and elongation (25 and 36.4%) efficiency in recombinant
P. pastoris carrying the 29D6E6D5 were higher
than that (desaturation, 6.733.3%; elongation, 14.3

The recombinant P. pastoris carrying the 19D6E6D5

produced 0.15% ARA and 0.05% EPA of total fatty
acids by co-expression of D6 and D5-desaturases, and
D6-elongase genes from Ph. tricornutum. However,
recombinant P. pastoris co-expressing the D6, D5desaturase, and D6-elongase genes from Marchantia
polymorpha produced 0.1% ARA and 0.03% EPA of
total fatty acids (Kajikawa et al. 2004). ARA and
EPA in Ph. tricornutum can accumulate up to 30% of
total fatty acids (Domergue et al. 2002), whereas their
content is only about 11% of total fatty acids in
M. polymorpha (Chiou et al. 2001). This indicates
that the genes encoding desaturases and elongases
from organisms which have high long-chain PUFAs
contents may be suitable for producing desirable
PUFAs by genetic manipulation.
The proportion of ARA and EPA accumulated in
the recombinant P. pastoris carrying the 29D6E6D5
was higher than that in the recombinant P. pastoris
carrying the 19D6E6D5. The result indicates that an
increase in copy number of the desaturase and
elongase gene can significantly improve the yield of
ARA and EPA. Thus, an increase in the copy number
of expression cassettes results in the production of
higher levels of heterologous mRNAs as well as
enzymes (Sreekrishna et al. 1997), and enhancement
of the content of ARA and EPA in the P. pastoris.
The result of the study showed that increasing in
copy number of the desaturase and elongase gene can
significantly improve the yield of ARA and EPA and
the genes encoding D6, D5-desaturase, and D6elongase genes from P. tricornutum are effective to
enhance the yield of ARA and EPA in recombinant
P. pastoris. The total lipid content of Pichia is low
(\8%) because it is non-oleaginous yeast. In other
words, it does not have the biosynthetic capability of
accumulating high levels of lipid, as are found in the
oleaginous yeasts, and for which the active participation of ATP: citrate lyase is essential. In the next
step, we will transform the three genes into PUFAsproducing microbes which have high content of lipid,
such as Pythium ultimum, to obtain the transgenic

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Biotechnol Lett (2009) 31:10111017

Table 1 Fatty acid composition (% w/w) of total fatty acids from transgenic P. pastoris
Fatty acid

P. pastoris

Transgenic P. pastoris
pAO815

pAO19D6E6D5

pAO29D6E6D5
14.1 0.4

PAa

16:0

10.7 0.2b

9.6 0.3

12.2 0.2

POA

16:1

5.7 0.1

6.2 0.2

3.2 0.1

2.0 0.2

SA

18:0

4.8 0.3

5.6 .2

10.6 0.2

14.3 0.4

OA

18:1

45.9 0.4

48.2 0.5

51.8 0.6

53.2 0.6

LA

18:2(n-6)

27.8 0.3

25.6 0.5

11.9 0.4

8.2 0.3

GLA

18:3(n-6)

NDc

ND

3.5 0.2

4.2 0.2

DGLA

20:3(n-6)

ND

ND

1.4 0.1

2.4 0.2

ARA

20:4(n-6)

ND

ND

0.1 0.0

0.3 0.1

ALA

18:3(n-3)

5 0.2

4.8 0.3

2.3 0.3

1.1 0.2

SDA

18:4(n-3)

ND

ND

0.6 0.1

0.6 0.1

ETA

20:4(n-3)

ND

ND

0.1 0.0

0.2 0.1

EPA

20:5(n-3)

ND

ND

0.05 0.0

0.1 0.0

6.8 0.4d

7.0 0.5

7.3 0.4

7.2 0.6

Total lipid

a
PA Palmitic acid, POA palmitoleic acid, SA stearic acid, OA oleic acid, LA linoleic acid, ALA a-linolenic acid, GLA c-linolenic
acid, DGLA dihomo-c-linolenic acid, ARA arachidonic acid, SDA stearidonic acid, ETA eicosatetraenoic acid, EPA eicosapentaenoic
acid
b

Each value is the mean SD from three independent experiments

Not detected

mg lipid g-1 dry cells

Table 2 Conversion efficiency (%) of P. tricornutum LCPUFAs biosynthetic enzymes in transgenic P. pastoris
Transgenic P. pastoris

PpDEDb
Pp2D2E2Dc

D6-des

D6-elo

D5-des

LA ? GLA

ALA ? SDA

GLA ? DGLA

SDA ? ETA

DGLA ? ARA

ETA ? EPA

22.7a
33.9

20.7
33.3

28.6
36.4

14.3
25

6.7
11.1

33.3
33.3

The conversion efficiency of each step was calculated as 100 9 product/(substrate ? product) (%)

PpDED: recombinant P. pastoris carrying the 19D6E6D5

Pp2D2E2D: recombinant P. pastoris carrying the 29D6E6D5

microbes strains with high yield of n3-very long

chain PUFAs.
Acknowledgements This work was supported by National
High-Tech Research and Development Plan (Grant No. 2007
AA10Z120 and 2007AA100402). We thank Mrs Xiaoman Gu
and Hong Cheng of the Analysis and Testing Center of
Huazhong University of Science and Technology for GC-MS
analysis.

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