Microbiological Research 167 (2012) 179–186

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Microbiological Research
journal homepage: www.elsevier.de/micres

Agrobacterium tumefaciens mediated transformation of marine microalgae

Schizochytrium
Rubin Cheng a,b , Ruijuan Ma a , Ke Li a , Hui Rong a , Xiangzhi Lin a,∗ , Zhaokai Wang a ,
Shanjun Yang a , Yong Ma a
a
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, Fujian Province,
People’s Republic of China
b
College of Oceanography and Environment, Xiamen University, Xiamen 361005, Fujian Province, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Schizochytrium was a known docosahexaenoic acid producing marine microalgae. In this study, we have
Received 11 April 2011 developed a novel transformation approach of Schizochytrium using the Agrobacterium tumefaciens (A.
Received in revised form 6 May 2011 tumefaciens) binary vector system. After co-cultivation of Schizochytrium protoplasts with A. tumefaciens
Accepted 8 May 2011
harboring pCAMBIA2301 containing the neomycin phosphotransferase II (NPT II) gene as the selectable
marker which confers resistance to G418, the Schizochytrium transformants were successfully obtained
Keywords:
on the G418-containing plates. The integration and expression of the transgenes were confirmed by
Schizochytrium
PCR analysis and GUS activity assay. To further validate the transformation system, pCAMBIA2301-EGFP
Agrobacterium tumefaciens
Transformation system
containing the egfp gene was introduced into Schizochytrium. The following results demonstrated that the
Fatty acid exogenous egfp gene has been successfully incorporated into the genome of Schizochytrium. In addition,
the introduced egfp gene expressed efficiently according to the Western blot and fluorescence assay
results. More importantly, the majority of the transformants displayed similar biomass and fatty acid
production comparing with the wild type strain. Our results demonstrated that exogenous genes could be
expressed efficiently in transgenic Schizochytrium, suggesting that genetically engineered Schizochytrium
could be explored by this system.
© 2011 Elsevier GmbH. All rights reserved.

Introduction
Schizochytrium sp. is a microalgae belonging to the Stra-
menopiles, a kingdom consisting of photosynthetic diatoms, brown
algae and other algae commonly referred to as chromophytes
(Cavalier-Smith et al. 1994). It can produce large amounts of oil (up
to 55% of the cell dry weight) in which DHA can comprise as much
as 45% of the total fatty acids (Cheng et al. 2011). Schizochytrium sp.
has been utilized for commercial production of DHA-rich oil and
dried powder used as a source of DHA in foods, feeds and nutritional
supplements (Fan et al. 2007).
Agrobacterium tumefaciens (A. tumefaciens) is widely used to
transform plant cells (Gelvin 1998). It has the natural ability to
transfer a segment of DNA from its Ti plasmid, known as T-DNA,
into plant so that the T-DNA integrates at random into the nuclear
chromosomes (Bundock et al. 1995; Piers et al. 1996; de Groot
et al. 1998). As previously reported, A. tumefaciens mediated trans-
formation (ATMT) also leads to homologous recombination and
∗ Corresponding author. Tel.: +86 592 2195562; fax: +86 592 2195770.
E-mail address: lxz8848@263.net (X. Lin).
facilitates gene knock-out (Bardiya and Shiu 2007). Recently, it has
been shown that the host range of A. tumefaciens can be extended
to 80 non-plant organisms, mainly fungi including yeasts but also
mammalian cells, microalgae and prokaryotic cells (Kunik et al.
2001; Kumar et al. 2004; Soltani et al. 2008; Kathiresan et al.
2009).
Although the development of methods for Schizochytrium
transformation has advanced significantly in the past few years
(Lippmeier et al. 2009; Metz et al. 2009; Cheng et al. 2011), it
remains unknown whether A. tumefaciens could mediate trans-
formation in Schizochytrium. In this study, we have developed the
A. tumefaciens mediated transformation system for Schizochytrium.
Genetically modified Schizochytrium strains with neomycin phos-
photransferase II (NPT II) selectable marker genes which confer
resistance to G418 were successfully obtained. Molecular charac-
terizations confirmed that the integration and expression of the
exogenous genes in the transgenic Schizochytrium. To our knowl-
edge, this represents the first report in which stable transgenic
Schizochytrium have been routinely produced using an A. tume-
faciens binary vector system. This strategy makes it possible to
explore genetically modified Schizochytrium to produce higher fatty
acids and DHA.

0944-5013/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2011.05.003
180 R. Cheng et al. / Microbiological Research 167 (2012) 179–186
Materials and methods
Strains and culture media
The microalgae Schizochytrium sp. TIO1101 was isolated using
pine pollen as bait. This strain has been deposited in China General
Microbiological Culture Collection Center (CGMCC No. 4603). It was
cultivated in YPD medium with a salinity equivalent to 50% that of
seawater at 28 ◦ C. For the selection and maintenance of transfor-
mants, YPD medium was supplemented with G418 (Invitrogen) at
300 ␮g/mL. A. tumefaciens strains LBA4404 and EHA105 were kind
gifts from Mr. Haifeng Li (Agricultural Science Institute of Zhejiang
Province).
Plasmid construction
The binary vector pCAMBIA2301-EGFP was constructed by
inserting a 1.6-kb EGFP expression cassette fragment into the
HindIII–BamHI site of pCAMBIA2301 (Fig. 1). The egfp gene was
cloned from the pEGFPN1. The TEF1 promoter and CYC1 termi-
nator were cloned from the pGAPZ␣A. In pCAMBIA2301-EGFP,
the expression of egfp gene was under the control of the con-
stitutive TEF1promoter which has been proved to be effective in
Schizochytrium (Cheng et al. 2011). The GUS gene was under the
control of CaMV35S promoter (Fig. 1).
Preparation of Schizochytrium protoplasts
Schizochytrium sp. TIO1101 were cultured overnight in YPD
medium, inoculated to fresh medium and grown to a logarithmic
phase. Cells were harvested by centrifugation at 4000 × g for 5 min
at 4 ◦ C. The collected cells were washed twice with sterile water
and re-suspended in 20 mM phosphate buffer (pH 5.8) containing
50 mM DETA and 25 mM DTT. The suspension was then incubated at
28 ◦ C for 30 min with shaking at 180 rpm. Subsequently, cells were
collected and placed into a 50 mL tube containing 10 mL enzyme
medium. This medium consisted of 2% (w/v) cellulase, 2% snailase,
0.7 M KCl in phosphate buffer (20 mM, pH 5.8). Following incuba-
tion with gentle shaking for 4–5 h at 28 ◦ C, the protoplasts cells
were collected and resuspended in the induction medium (IM) con-
taining 0.7 M KCl. The protoplasts cells were plated on YPD plates
containing 0.7 M KCl to regenerate the cell wall at 28 ◦ C.
Agrobacterium-mediated Schizochytrium transformation
The A. tumefaciens strains LBA4404 and EHA105, each harboring
pCAMBIA2301 and pCAMBIA2301-EGFP were cultivated at 28 ◦ C
in YEB medium in the presence of the proper selective antibi-
otics to an OD600 of 0.8. A. tumefaciens cells were then collected
by centrifugation at 4000 × g for 5 min at 4 ◦ C, and resuspended
by induction medium (containing 200 ␮M acetosyringone and
50 ␮g/mL kanamycin) to an OD600 of 0.3–0.4. The A. tumefaciens
strains were cultured for additional 4 h at 28 ◦ C to pre-induce the
virulence of A. tumefaciens. For transformation, Schizochytrium pro-
toplasts cells were incubated with A. tumefaciens in IM medium for
12 h and plated onto selective YPD medium containing 0.7 M KCl at
28 ◦ C.
PCR analysis to determine the incorporation of transferred genes
To confirm the genome integration of transferred genes,
PCR analysis was performed. The genomic DNA of the trans-
formed Schizochytrium was isolated (Lippmeier et al. 2009; Cheng
et al. 2011). Primers NPT-F (5 -TCACTGAAGCGGGAAGGGACT-
3 ) and NPT-R (5 -GCGGCGATACCGTAAAGCAC-3 ) for amplifi-
cation of the neomycin phosphotransferase II (NPT II) gene
were designed to confirm the incorporation of NPT II gene.
Primers GUS-F (5 -GACTCGTCCGTCCTGTAGAAACC-3 ) and GUS-
R (5 -AAAGTCCCGCTAGTGCCTTGTC-3 ) were designed to con-
firm the incorporation of gus gene. The amplification of the
egfp gene (760 bp) from the genome was performed with
primers EGFP-F (5 -GCCACCATGGTGAGCAAG-3 ) and EGFP-R (5 -
TTACTTGTACAGCTCGTC-3 ). PCR reaction parameters were as
follows: 94 ◦ C for 3 min, followed by 30 cycles of 94 ◦ C for 30 s, 58 ◦ C
for 30 s and 72 ◦ C for 1 min, and followed by a final extension of
10 min at 72 ◦ C.
ˇ-Glucuronidase (GUS) activity assay
The activity of ␤-glucuronidase was assayed using the 4-methyl-
umbelliferyl-␤-d-glucuronide (4-MUG) as substrate (Wickes and
Edman 1995; Ory et al. 2004). The Schizochytrium cells were col-
lected by centrifugation at 4000 × g for 5 min at 4 ◦ C, washed and
re-suspended in 500 ␮l of GUS assay buffer. Cells were disrupted
with Vcx750 watt ultrasonic processor (Sonics and Materials, Inc.,
USA) at 0 ◦ C and 9 kHz for 2 min, and the supernatant was obtained
by centrifugation at 12,000 × g and 4 ◦ C for 15 min. The protein con-
centration was assayed with BCA Protein Assay kit (Pierce, USA). For
each sample, 200 ␮g proteins were added into a tube with final vol-
ume adjusted with GUS assay buffer to 400 ␮l. Then, 100 ␮l 5 mM
MUG was transferred to each tube. After reaction at 37 ◦ C for 1 h,
50 ␮l of the mixture was transferred into a new tube, mixed with
900 ␮l of stop buffer. The fluorescence was detected using Spec-
traMax M5 with excitation filter at 388 nm and emission filter at
480 nm. The activity of ␤-glucuronidase was indicated directly with
the fluorescence value.
Southern blot analysis
Southern hybridization was performed under conditions recom-
mended for the digoxigenin (DIG) hybridization system according
to kit instructions (Roche Diagnostics, Mannheim, Germany). 10 ␮g
genomic DNA was digested with the restriction enzyme SalI, sep-
arated on 0.8% agarose gel, and transferred to Hybond N+ nylon
membranes. The PCR-amplified 760-bp-long fragment of the egfp
gene was labelled as probe to detect the T-DNA integrated into the
genome. Pre-hybridization, hybridization and chemiluminescent
detection of the blots were performed following the manufacturer’s
instruction.
Western blot analysis
The Schizochytrium cells were collected, washed and re-
suspended in 500 ␮l of phosphate-buffered saline (50 mM, pH 7.0).
Cells were ultrasonically disrupted at 0 ◦ C and 9 kHz for 2 min,
and then the cell free extract was obtained by centrifugation at
12,000 × g and 4 ◦ C for 15 min. The protein concentration was
assayed with BCA Protein Assay kit. For each sample, 100 ␮g of pro-
teins was separated and electrophoretically and transferred onto
polyvinylidene difluoride membranes (PVDF). Membranes were
immunoblotted overnight at 4 ◦ C with anti-GFP antibody (Clon-
tech) and incubated for 1 h at room temperature. The proteins were
detected by an enhanced chemi-luminescence (Pierce).
Fluorescence assay
The wild type Schizochytrium and transformants were cultured
overnight in YPD medium. The collected cells were disrupted and
the protein concentrations were determined. Then, 200 ␮g pro-
teins were transferred to a 96-well microtitre plate with the final
volume adjusted with phosphate-buffered saline to 200 ␮l. Fluores-
 R. Cheng et al. / Microbiological Research 167 (2012) 179–186
Fig. 1. Schematic representation of the binary vector pCAMBIA2301 and pCAMBIA2301-EGFP.
cence was measured using SpectraMax M5 with excitation filter at
474 nm and emission filter at 515 nm.
Biomass and lipid production analysis
For biomass and lipid production analysis, the wild type
Schizochytrium and transformants were cultivated in a improved
GPY medium (4% glucose, 1.5% peptone, 0.5% yeast extract) with
a salinity equivalent to 50% that of sea water in 500 mL flask at
200 rpm and 28 ◦ C for three days. Then, the biomass was deter-
mined after freeze-drying. The lipid was extracted by the Bligh
and Dyer method (Bligh and Dyer 1959). The lipid production was
determined gravimetrically after evaporating a measured aliquot
of the combined chloroform phase to dryness under nitrogen.
The extracted free fatty acids were transmethylated with 0.5 M
NaOH/MeOH solution at 50 ◦ C for 10 min, and then the resultant
fatty acid methyl esters were extracted with hexane, concentrated
and analyzed by GC–MS. The column temperature was maintained
at 240 ◦ C during the analysis process.
181
Results
Optimization of selection conditions for Schizochytrium
transformants
In order to select appropriate binary vectors for transformation,
we first determined the antibiotic sensitivity of Schizochytrium sp.
TIO1101. The results revealed that 300 ␮g/mL G418 was fully able
to inhibit the Schizochytrium growth on solid plate. Therefore, G418
was expected to be a suitable selective agent and the binary vec-
tor pCAMBIA 2301 encoding the neomycin phosphotransferase II
(NPT II) gene which confers resistance to G148 was chosen for
transformation in this study (Fig. 1). The growths of A. tumefa-
ciens LBA4404 and EHA105 were significantly blocked by ampicillin
and chloromycetin at low concentration. Therefore, the putative
Schizochytrium transformants could be obtained on the selective
YPD plates containing these three antibiotics G418, ampicillin and
chloromycetin. The latter two antibiotics were used to discriminate
the A. tumefaciens strains from transgenic Schizochytrium during the
selecting process.
182 R. Cheng et al. / Microbiological Research 167 (2012) 179–186

Fig. 2. Agrobacterium tumefaciens harboring pCAMBIA2301 mediated transformation of Schizochytrium. (A) Aggregates of Schizochytrium protoplast cells after incubation
with Agrobacterium (bars = 100 ␮m). (B) The number of transfomrants generated by Agrobacterium LBA4404 and EHA105. (C) The putative Schizochytrium transformants
tested on a second G418-containing plate. The wild type strain was used as the control.

Agrobacterium-mediated Schizochytrium transformation
A. tumefaciens strains LBA4404 and EHA105 harboring pCAMBIA
2301 were used as the donor strains for Schizochytrium trans-
form, respectively (Fig. 1). Acetosyringone was added to induce
virulence gene expression to initiate the process of transfer and
integration of the T-DNA into the recipient’s genome. The contact
between the host cell and A. tumefaciens is an essential prereq-
uisite for the T-DNA transfer. The previous studies demonstrated
that Agrobacterium attaches to host cells, enmeshing itself and the
host cell into aggregates (Matthysse et al. 1978, 1996; Kunik et al.
2001). Then we detected whether the form of Schizochytrium pro-
toplasts has any changes after incubation with Agrobacterium. As
shown in Fig. 2A, Schizochytrium protoplast cells joined and aggre-
gated each other after incubation with Agrobacterium, indicating
that Agrobacterium strains could attach to Schizochytrium proto-
plast. In addition, the aggregates of Schizochytrium infected by A.
tumefaciens LBA4404 was much larger than that of EHA105, sug-
gesting that LBA4404 has a higher binding affinity to Schizochytrium
cells. Co-cultivation of Schizochytrium protoplasts with A. tumefa-
ciens resulted in positive colonies on the selective plates, whereas,
no transformants were detected when Schizochytrium protoplast
cells were incubated with A. tumefaciens strains without pCAM-
BIA2301. However, the transformation efficiency of the two donor
strains LBA4404 and EHA105 differed obviously. Agrobacterium
strain LBA4404 yielded much more transformants than EHA105
(Fig. 2B). When randomly selected transformants were subse-
quently tested on G418-containing plates, all of the colonies could
grow successfully, indicating that the protocol used in this study
was a rapid and effective screening system for Schizochytrium trans-
formation (Fig. 2C).
To confirm the presence of the transgenes in the putatively
transformed clones, genomic DNAs were extracted for PCR ampli-
fication. PCR reactions with NPT II and gus genes specific primers
yielded the expected amplification production in each transfor-
mant analyzed, suggesting that the exogenous genes has been
incorporated into the genome (Fig. 3A and B). Then the expression
level of gus gene was examined by detecting the activity of beta-
glucuronidase. The majority of the transformants exhibited a much
higher GUS activity than the wild type strain, indicating that the
exogenous gus gene successfully expressed in the Schizochytrium
transformants (Fig. 3C). These results demonstrated that A. tume-
faciens mediated transformation (ATMT) was an efficient tool to
transfer exogenous gene into Schizochytrium.
Transformation of egfp gene into Schizochytrium
The above results indicated that ATMT system could be applied
to introduce functional exogenous genes into Schizochytrium.
To further validate this conclusion, the enhanced green fluo-
rescent protein (EGFP) expression cassette was inserted into
the binary vector pCAMBIA2301, yielding recombinant plasmid
pCAMBIA 2301-EGFP (Fig. 1). A. tumefaciens strains containing
pCAMBIA2301-EGFP were co-cultivated with Schizochytrium pro-
toplasts and G418-resistance transformants were obtained and
analyzed subsequently. PCR was first carried out to confirm the
integration of the egfp gene into the genome of Schizochytrium
transformants. As shown in Fig. 4A, reactions with egfp-specific
primers yielded the expected amplification products in the trans-
formants respectively, but not in the parental strain Schizochytrium
sp. TIO1101. This result suggested that the exogenous egfp gene has
integrated into the genome of Schizochytrium. In order to further
 R. Cheng et al. / Microbiological Research 167 (2012) 179–186 183

Fig. 3. Analysis of the putative Schizochytrium transformants. (A) PCR products of NPT II gene of the genomic DNA of Schizochytrium transformant. (B) PCR products of gus
gene of the genomic DNA of Schizochytrium transformant. The sizes of the bands were labelled. Genomic DNA of the wild type Schizochytrium was used as the negative control.
(C) The expression level of gus gene in each Schizochytrium transformants.

confirm the integration and copy number of egfp gene, South-
ern blot analysis was performed for the above six transformants.
Genomic DNA from each transformant was digested with SalI and
probed with a 760-bp egfp gene fragment (Fig. 4B). The result
revealed that all of the transformants harbored a single copy of
egfp gene integrated randomly in the transgenic Schizochytrium
genome. Similar results have been reported in other species where
a majority of transformants contained a single copy of T-DNA in
the genome (Gouka et al. 1999; Kunik et al. 2001; Meyer et al.
2003). To verify the expression of the introduced egfp gene in the
transformants, the expression level of egfp gene was analyzed by
Western blot firstly. The result demonstrated that the introduced
egfp gene was highly expressed in the transformants (Fig. 4C). Then
the fluorescence of the transformant was measured with a fluores-
cence microplate reader, at excitation and emission wavelengths
of 474 and 515 nm, respectively. The fluorescence assay clearly
demonstrated that all transformants showed much higher intense
fluorescence than the wild type strain (Fig. 4D). Finally, the trans-
184 R. Cheng et al. / Microbiological Research 167 (2012) 179–186

Fig. 4. Molecular analysis of integration of egfp gene into the genome of Schizochytrium transformants. (A) PCR products of egfp gene of the genomic DNA of Schizochytrium
transformant. The sizes of the bands were labelled. Genomic DNA of the wild type Schizochytrium was used as the negative control. (B) Southern blot analysis with a DIG-
labelled egfp gene probe. The genomic DNA isolated from wild type Schizochytrium was used as a negative control. (C) Western blots analysis of Schizochytrium transformants.
Proteins from wild type Schizochytrium were used as a negative control. (D) Fluorescence assay of the Schizochytrium transformant. The wild type strain was used as the
control. (E) Fluorescence microscopic analysis of the Schizochytrium transformant (bars = 100 ␮m).

formants were inspected by fluorescence microscopy. The results

revealed that stable and efficient egfp expressed successfully in
the transformants, confirming the expression of egfp gene in the
transgenic Schizochytrium (Fig. 4E).
To further evaluate the feasibility of ATMT approach to explore
genetically modified Schizochytrium, the biomass, total lipid and
lipid composition of the transformants were determined. As shown
in Fig. 5, the majority of the transformants exhibited similar
biomass and lipid with the wild type strain, demonstrating that
the ATMT strategy could be used to introduce functional genes
into Schizochytrium and explore genetically modified strain. Unex-
pectedly, the transformants E3 showed a significant decrease in
the biomass and total lipid. Since the random insertion of T-
DNA in Schizochytrium genome, the reduction on the biomass
and lipid of E3 was hypothesized to be the inactivation of genes
related to growth and fatty acid biosynthesis. The compositions
of the lipid fraction from the wild type strain and the transfor-
mants were subsequently determined by GC–MS. Both the wild
type strain and the transformants accumulated tetradecanoic acid
(C14:0), hexadecanoic acid (C16:0), 9-hexadecenoic acid (C16:1),
Oleic Acid (C18:1), docosapentaenoic acid (DPA n-6) and DHA. This
result suggested that ATMT did not alter the fatty acid profile of
Schizochytrium. Taken together, A. tumefaciens mediated transfor-
Fig. 5. Biomass and fatty acid content analysis of the Schizochytrium transformants.
mation was an efficient approach to transfer foreign genes and (A) The biomass of the Schizochytrium transformants. (B) The fatty acid content of
process genetic engineering in Schizochytrium. the Schizochytrium transformants. The wild type strain was used as the control.
 R. Cheng et al. / Microbiological Research 167 (2012) 179–186
Discussion
In this study, we have developed a novel approach to produce
transgenic marine microalgae Schizochytrium via A. tumefaciens
mediated transformation (ATMT). The exogenous gene could be
integrated into the genome of Schizochytrium during the T-DAN
transfer process. Southern blot analysis revealed that integration
occurred at different chromosomal sites in each transformants. Fur-
thermore, the exogenous gene could be expressed effectively. The
green fluorescent protein (GFP) and its variants are now some of
the most widely used reporters for monitoring gene expression in
many microbes (Lorang et al. 2001; Lissemore et al. 2009). GFP
fluorescence is stable, species-independent, and can be detected
non-invasively in living cells by either fluorescence microscopy or
macroscopic imaging techniques. As the GFP signal can be detected
easily and conveniently without manipulation of the samples, the
application of GFP technology in selection procedures and genetic
screens is emerging as perhaps the most promising approach in
the era of functional genomics (Lorang et al. 2001). The use of GFP
to identify DNA-tagged transformants will allow rapid enrichment
of the desired mutants, facilitating the development of efficient
genetic transformation systems to carry out large-scale insertional
mutant screens (Ding et al. 2010; Zhong et al. 2010). The majority of
Schizochytrium transformants with GFP expression exhibited simi-
lar biomass and lipid production in this study, indicating that GFP
could be also used as the reporter to detect the expression levels of
functional genes in Schizochytrium.
Contact between recipient and Agrobacterium is an essential
prerequisite for the T-DNA transfer. The previous reports have
demonstrated that Agrobacterium cells attached to many species,
including plant protoplasts, fungal cells and Hela cells (Matthysse
et al. 1978; Kunik et al. 2001; Sharma and Kuhad 2010). Our
results indicated that Agrobacterium cells could also attach to
microalgae Schizochytrium cells. The ability of Agrobacterium cells
to associate with the recipient cells was presumed to be criti-
cal for the transformation efficiency. In this study, A. tumefaciens
strain LBA4404 strains showed a significant higher binding affin-
ity to Schizochytrium cells than EHA105. The relative lower affinity
of EHA105 strain to host cells may lead to the relative lower
transformation efficiencies. To transfer its T-DAN into the host
cells, the attached Agrobacterium uses complex transport machin-
ery encoded by the vir region and specifically activated by signal
molecules. The best studied inducer of vir gene expression is ace-
tosyringone (AS), a monocyclic phenolic molecular (Stachel et al.
1985). Although it has been reported that Agrobacterium infection
of the unicellular algae C. reinhardtii (Kumar et al. 2004) and H. plu-
vialis (Kathiresan et al. 2009) was feasible without the inclusion
of AS, we observed that un-induced Agrobacterium lost the ability
to transform Schizochytrium cells in this study, suggesting that AS
was necessary for ATMT of microalgae Schizochytrium. Up to now,
only four microalgae species Chlamydomonas reinhardtii (Kumar
et al. 2004), Haematococcus pluvialis (Kathiresan et al. 2009), Nan-
nochloropsis sp. (Cha et al. 2011) and Dunaliella bardawil (Anila et al.
2011) have been successfully transformed by the ATMT approach.
The Agrobacterium carrying a neomycin resistance gene generated
G418-resistance Schizochytrium transformants expands its poten-
tial host range in microalgae.
The delivery of NDA into the nuclear genome of Schizochytrium
can be achieved using various techniques. Gene targeting by
homologous recombination is a genetic tool that permits modi-
fication of cellular genes in a precise and predetermined fashion.
Both of particle bombardment and electroporation transformation
approaches require homologous sequences from Schizochytrium
genome (Lippmeier et al. 2009; Metz et al. 2009; Cheng et al. 2011).
Although illegitimate recombination requiring little or no homol-
ogy to the host genome has been occurred in other microalgae
185
(Zaslavskaia et al. 2000), similar ectopic integrations of introduced
DNA have not been reported for Schizochytrium via the above
transformation strategies. Up to now, the particle bombardment
and electroporation transformation approaches could only medi-
ated homologous integration of transferred DNA into the genome
of Schizochytrium. Thus, the application of the above two trans-
formation strategies was restricted to a certain extent since the
little knowledge of Schizochytrium genome. Our results demon-
strated that A. tumefaciens mediated transformation could result in
non-homologous recombination for Schizochytrium, indicating that
ectopic integration would also work in this microalga. The previous
studies showed that ATMT could also lead to homologous recom-
bination (Bardiya and Shiu 2007). Thus, ATMT was hypothesized to
result in homologous recombination in Schizochytrium with addi-
tional homologous sequences in the binary vector. Then, it is easy
to know where the integration took place in the algal genome.
The high transformation frequency, together with the preci-
sion and simplicity of T-DNA integration, makes T-DNA a suitable
element for genome mutagenesis approaches, which can also
be exploited in different biotechnological applications (Ruiz-Diez
2002; Sharma et al. 2006). Genomic sequences flanking T-DNA
insertions can be isolated by PCR-based methods such as TAIL-
PCR, representing a powerful tool for insertional mutagenesis and
subsequent reverse genetic analysis of transformants (Combier
et al. 2003). Experience gained in filamentous fungi has high-
lighted T-DNA as the preferred insertion mutagen for generating
large libraries of fungal mutants to perform functional genomics
(Jeon et al. 2007). Although the DHA synthesis in Schizochytrium
was accomplished in part by the PKS enzymes, the exact biosyn-
thetic mechanism of PKS pathway has a long way to go before it is
finally unraveled (Metz et al. 2001). In addition, the mechanism of
Schizochytrium binary cell division requires further study. Genome
research in Schizochytrium would certainly expand our knowledge
on these aspects. However, ATMT strategy provides a rapid and
cost-efficient mean of exploring the precise mechanism of DHA
biosynthesis and binary cell division in Schizochytrium according
to our results. The transformants E3 showed a significant reduc-
tion in the biomass and fatty acid production. Then isolation of the
T-DNA-Schizochytrium genome DNA junction from E3 would facil-
itate the clarification of biomass and lipid production. Similarly,
screening Schizochytrium mutants in binary cell division and DHA
synthesis by ATMT approach and amplifying of T-DNA junction to
investigate the chromosomal DNA flanking the T-DNA insert would
further understand the mechanism in Schizochytrium.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgment
This work was supported by the Ocean Public Welfare Scientific
Research Special Appropriation Project (grant no. 200805041).
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