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ORAL PAPERS: GENOMICS & BIOTECHNOLOGY

Proceedings of the 8
th
Malaysia Congress on Genetics, 4-6 August 2009, Genting Highlands, Malaysia

190
OC03

EXPRESSION STUDIES OF JESSENIA BATAUA Β-KETOACYL-ACP SYNTHASE II IN
E.COLI

Teh Ooi Kock
1
and Umi Salamah Ramli



1
Malaysian Palm Oil Board (Advanced Biotechnology and Breeding Centre) No.6, Persiaran Institusi, Bandar
Baru Bangi, 43000 Kajang Selangor
§
Corresponding author: umi@mpob.gov.my

Abstract

Genetic modification of oil crops to produce edible oils with desirable fatty acid composition
is challenging. As part of the integrated effort to produce high-oleic oil palm, we studied the
fatty acid biosynthesis pathways of an Amazonian palm Jessenia bataua. Jessenia produces
oil with fatty acid composition similar to olive oil, which is largely oleic acid (70-80%). We
dissected the fatty acid biosynthesis pathways by isolating genes that may act along the oleic
acid biosynthesis pathway. One of the candidate genes is Ketoacyl ACP Synthase (KAS) II.
KASII is a condensing enzyme that elongates the pamitoyl ACP (C16:0) to stearoyl-ACP
(C18:0), which serves as precursors for oleic acid production. We have isolated a putative
KASII-encoding cDNA from the Jessenia mesocarp tissues. The KASII cDNA has a putative
Open Reading Frame (ORF) of 488-amino acid and possesses 2 conserved domains which are
essential for KASII activities. We expressed the Jessenia KASII (JbKASII) as epitope tagged
fusion protein in E.coli. Overexpression of JbKASII at 37
o
C led to inclusion bodies formation
and JbKASII migrated as a 75kDa protein on SDS-PAGE. To render the JbKASII soluble we
expressed the JbKASII at 28
o
C and 22
o
C but JbKASII remained insoluble. Gas
chromatography analysis showed that JbKASII over-expression in E.coli did not significantly
increase the C18:0 composition and thus supported the notion that insoluble JbKASII is not
functional. We are constructing a JbKASII GFP-fusion to be expressed in Arabidopsis KASII
mutant fab1. Genetic complementation of fab1 by JbKASII will ascertain the in vivo
enzymatic activities of JbKASII.

Introduction

MPOB has assembled the world’s largest oil palm germplasm collection in collaboration with
public and private organisations in different countries. The programme covers all aspects of
germplasm collection, conservation, evaluation/documentation and utilization. Jessenia
bataua is among the species brought into Malaysia during the early expedition by PORIM
from different parts of the world to collect oil palm genetic resources (Rajanaidu et al., 1991).
Jessenia was planted in the form of open-pollinated family, mainly at MPOB Research
Stations, as exotic palms. Compared to other oil palm breeding materials, Jessenia has
received very little attention despite the fact that the oil fatty acid composition resembles olive
oil , which contains high oleic acid (~80%) (Balick and Gershoff, 1981). However, the
number of fruit sets and oil content are extremely low hence rendering this valuable genetic
trait unavailable for exploitation at commercial scale. Here we report the isolation of cDNA
which encodes a fatty acid biosynthesis enzyme, β-Ketoacyl ACP Synthase (KAS) II. KAS II
is known to be one of the key enzymes exploited for changing fatty acids composition in palm
oil through genetic manipulation (Parveez et al., 2005). KAS II enzymatic activity was
reported to convert palmitoyl ACP (C16:0-ACP) to stearoyl ACP (C18:0-ACP) in plants
ORAL PAPERS: GENOMICS & BIOTECHNOLOGY

Proceedings of the 8
th
Malaysia Congress on Genetics, 4-6 August 2009, Genting Highlands, Malaysia

191
(Voelker and Kinney, 2001). In this report we present evidence that overexpression of
JbKASII did not significantly increase the C18:0 composition in E.coli.

Materials and Methods

End-to-End PCR of JbKASII Full Length cDNA
RT-PCR was performed in a PE 9600 thermocycler (Perkin Elmer) using SMART
TM
RACE
cDNA Amplification Kit (Clontech) or GeneRacer™ Kit (Invitrogen) to amplify 3’ and 5’
partial fragments of KASII cDNA. First strand cDNA was synthesized from 1 µg of the total
RNA according to the manufacturers’ instructions. The RACE-ready cDNA served as
template. Forward primers gKASF4 (5’ TGA GGA GAA GGA TAT AAA TGG GCA GTT
CCC 3’) or gKASF6 (5’ TCT ATT CTC CTC TCC TTC TCT TCT TTC TCC 3’) was used
together with a reverse primer gKASR6 (5’ ACC TTT GCA TCA TTC AGC TAG AAG
TAA AAC 3’) to amplify the full length KASII cDNA.

A 25μl PCR reaction was set up with 1X Advantage
®
2 PCR Buffer, 0.3μM primer
gKASF4 or primer gKASF6, 0.3μM primer gKASR6, 0.3mM dNTP Mix, 2% DMSO, 100ng
cDNA and 0.5μl Advantage
®
2 Polymerase Mix. The amplification was performed on a T-
Professional thermalcycler (Biometra

)

with the following cycling parameters: 94
o
C, 2 mins;
seven cycles of [94
o
C, 25 sec; 72
o
C, 2 mins 30 sec]; 32 cycles of [94
o
C, 25 sec; 67
o
C 2 mins
30 sec] and final elongation at 72
o
C for 3 mins.

In silico analysis of DNA and Protein Sequences
Full length cDNA was cloned into a sequencing vector, pTOPO and sequenced in both
directions. The DNA sequences were translated into 6 frames protein sequences and blasted
against Genebank Plant Sequences using SDSC Biology Workbench, with default settings.
Multiple Sequence Alignment was then performed on positively-hit proteins together with
putative JbKASII, using CLUSTALW. To determine the Open Reading Frame (ORF) of
JbKASII, full length cDNA sequences were analysed using NCBI ORF Finder programme.
The longest reading frame generated was taken as the putative ORF for JbKASII.

Protein overexpression and immuno-detection of JbKASII in E.coli
JbKASII was cloned into pET32a (Novagen) as BamHI-NotI fragment to generate
pET32a:His-JbKASII-His. Protein expression in E.coli culture was induced by 1mM IPTG
and incubated at either 37
o
C, 28
o
C or 22
o
C for 4~16 hours. Total protein was extracted from
cell pellets by repeated freezing-thawing cycles. Soluble and insoluble protein fractions were
resolved on SDS-PAGE and subjected to immuno-detection by His-Tag
®
monoclonal
antibody (Novagen) on PVDF membrane.

Fatty acid extraction and gas chromatography analysis
Total fatty acid was extracted from E.coli cultures and converted to fatty acid methy ester
(FAME) according to Kates (Kates, 1982) with minor modifications. FAME samples from
control and induced E.coli cultures were separated on a 30m x 0.25mm DB Wax capillary
column on a Perkin Elmer Clarus 500 chromatograph. Relative quantity of all compounds
were calculated based on external standard run concomittantly with each analysis.





ORAL PAPERS: GENOMICS & BIOTECHNOLOGY

Proceedings of the 8
th
Malaysia Congress on Genetics, 4-6 August 2009, Genting Highlands, Malaysia

192
Results and Discussion

Using 5’ and 3’ ends partial sequence information (Ramli et al, 2007), primers were designed
to amplify the KASII full length cDNA. Six pairs of primers, namely gKASF1-gKASF6 and
gKASR1- gKASR6 were designed (not shown). We tested all possible 36 primer
combinations and only primer combinations gKASF4-gKASR6 and gKASF6-gKASR6
produced single PCR product of expected size, which was ~2kb (Figure 1A). The putative
JbKASII cDNA was cloned into sequencing vector pTOPO and sequenced from both
directions. Sequencing results demonstrated that the putative JbKASII cDNA was 1874bp in
length, agreeing with previously reported KAS II cDNA from other species such as
E.guineensis (Ramli et al., 1996).

The putative JbKAS II cDNA was translated into 6 fames protein sequences and
blasted against Genebank Plant Sequences database. Many positive hits were obtained. We
have selected a few KAS II cDNA sequences of closely related monocots and dicots to
demonstrate their sequence similarities (Figure 1B). Multiple sequence alignment of these
protein sequences revealed a very interesting feature of JbKASII protein structure. JbKASII
possesses 2 highly conserved protein domains, namely N-terminal KAS II domain and C-
terminal KAS II domain. Protein structure analysis suggested that the enzymatic activity of
KAS II protein lies within these 2 domains (Huang et al., 1998).

To further ascertain the in vivo function of JbKASII, we constructed a His-tagged
cDNA fusions of JbKASII. The construct, pET32a:His-JbKASII-His, contains an poly-
Histidine epitope tag at both N- and C-termini to allow immuno detection. To enhance protein
solubility, we expressed the construct in 6 E.coli strains [BL21, BL21(DE3),
BL21(DE3)pLysS, Rosetta-gami, Rosetta-gami B(DE3)pLysS and Origami(DE3)] and only
strains Rosetta-gami B(DE3)pLysS (Figure 1C) and Rosetta-gami (not shown) expressed
copious amount of insoluble protein at 37
o
C. It is known that accumulation of newly
synthesized polypeptide from recombinant protein may lead to aggregates formation and
therefore produce non-functional inclusion bodies (Mitraki and King, 1989). We attempted to
enhance JbKASII solubility by inducing the protein expression at lower temperatures
(Voelker and Davies, 1994) but was unsucessful (Figure 1D). Previously Garwin et al
reported that the enzymatic activities of E.coli KASII were significantly enhanced at lower
temperatures (Garwin et al, 1980). With this notion we next ask the question whether
JbKASII overexpression at 22
o
C alters the fatty acid profile in E.coli, particularly the relative
composition of C16:0 and C18:0. Gas chromatography analysis of FAME samples prepared
from E.coli cultures overexpressing JbKASII showed that the overall fatty acid profiles were
not significantly different from the control (Figure 1E). This observation is in line with the
notion that misfolded protein in inclusion bodies was dysfunctional and therefore JbKASII
could not exert enzymatic activities. Another possible explanation for the lack of condensing
activities of JbKASII is that the overproduction of KASII may lead to blockage of fatty acid
synthesis in E.coli (Subrahmanyam and Cronan, 1998). We will next perform mutant
complementation studies using Arabidopsis KASII mutants, fab1-1 and fab1-2. Two JbKASII
GFP fusion constructs have been made and will be used to transform fab1 as well as wild
type.

Conclusion

We conclude that JbKASII when overexpressed in E.coli will lead to inclusion body
formation with no detectable condesing enzymatic activities.
ORAL PAPERS: GENOMICS & BIOTECHNOLOGY

Proceedings of the 8
th
Malaysia Congress on Genetics, 4-6 August 2009, Genting Highlands, Malaysia

193
Acknowledgement

The authors thank the MPOB Director General’s permission to publish the findings. We are
grateful to the Breeding group for Jessenia fruit samples. Thanks to Dr Rajanaidu who
introduced the Jessenia palm to Malaysia.

References

Balick, M.J. and S.N. Gershoff. 1981. Nutritional Evaluation of the Jessenia bataua Palm:
Source of High Quality Protein and Oil from Tropical America. Eco Bot. 35:261.
Garwin, J.L., A.L. Klages and J.E. Cronan Jr. 1980. Structural, enzymatic and genetic studies
of β-ketoacyl-acyl carrier protein synthase II of Escherichia coli. J. Biol. Chem.
255:11949-11956.
Huang,, W., Jia, J., Edwards, P., Dehesh, K., Schneider, G. and Lindqvist, Y. 1998. Crystal
structure of β-ketoacyl-acyl carrier protein synthase II from E.coli reveals the
molecular architecture of condensing enzymes. EMBO Journal. 17(5): 1183-1191.
Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J. and King J. 1991. Global
suppression of protein folding defects and inclusion body formation. Science.
5:253(5015):54-58.
Parveez, G.K.A., Abrizah, O., Masani, A.M.Y., Umi Salamah, R., Sambanthamurthi, R.,
Baharia, B., Nur Fariza, M.S., Yuen, L.H., Nur Hanim, A., Tarmizi, A.H., Zamzuri, I.,
Cheah, S.C., Kushairi, A.D., Basri, M.W., Gregory, Y. and Yeong, B.J. 2005. Value
addition of oil palm through genetic engineering. Proceedings of the 2005 Conference
on Biotechnology of Plantation Commodities.pp, 198-210.
Rajanaidu, N., Jalani, B.S., Ras, V. and Kushairi, A. 1991. New exotic palms for plantations.
In: Proceeding of PORIM International Palm Oil Conference- Agricultu. pg19-27.
Ramli, U.S. and Sambanthamurthi, R. 1996. β-ketoacyl ACP Synthase II in the oil palm
(Elaeis guineensis Jacq) mesocarp. In Physiology, Biochemistry and Molecular
Biology of Plant Lipids. Williams, J.P., Khan, U.M. and Lem, N.W. (eds). Kluwer
Academic Publishers. Toronto. pp67-71.
Ramli, U.S., Seman, N.A. and Rahim Mohd., A. 2007. Isolation of β-ketoacyl ACP Synthase
(KAS) II gene from Jessenia bataua. In Proceedings of Agriculture, Biotechnology &
Sustainability Conference Vol.1
Subrahmanyam, S. and Cronan Jr., J.E. 1998. Overproduction of a functional fatty acid
biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli. J. Bacteriol.
180(17): 4596-4602
Voelker, T. and Kinney, A.J. 2001. Variations in the biosynthesis of seed-storage lipids.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:335-361



ORAL PAPERS: GENOMICS & BIOTECHNOLOGY

Proceedings of the 8
th
Malaysia Congress on Genetics, 4-6 August 2009, Genting Highlands, Malaysia

194
(A) (C)








(B)





(D)


(E)

Figure 1 Isolation and expression of
JbKASII in E.coli
(A) RACE-PCR of JbKASII. A, B, C, D and
E are different RACE-ready cDNA aliquots.
Lanes 1 and 2 are amplification of primer
combination gKasF4-gKASR6 and gKasF6-
gKasR6 respectively. M, Fermentas 1kb
DNA ladder.

(B) Multiple sequence alignment of KASII
homologs. Conserved amino acids are highlighted in black. The accession numbers are
E.guineensis (AF220453), J.curcas (DQ987700), C.wrightii (U67317), A.thaliana
(NM106154), G.max (AF244518) and O.sativa (NM001066809). (C) Pellet and soluble
protein fractions from strain Rosetta-gami B(DE3)pLysS resolved on SDS-PAGE. A 75kDa
protein was observed in induced pellet fraction. (D) Anti-His immuno blot of JbKASII.
Protein was extracted from E.coli cultures {Strains Rosetta-gami B(DE3)pLysS (RpLyS) and
Rosetta-gami (R)} incubated at 37
o
C, 28
o
C and 22
o
C. (E) Fatty acid profile of strain Rosetta-
gami B(DE3)pLysS incubated at 22
o
C. Values shown are mean relative percentage of major
fatty acids from 4 experiments. Error bars are standard deviations.