Regulation of key enzymes involved in the fatty acid biosynthesis
in Crambe abyssinica
R. Guan, X.Y. Li, P. Hofvander, D.N. Wang, I. Lager, S. Stymne and L.H. Zhu*
Department of Plant Breeding and Biotechnology,
Swedish University of Agricultural Sciences. *E-Mail: [email protected]
Alteration of oil composition by manipulating
PDAT and LPCAT & PDCT
Introduction
Erucic acid (22:1) is a major feedstock for the oleochemical industry. Crambe
abyssinica is a novel oilseed crop containing ca. 60% of 22:1, but further
increasing 22:1 will be of great commercial interest. Through manipulating
three genes involved in the fatty acid biosynthesis, we have developed
transgenic crambe lines with 22:1 of 73% in seed oil. The aim of this study is,
by modifying another three key genes involved in the fatty acid biosynthesis, to
further increase the 18:1-CoA, the precursor of 22:1 biosynthesis.
The preliminary results have showed the trend that the Inhibition of PDAT
(phospholipid:diacylglycerol acyltransferase) had led to a drastic increase
of 18:2 in PC and moderate increase of 18:2 in TAG, while no clear
changes in the levels of other fatty acids. Lines with RNAi targeting
LPCATs
(lysophosphatidylcholine
acyltransferase)
and
PDCT
(phosphatidylcholine:diacylglycerol cholinephosphotransferase) led to
significant increase in 18:1, 16:0 and 20:1 and a decrease in 18:2 and
22:1 in TAG. In PC there was a moderate increase in 18:2 and a drastic
increase in 18:3 (Fig 1).
Fatty acid (%)
80
Wt
TAG
RNAi PDAT
60
RNAi LPCAT PDCT
40
20
0
16:0
Fatty acid (%)
60
18:1
18:2
18:3
PC
20:1
22:1
Wt
50
RNAi PDAT
40
RNAi LPCAT PDCT
30
20
10
0
16:0
18:1
18:2
18:3
20:1
22:1
Fig 1. Fatty acid compostion of TAG and PC from pooled seeds of the transgenic lines
and WT.
Demonstration of relative activities of enzymes in
the Kennedy Pathway in microsomes
from the high erucic acid transgenic crambe lines
Previously introduced LdLPAAT gene enabled efficient acylation of 22:1-CoA
to the sn-2 position of 22:1-LPA in microsomal preparation from transgenic
seeds, resulting in accumulation of PA, DAG and TAG (Fig 2A). In WT
microsomes, the LPA substrate was hardly acylated, but was partially
metabolised to monoacylglycerols (MAG).(Fig 2B)
Comparison of the relative activities of
enzymes in the Kennedy Pathway in
microsomes from WT crambe and safflower
The DAG-PC equilibration as measured
in the microsomes of
developing seeds from WT crambe has low activity and resulted in very
little amount of PC accumulation from [14C] glycerol-3-phosphate,
whereas safflower microsomes catalysed a major flow of glycerol
backbone into PC (Fig 3).
A
Substrate (nmol)
6
B
TAG
DAG
MAG
A
Crambe
5
TAG
4
DAG
3
PA
2
PC
1
0
0m
PA
20m
40m
80m
Incubation time
5
Percenta ge of
total lane(%)
3G7-13
Safflower
4
TAG
3
DAG
2
PA
Substrate (nmol)
LPA
80
B
WT
60
40
PC
1
20
0
TAG
DAG
MAG
PA
LPA
0
0m
Fig 2. Autoradiogram of 14C-lipid separation by TLC showing the different utilization of
[14C] 22:1-LPA in microsomes prepared from developing seeds of high erucic acid
transgenic line 3G7-13 (A) and WT (B) when using 22:1-CoA as acyl donor. Incubation
time was 80 min.
20m
40m
80m
Incubation time
Fig 3. The formation of 14C-lipids from 18:1-CoA and [14C] glycerol3-phosphate in WT crambe (A) and safflower micromsomes (B).
Acknowledgements: Financial support to this study from FORMAS, EU-ICON and Vinnova is highly acknowledged