Download Manipulating flux through plant metabolic pathways Anthony J Kinney

173
Manipulating flux through plant metabolic pathways
Anthony J Kinney
The past two years have seen a marked increase in patent
applications for novel methods of altering the level and
spectrum of commercially important products in plants.
Results from these studies have proven surprising, showing
that in many cases those enzymes traditionally thought of as
flux-controlling have no impact on product formation when
they are directly altered by genetic manipulation. In many
cases, successful induction of increased flux throughout an
entire pathway has been achieved by targeting one of the
terminal enzymes in the pathway.
Addresses
DuPont Experimental Station, P.O. Box 80402 Wilmington,
DE 19880-0402, USA; e-mail: [email protected]
Current Opinion in Plant Biology 1998, 1:173–178
http://biomednet.com/elecref/1369526600100173
 Current Biology Ltd ISSN 1369-5266
Introduction
The manipulation of metabolic pathways, an art restricted
to microbes only a few years ago, is rapidly becoming
commonplace in transgenic plants. The recent advent
of rapid sequencing of cDNA libraries has led to the
discovery of many new plant genes related to primary,
secondary and energy metabolism. Coupled with improvements in the transformation efficiency of plants, this
technology can now be used to produce new traits in
agriculturally important crops. These traits include the
improvement of human food or animal feed quality [1,2],
the production of industrially useful molecules in plants
[3,4•], the production of pharmaceuticals in plants [5••,P1]
and the manipulation of a plant’s ability to defend itself
against pathogens (as reviewed in [6]).
The ability to manipulate plant metabolism at will is,
however, dependent upon an understanding of metabolic
regulation. Generally, increased or reduced expression of
a gene encoding a metabolic enzyme will produce the
desired phenotype only if that enzyme has significant
control of the flux through the entire pathway in question.
This review will demonstrate that enzymes previously
regarded as the regulatory step in a given pathway may not
prove to be the most efficient point at which to manipulate
the pathway to increase product levels. I will illustrate
this point by comparing some different strategies used in
attempts to increase flux through a variety of important
branches of metabolism. I will then go on to describe the
flipside of these strategies, where increased product levels
can be suppressed by the resultant increase in catabolism,
or can be maintained by the direct downregulation of
catabolic enzymes. Finally, I will discuss the potential for
producing novel classes of compounds in plants by genetic
engineering.
Early targets in flux control
An example of phenotype alteration may be found in
fatty acid biosynthesis in developing oilseeds [4•]. Tenfold
overexpression of an enzyme involved in the synthesis of
palmitoyl-ACP, a beta-ketoacyl-ACP synthetase I, did not
result in increased palmitate in the oil of transgenic rape,
soy or tobacco. On the other hand, twofold overexpression
of a palmitoyl-ACP thioesterase in rape and soy plants
resulted in a doubling of oil palmitate content in those
plants. Thus, it is apparent that the thioesterase has a
relatively high control co-efficient in plant oil biosynthesis.
This experiment also illustrates an effective method
which improves our understanding of the regulation of a
metabolic pathway: manipulating the expression levels of
potential control enzymes in the pathway and analyzing
the resulting changes in flux or pathway product.
When this kind of analysis is done the results are often
surprising. Thus, for example, the action of phosphofructokinase (PFK) is cited in many text books as a regulatory
step in glycolysis. Genetically engineered plants, however,
that overexpress genes encoding this enzyme do not
usually display any change in glycolytic or respiratory flux
[7••]. One obvious explanation of this observation is that
some other step has become rate-limiting, resulting in
an accumulation of substrate at some other point of the
pathway. Biochemical analysis of the transformed tissue
suggests that this is not the case, however. Rather there
appears to be an attenuation of the increased substrate
concentration caused by PFK overexpression as the flux
passes down the pathway [8••]. Thus, there was little or no
increase in substrate concentration of the terminal enzyme
of the pathway, and hence no change in pathway product.
The conclusions the authors drew from these experiments
[8••] is that over-expression of single enzymes early in
metabolic pathways may be of limited effectiveness in
increasing overall flux through that pathway. One possible
corollary of this hypothesis is that, in some cases, changes
in the expression level of genes encoding enzymes close
to the terminal reaction of a pathway may have more
effect on pathway product than changes in the expression
of genes encoding enzymes normally thought of as flux
generating or as committing steps for a particular branch of
that pathway. Although this seems counter-intuitive, there
are some recent examples in plants which may support this
postulate.
In developing oilseeds the fatty acid chains which
constitute the triacylglycerol product are synthesized
in the plastid (Figure 1). They are released into the
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Plant biotechnology
cytoplasm as acyl-CoA moieties which are then assembled
onto the glycerol backbone by fatty acyltransferases. There
is a unique acyltransferase for placing a fatty acyl chain on
each of the three carbons on the glycerol backbone.
The reaction which is often cited as the flux-generating
or committing step in the synthesis of fatty acids, and
hence of triacylglycerol, is the conversion of acetyl-CoA
to malonyl-CoA [4•]. This reaction is catalyzed by the
Figure 1
0R
18:1-0
0-MGDG
Plastid
C3:0-ACP
C3:0-CoA
C4:0-ACP
C2:0-CoA
ACCase
C6:0-ACP
*
0R
18:2-0
0-MGDG
C8:0-ACP
C10:0-ACP
C12:0-ACP
KAS 1
C14:0-ACP
C16:0-ACP
C18:0-ACP
C18:1-ACP
0R
18:3-0
FAT B
(16:0 thioesterase)
0-MGDG
FAT A
(18:1 thioesterase)
*
C16:0-CoA
C18:1-CoA
18:1-DAG
18:3-DAG
DAG
✽
LPA
acyl-CoA pool
PA
DAG
TAG
SLC1
C18:2-CoA
C18:3-CoA
0R
18:3-0
0R
Fad 2
0R
18:1-0
18:2-0
0-PdtC
Endoplasmic
reticulum
C18:1-CoA
0-PdtC
✽
0-PdtC
Current Opinion in Plant Biology
Oil biosynthesis in developing seeds. Potential flux control points are marked with asterisks. Fatty acids are synthesized by acetyl-CoA in
the plastid. The acyl chains are extended by the condensation of the growing acyl-ACP with malonyl-ACP, reactions catalyzed by β-ketoacyl
synthetases (e.g. KAS I). Each condensation is a four step process and the condensations are the rate limiting steps for elongation of the
chain. Condensing enzyme activities, however, do not have high control coefficients for acyl pool composition. The acyl chains are released
into the cytoplasm by acyl-ACP thioesterases. The activity of these thioesterases, especially the 16:0-thioesterase (FAT B), relative to the
flux through the pathway, determines the acyl-CoA composition of the cytoplasm. Acyl CoAs are incorporated into membrane phospholipid
where the second and third double bonds are added by phospholipid desaturases. Desaturated acyl-CoAs are returned to the cytoplasmic
pool. The desaturation step is the rate limiting step in this process and increases in desaturase activity, especially Fad 2, result in increased
desaturation in the final product. Acyl-CoAs are attached to the glycerol backbone by three separate acyltransferases (e.g. lysophosphatidic
acid acyltransferase). Small changes in the activities of FAT B and Fad 2 result in corresponding changes in the fatty acid composition of the
oil. Changes in lysophosphatidic acid acyltransferase activity (demonstrated by expressing a yeast lysophosphatidic acid acyltransferase, SLC1,
in seeds) result in increased product (oil) content.
Manipulating flux through plant metabolic pathways Kinney
enzyme acetyl-CoA carboxylase. It has not yet been
possible, however, to demonstrate any significant changes
in the total oil content of transgenic oilseeds overexpressing genes for acetyl-CoA carboxylase, the supposedly
flux-generating step of fatty acid biosynthesis [3].
Late targets in flux control
The yeast gene SLC 1-1 was isolated as a variant of the
yeast gene SLC 1, which itself was isolated from a mutant
unable to make sphingolipids [9]. SLC 1-1 was able to
complement the mutant and it was suggested that its
function was to transfer long-chain acyl groups to the sn-2
position of phosphatidylinositols. The mutant gene, slc 1,
had a single base change which caused it to lack this
function. Recently it was shown that the SLC 1-1-gene was
also able to transfer acyl groups to the sn-2 position of plant
triacylglycerols when it was expressed in Arabidopsis and
oilseed rape seeds [10••]. Furthermore, transgenic seeds
over-expressing SLC 1-1 had an increased total oil content.
Thus, in this instance, it appears that increased activity of
a terminal reaction resulted in increased flux through the
entire pathway. It will be interesting to determine if this
is an isolated case or if similar results will be obtained for
other oilseed plants or for other metabolic pathways.
Another example of increasing flux into product by
increasing the activity of a near-terminal enzyme comes
from the isoprenoid pathway. This is a highly branched
pathway which originates with 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) [11]. From the essentially
linear conversion of HMG-CoA to ubiquinone there
are many branches to important metabolites such as
cytokinins, squalene, sterols, carotenoids and tocopherols.
The activity of the enzyme HMG-CoA reductase was
for many years believed to be a rate-limiting step in
sterol biosynthesis and possibly that of other isoprenoids.
Increasing the activity of this enzyme was then an
obvious target for increasing flux through the entire
pathway and thus the concentration of useful isoprenoid
end-products. A few years ago, however, it was shown that
increasing the catalytic activity of HMG-CoA reductase
in transgenic tobacco, corn and tomatoes resulted in an
accumulation of only cycloartenol rather than sterols or
other isoprenoids [P2]. More recently a number of groups
[P3••,P4••,P5•] have demonstrated that overexpression of
phytoene desaturase, an enzyme close to the terminal
reactions of carotenoid biosynthesis, resulted in a large
increase in the concentration of the orange pigment
lutein in various transgenic plants — thus supporting the
hypothesis that increased levels of terminal enzymes in
a pathway may indeed have more effect on pathway
products than directly manipulating those enzymes that
were traditionally thought of as rate-limiting. The results
of metabolic flux experiments in transgenic plants must
be interpreted with care if they are to be used to devise
a strategy for the increased production of products of
commercial value.
175
Catabolic targets in flux control
In some cases the flux through a pathway may be
greatly increased as a result of increased expression of
one or more enzymes in that pathway. The steady-state
concentration of pathway product, on the other hand,
may not change because of some other effect, such as
an increased rate of catabolism of the product. Attempts
to increase the free lysine content of seeds are a good
example of this. A key reaction in the biosynthesis of
free lysine in plant seeds is the conversion of aspartic
semialdehyde to 2,3-dihydrodipicolinate, catalyzed by
the enzyme dihydrodipicolinate synthase. In plants, the
activity of this enzyme is inhibited by free lysine which
regulates the flux through the synthetic pathway. The rate
of lysine synthesis has been greatly increased in transgenic
tobacco, soy, corn and barley meal by the expression of a
gene encoding a feedback-insensitive dihydrodipicolinate
synthase from either E. coli or Corynibacerium glutamicum.
[12,13]. In a number of these experiments, however, the
steady-state level of lysine remained constant even though
the flux through the entire pathway had been significantly
increased. This was because lysine catabolism had also
increased in these transgenic plants [13]. Increases in
lysine were only observed when the increase of flux into
lysine was so great that the activity of the first lysine
catabolic pathway member, lysine ketoreductase, became
saturated [12,13].
One of the most effective ways of changing flux through
any pathway is by silencing the expression of genes
which encode enzymes at or beyond branch points in
the pathway. In many cases the resultant phenotype
of such experiments is similar to that predicted. For
example, again in the developing oilseed, the synthesis
of polyunsaturated fatty acids may be regarded as a
branch pathway in oil biosynthesis. Oleoyl-CoA may be
incorporated into phosphatidylcholine (PC) for desaturation to linoleoyl-PC and linolenyl-PC, or it may be
incorporated directly into triacylglycerol. Silencing the
gene which encodes the omega-6 desaturase responsible
for the conversion of oleoyl-CoA to linoleoyl-CoA acid in
soybean results in a large increase in the oleic acid content
of the triacylglycerol with a corresponding reduction
in polyunsaturated fatty acid content [4•]. Indeed, this
particular example of pathway engineering has resulted in
the commercial production of oxidatively stable vegetable
oil from transgenic soybeans [14•].
Production of novel compounds
One of the more exciting prospects in pathway engineering is the diversion of flux into novel products by the
expression of genes for enzymes not normally present in
a pathway. It is conceivable that whole new pathways
in plants could be engineered by the expression of a
multitude of transgenes. From a plant breeding and
commercial product development viewpoint, however, the
fewer additional genes added to a pathway the better.
The triacylglycerols of oilseed plants provide a convenient
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Plant biotechnology
Figure 2
HMG-Coa
*
HMG-CoA
reductase
6
st
ep
s
Mevalonic acid
Squalene
Farnesyl pyrophosphate
2 steps
2 steps
1 step
*
Cycloartenol
1 step
Geranylgeranyl pyrophosphate
1 step
*
Methylated cycloartenol
Solenesyl pyrophosphate
2 or more
steps
Many steps
Many steps
Plant methylated sterols
Plastiquinones, ubiquinones, rubber
Phytoene
4 steps
1 step
Lutein
*
β-Carotene
Abscisic
acid
1 step
1 step
Lycopene
Phytoene
desaturase
Phytols
Tocopherols
Diterpenes
γ-Carotene
α-Carotene
Current Opinion in Plant Biology
Overview of isoprenoid metabolism in plants. The pathway must be envisioned as two main branches: the sterol and terpenoid pathways. The
six reactions leading to squalene (via geranyl pyrophosphate), however, are common to both branches and thus sterol and lutein biosynthesis
may also be regarded as branches of a single linear pathway from HMG-CoA to ubiquinones. All plants contain both sterol and terpenoid
pathways but the specific products synthesized depend upon the plant species and tissue type. Potential flux control steps are marked with
asterisks. The pathway begins with mevalonic acid from acetyl-CoA. The rate limiting step in this 3-step pathway is thought to be HMG-CoA
reductase. Increasing HMG-CoA reductase activity in tobacco leaves, however, resulted in an increased cycloartenol content, suggesting that
geranylgeranyl pyrophosphate synthesis is rate-limiting but squalene synthesis is not. A near-terminal reaction, δ-24 methylation, must have a
high control coefficient for this sterol branch of the pathway. Increasing phytoene desaturase in a number of plants resulted in a large increase
in lutein, suggesting that this enzyme has a high control coefficient in the terpenoid branch of the pathway.
source of aliphatic carbon compounds, although current
major crop plants produce only a handful of different fatty
acids with limited nonfood applications. Triacylglycerol
synthesis, however, is an attractive target for modification
for a number of reasons. First, for the high level oleic
acid soybean example above, it has been shown that
large changes in the types of fatty acids found in the
triacylglycerol can be made without affecting the viability
of the crop plant or seed germination [14•]. Secondly,
nature has already provided a precedent for producing
unusual fatty acids in seed oil, over 400 different kinds of
fatty acids have been observed in the oils of exotic oilseed
plants. And lastly, it is theoretically possible to produce
plant oils with entirely new functionality by expressing
only one or two heterologous genes [4•].
A recent example of this is the production of acetylenic
fatty acids in the oils of transgenic seeds [P6••]. Acetylenic
acids, such as crepenynic acid, are industrially useful in
the production of paint and other coatings, as well as
plastics and lubricants. Crepenynic acid is found in the
triacylglycerols of a number of exotic oilseed plants such as
Crepis alpina. It is produced by the action of an acetylase
enzyme on linoleic acid, probably while the linoleate is
Manipulating flux through plant metabolic pathways Kinney
also catalyze related mono-oxygenase reactions, such as
hydroxylation [16••]. Being able to produce fatty acids with
a desired functional group at a specific position in the acyl
chain opens up, among other things, the possibilities of
new polymers and composites, improved PVC plasticizers
and better paints.
Figure 3
Acetyl-CoA
Aspartate
many steps
✽
Conclusions
β-aspartyl phosphate
The future of pathway engineering, therefore, may well
be in protein engineering. Pathway enzymes do not
always catalyze the desired reaction at the required rate
to change flux through the pathway, or they may not
catalyze precisely the desired reaction. The ability to
engineer enzymes with precisely the right catalytic activity
to produce novel pathway products or increased amounts
of existing products may well be the key to long term
success in plant metabolic pathway engineering.
Aspartic
β-semialdehyde
DHDPS
✽
2-3 dihydrodipicolinate
Threonine
Methionine
177
5 steps
Lysine
References and recommended reading
✽
LKR
many steps
Acetyl-CoA
Saccharopine
Current Opinion in Plant Biology
Lysine turnover in plants. In common with oil and isoprenoid
biosynthesis, lysine synthesis originates with acetyl-CoA via the
TCA cycle. Lysine can also be catabolised back to acetyl-CoA.
Potential flux control steps are marked with asterisks. The enzyme
dihydrodipicolinate synthase (DHDPS) is strongly down-regulated
by free lysine. Removing this feedback regulation by expressing a
prokaryotic DHDPS gene in plants can result in large increases
in free lysine in transgenic plants, confirming that this enzyme has
a high control coefficient for product synthesis. Large increases
in saccharopine were also seen in transgenic high lysine plants,
demonstrating that the catabolic part of the pathway is very active
and that lysine ketoglutarate reductase (LKR) is rate-limiting for this
catabolism.
still esterified to phosphatidylcholine. The acetylase is
very closely related to known plant fatty acyl-phospholipid
desaturases. Recently the C. alpina gene encoding this
acetylase was expressed in transgenic Arabidopsis plants,
which subsequently were shown to produce low amounts
of crepenynic acid in their seed oil [P6••]. Thus the
addition of a single enzyme created a whole new
branch point in fatty acid metabolism and triacylglycerol
biosynthesis. Additional genes, perhaps encoding for
acyltransferases which are more efficient at attaching
novel fatty acyl-CoAs to the glycerol backbone, may
be needed before commercially significant concentrations
of acetylenic fatty acid end-products will be made in
domestic crop plants.
Acetylases are members of the mixed-function monooxygenase family, which as well as desaturases includes
enzymes that catalyze the formation of epoxy, hydroxy and
keto groups [15•]. The catalytic function of these enzymes
is similar enough to fatty acid desaturation that it has
been possible to re-engineer desaturases so that they will
Papers of particular interest published within the annual period of review
have been highlighted as:
•
••
of special interest
of outstanding interest
1.
Kinney AJ: Improving soybean seed quality. In Induced
Mutations and Molecular Techniques for Crop Improvement.
Vienna: International Atomic Energy Agency: 1995:101-113.
2.
Herbers K, Sonnewald U: Manipulating metabolic partitioning in
plants. Trends Biotech 1996, 14:198-205.
3.
John ME, Keller G: Metabolic pathway engineering in cotton:
biosynthesis of polyhydroxybutyrate in fiber cells. Proc Natl
Acad Sci USA 1996, 93:1268-1277.
4.
•
Kinney AJ: Genetic engineering of oilseeds for desired traits.
In Genetic Engineering. Edited by Setlow JK. New York: Plenum
Press 1997, 19:149-166.
Comprehensive review of the metabolic engineering of fatty acid biosynthetic
pathways in plants.
5.
Hezari M, Croteau R: Taxol biosynthesis: an update. Planta
••
Medica 1997, 63:291-295.
An update on the biosynthesis of taxol, a diterpenoid chemotherapeutic
agent, with useful discussion on target for pathway engineering to increase
production. A good example of the potential for engineering secondary
metabolism to produce a pharmaceutically important chemical.
6.
Dixon RA, Lamb CJ, Masoud S, Sewalt VJH, Paiva NL: Metabolic
engineering: prospects for crop improvement through the
genetic manipulation of polypropanoid biosynthesis and
defense responses. Gene 1996, 179:61-71.
7.
••
Thomas S, Mooney PJF, Burrell MM, Fell DA: Finite change
analysis of glycolytic intermediates in tuber tissue of lines
of transgenic potato overexpressing phosphofructokinase.
Biochem J 1997, 322:111-117.
Using transgenic plant lines for metabolic control analysis. With its companion paper (see [8••]) postulates novel view of flux generating steps in
metabolic pathways. A different and interesting view of metabolism.
8.
••
Thomas S, Mooney PJF, Burrell MM, Fell DA: Metabolic control
analysis of glycolysis in tuber tissue of potato (Solanum
tuberosum): explanation for the low control coefficient of
phosphofructokinase over respiratory flux. Biochem J 1997,
322:111-117.
With its companion paper [7••] expands on a novel view of flux-generating
steps in metabolic pathways with the postulation that flux-generating steps
may not be ideal targets for pathway flux manipulation.
9.
Nagiec MM, Wells GB, Lester RL, Dickson RC: A suppressor
gene that enables Saccharomyces cerevisiae to grow without
making sphingolipids encodes a protein that resembles an E.
coli fatty acyltransferase. J Biol Chem 1993, 268:22156-22163.
10.
••
Zou J, Katavic V, Giblin EM, Barton DL, MacKenzie SL, Keller
WA, Hu X, Taylor DC: Modification of seed oil content and acyl
178
Plant biotechnology
composition in the Brassicaceae by expression of a yeast sn-2
acyltransferase gene. Plant Cell 1997, 9:909-923.
First demonstration that flux through oil biosynthetic pathways in plants may
be manipulated by increasing the catalytic activity of a terminal reaction, in
this case a yeast acyl-transferase. Expressing the yeast gene in plants results
in an increased seed oil content. If this result turns out to be a general
effect then this would be a viable way of increasing the world’s vegetable
oil production.
hydroxy fatty acids. This is a good example of manipulating the direction of
a pathway flux by expressing a heterologous gene.
11.
•
P1.
Maloney M: Expressing recombinant polypeptide as
fusion with oil body protein. International patent application
December/21st/1995 WO9621029.
P2.
Chappel J, Cuellar RE, Saunders CA, Wolf FR: Plant sterol
accumulation and pest resistance by increasing copy number
of HMG-CoA reductase gene in tobacco, tomato and corn.
European patent application October/10th/1991 EP480730.
Scolnik PA, Bartley GE: A table of some cloned genes involved
in isoprenoid biosynthesis. Plant Mol Biol Rep 1996, 14:305319.
State of the art of our understanding of plant isoprenoid metabolic pathways.
A very good, compact review with a lot of information.
12.
Falco SC, Guida T, Locke M, Mauvais J, Sanders C, Ward RT,
Weber P: Transgenic canola and soybean seeds with increased
lysine. Biotechnology 1995, 13:577-582.
13.
Brinch-Pedersen H, Galili G, Knudsen S, Holm PB: Engineering
of the aspartate family biosynthetic pathway in barley. Plant
Mol Biol 1996, 132:611-620.
14.
•
Kinney AJ, Knowlton S: Designer oils: the high oleic acid
soybean. In Genetic Engineering for the Food Industry. Edited by
Harlander S, Roller S. London: Blackie Academic; 1997:193-213.
Review of the development of a soybean line with genetic modified metabolic
pathway. From gene cloning to product analysis, commercialization and
safety issues. The other chapters in this book are also well worth reading, especially the ones concerning the safety and public acceptance of genetically
engineered plants.
15.
•
Shanklin J, Cahoon EB, Whittle E, Lindqvist Y, Huang W,
Schneider G, Schmidt H: Structure-function studies on
desaturases and related hydrocarbon hydroxylases. In
Physiology, Biochemistry and Molecular Biology of Plant Lipids.
Edited by Williams JP, Kahn MU, Lem NW. Dordrecht: Kluwer
Academic; 1997:6-10.
Good overview of mono-oxygenase type desaturase-related enzymes.
16.
••
Broun P, Somerville CR: Accumulation of ricinoleic, lesquerolic,
and densipolic acids in seeds of transgenic Arabidopsis plants
that express a fatty acyl hydroxylase cDNA from castor bean.
Plant Physiol 1997, 113:933-942.
This paper reports important ground work for re-engineering of pathway enzymes to produce novel pathway products. By inserting a gene from castor
bean into Arabidopsis, the authors report transgenic plants containing novel
Patents
• of special interest
•• of outstanding interest
P3.
••
Brinkhaus FL, Englisah J, Eschenfeldt WH, Hauptman R, Ausich R,
Mukharji I, Poroffitt JH, Yarger JG, Yen HB: Accumulating colored
native carotenoids in transgenic plants. International patent
application October/27th/1995 WO9613149.
This paper demonstrates that the manipulation of terminal enzymes of isoprenoid metabolism, in this case phytoene desaturase, can lead to an increased carotenoid content in plants.
P4.
••
Grierson D, John I, Karvouni Z, Taylor J, Turner A, Watson C:
New isolated DNA encoding melon phytoene desaturase.
International patent application July/6th/1995 WO9602650.
A similar study to that cited in [P3••]. Manipulation of terminal enzymes of
isoprenoid metabolism to increase carotenoid content of plants.
P5.
•
Braun CJ, Trulson AJ: Visual identification of transgenic plant
material by production of carotenoid pigment encoded by
cassette containing Erwinia phytoene desaturase. International
patent application March/29th/1997 WO9714807.
Use of technology in [P3••] and [P4••] as a selection for transgenic plant tissue with engineered metabolism. Makes good use of a genetic manipulation
which results in a colored phenotype, in this case as a selectable marker.
P6.
••
Gummeson P, Lee M, Lenman M, Sjoedahl M, Stymne S: New
acetylase used for production of crepenynic acid from
linoleic acid. International patent application February/14th/1997
WO9737033.
An exciting report of a desaturase-related enzyme which inserts a triple bond
into fatty acids and genetic engineering of plants to produce these novel fatty
acids. The enzyme — an acetylase — is structurally and functionally similar to
fatty acid desaturases.