The importance of carotenoids, both in plants and organisms that
consume plants in their diets, has fueled efforts in metabolic engineering of the biosynthetic pathway [2] and instilled future interest
in use of synthetic biology to capitalize on these multifunctional
molecules [3]. The long-standing efforts to manipulate the pathway have not always led to expected results [4] because fundamental understanding of pathway regulation, in particular the
organization of the multienzyme complex, is poorly understood
[5]. The carotenoid biosynthetic pathway enzymes are nuclearencoded and localize to chloroplasts and other plastids via targeting
mediated by N-terminal transit peptides that are cleaved upon
plastid import. Research to date indicates that the carotenoid biosynthetic enzymes can be found in various locations within plastids
[5]. The carotenoid biosynthetic pathway enzymes are part of
poorly described, high molecular weight complexes, and the pathway shows evidence of substrate channeling, as expected for a
multienzyme pathway structure. Enzymes such as phytoene
synthase (PSY) require membrane association for activity and PSY
isozymes show varying locations within the plastid, while other
downstream pathway enzymes are widely distributed within plastids [6]. However, there is a dearth of information on how the
complete biosynthetic machinery is assembled to form a fully functional complex. This gap in knowledge severely limits opportunities
for predictable engineering of the pathway, especially in the variable
genotypes of plants.
Early studies to explore carotenoid enzyme localization
entailed polyclonal antibodies raised against the biosynthetic
enzymes (for examples, see Refs. [7, 8]). Use of antibodies for
localizing the carotenoid enzymes requires high-level protein
expression in bacteria, followed by purification of large quantities
of the carotenoid enzymes needed to raise antibodies in animals.
This immunological approach is time consuming and fraught with
problems, including variable antigenic specificities. Furthermore,
antibodies may reveal location, but not enzyme–enzyme interactions in vivo. Recently, we utilized fluorescence microscopy and
transient expression in protoplasts for in vivo localization of carotenoid enzymes in maize and other plants [6, 8, 9] and tested
potential interactions between carotenoid enzymes [10]. This
approach skips the problematic step of high level expression of
carotenoid enzymes needed for immunolocalization experiments,
and also allows for the rapid (as compared to immunolocalization
or stable transformation) in vivo examination of carotenoid enzyme
interactions. In this method, plasmids encoding a fluorescent protein, most commonly Green Fluorescent Protein (GFP), that is
C-terminally fused to the carotenoid enzymes, are delivered to
isolated plant protoplasts. The sequence encoding the carotenoid
enzyme includes the native transit peptide needed for plastid targeting and import. After a period of incubation to allow protein
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Maria Shumskaya et al.
consume plants in their diets, has fueled efforts in metabolic engineering of the biosynthetic pathway [2] and instilled future interest
in use of synthetic biology to capitalize on these multifunctional
molecules [3]. The long-standing efforts to manipulate the pathway have not always led to expected results [4] because fundamental understanding of pathway regulation, in particular the
organization of the multienzyme complex, is poorly understood
[5]. The carotenoid biosynthetic pathway enzymes are nuclearencoded and localize to chloroplasts and other plastids via targeting
mediated by N-terminal transit peptides that are cleaved upon
plastid import. Research to date indicates that the carotenoid biosynthetic enzymes can be found in various locations within plastids
[5]. The carotenoid biosynthetic pathway enzymes are part of
poorly described, high molecular weight complexes, and the pathway shows evidence of substrate channeling, as expected for a
multienzyme pathway structure. Enzymes such as phytoene
synthase (PSY) require membrane association for activity and PSY
isozymes show varying locations within the plastid, while other
downstream pathway enzymes are widely distributed within plastids [6]. However, there is a dearth of information on how the
complete biosynthetic machinery is assembled to form a fully functional complex. This gap in knowledge severely limits opportunities
for predictable engineering of the pathway, especially in the variable
genotypes of plants.
Early studies to explore carotenoid enzyme localization
entailed polyclonal antibodies raised against the biosynthetic
enzymes (for examples, see Refs. [7, 8]). Use of antibodies for
localizing the carotenoid enzymes requires high-level protein
expression in bacteria, followed by purification of large quantities
of the carotenoid enzymes needed to raise antibodies in animals.
This immunological approach is time consuming and fraught with
problems, including variable antigenic specificities. Furthermore,
antibodies may reveal location, but not enzyme–enzyme interactions in vivo. Recently, we utilized fluorescence microscopy and
transient expression in protoplasts for in vivo localization of carotenoid enzymes in maize and other plants [6, 8, 9] and tested
potential interactions between carotenoid enzymes [10]. This
approach skips the problematic step of high level expression of
carotenoid enzymes needed for immunolocalization experiments,
and also allows for the rapid (as compared to immunolocalization
or stable transformation) in vivo examination of carotenoid enzyme
interactions. In this method, plasmids encoding a fluorescent protein, most commonly Green Fluorescent Protein (GFP), that is
C-terminally fused to the carotenoid enzymes, are delivered to
isolated plant protoplasts. The sequence encoding the carotenoid
enzyme includes the native transit peptide needed for plastid targeting and import. After a period of incubation to allow protein
224
Maria Shumskaya et al.