Diverse Activities of Carotenoid Cleavage Oxygenases - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 408

19.6 COMMERCIAL RELEVANCE OF APOCAROTENOIDS
Apocarotenoids play important roles in our daily lives as aroma compounds and pigments.
Production of aroma compounds and pigments and the development of new aroma compounds for
mostly food applications provide opportunities for the use of enzymatic processes (either in vivo or
in vitro ) for their synthesis. Cooxidation systems employing LOXs, peroxidases, or xanthin oxidases
are the current processes for enzymatic carotenoid cleavage to yield aroma compounds. The mecha-
nism behind these reactions is the generation of a free radical species from a cofactor which then
cleaves the carotenoid substrate. The free radical generation, however, results in a mixture of cleav-
age products because it lacks specii city. CCOs in general have broad substrate specii cities, but
cleave with high regioselectivity, making them appealing enzymes for the production of l avor and
fragrance compounds (Winterhalter and Rouseff 2002). However, because of poor in vitro activi-
ties of most CCOs described today, biosynthesis of apocarotenoids in engineered hosts expressing
recombinant CCOs appears presently to be the most feasible biotechnological application of this
class of enzymes. The utilization of CCOs in in vitro transformation reactions i rst requires a better
understanding of their enzymatic properties.

19.6.1 A POCAROTENOID B IOSYNTHESIS IN R ECOMBINANT H OSTS
The low threshold values, characteristic aroma notes, and potency of aroma volatiles derived from
carotenoids (C 40 ) has led to the isolation and structural elucidation of a wide variety of carote-
noid aroma compounds (mainly C 9 -C 13 ) from plant extracts (reviewed by Winterhalter and Rouseff
2002). Volatile 9-13 carbon carotenoid cleavage compounds such as b-ionone, a-ionone, dihydro-
actinidiolide, gerinol, damascenol, and eugenol serve in plants as pollinator attractants, antifungals,
or to deter pests (Pichersky and Gershenzon 2002). The C 13 ionones are found in many fruit l avors
(raspberry, blackberry, blackcurrant, peach, apricot, melon, tomato), plant odors (violet, black tea,
tobacco, carrot, vanilla), and mushrooms (Chanterelle). The structurally diverse aroma chemicals
derived from oxidative cleavage of carotenoid compounds result from the tremendous structural
diversity of carotenoid precursors (more than 800 known), cleavage site variations, and subsequent
oxidative modii cations and glycosylations of the cleavage products (Enzell 1985). The production
of volatile aroma and l avor compounds from recombinant carotenoid cleavage reactions is one
potential industrial application of CCOs (Marasco and Schmidt-Dannert 2003). Functional expres-
sion of CCO from all kingdoms has been achieved in recombinant E. coli coexpressing carotenoid
biosynthetic genes and found to exhibit carotenoid cleavage activity in vivo (von Lintig and Vogt
2000, Kiefer et al. 2001, Schwartz et al. 2001). Thus, coexpression of many of the available caro-
tenoid biosynthetic pathways together with different types of carotenoid cleavage enzymes opens
new avenues for the production of structurally diverse carotenoid aroma compounds in engineered
microbial or plant systems.
Two examples of important industrial apocarotenoid products include the pigment, bixin, and the
spice, saffron. Saffron, the most expensive spice in the world ($1000-2000 kg −1 ), is comprised of
water soluble apocarotenoid glycosides and found in the dry stigma of Crocus sativus . The majority
of the color derives from crocetin esters which are created by the cleavage of zeaxanthin (Tarantilis
et al. 1995). This cleavage reaction is catalyzed by a zeaxanthin-specii c 7,8 (7 ) cleavage dioxy-

genase ( Cs ZCD) (see Section 19.3). The heterologous expression of both the Cs ZCD and a suitable
glucosyl transferase together with a carotenoid biosynthesis pathway would lead to a competitive
alternative to natural crocin production (which is extremely tedious as it requires manual picking of
Crocus stigmata) by production in hosts such as E. coli or yeast. Similar strategies using recombi-
nant enzymes to produce bixin (an important colorant for dairy products) in a heterologous host has
industrial implications. Unlike crocetin ester biosynthesis, all genes necessary for bixin biosynthe-
sis have recently been cloned and expressed in E. coli making it feasible to develop a biotechnologi-
cal production process (Bouvier et al. 2003).

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