plant organ and thus enhancing the sweet component of the flavor. S-Lin-alool synthase from Clarkia breweri (Onagraceae), an annual plant native to California, was the subject of another patent specification. By expressing the respective nucleic acid sequence in appropriate host plants, enhancement of their scent production has been attempted (18).
1-Aminocyclopropane-l-carboxylic acid oxidase (ACO) is an enzyme involved in the biosynthesis of ethylene, a plant growth regulator initiating fruit ripening. To improve the storage and handling characteristics of cantaloupe charentais melon (Cucumis melo var. cantalupensis, Naud. cv Ved-randais), a cantaloupe melon line was transformed with an ACO antisense gene. A strong reduction of ethylene synthesis and, as a consequence, delayed ripening were achieved. The total quantity of volatiles detected in the antisense fruit was only 20 to 40% of that in the control fruit, indicating that biosynthesis of flavors is strongly controlled by ethylene. Exogenous ethylene was able to restore a qualitative and quantitative aroma profile very similar to that of the control fruit without antisense ACO (19).
Ultimately aiming at the modification of the essential oil composition of aromatic plants by genetic transformation, Faure et al. (20) developed an efficient in vitro shoot regeneration method from spearmint (Mentha spicata L.) and peppermint (Mentha X piperita) leaf disks. On the basis of these results, Diemer et al. (21,22) established an Agrobacterium tumefaciens-mediated transformation procedure for peppermint, spearmint, and cornmint (Mentha arvensis L.). The stable integration and expression of two reporter genes was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription PCR (RT-PCR), and a histoenzymatic assay (21,22).
Although microbial enzymes are already widely applied in industrial flavor production (e.g., Refs. 23,24), the failure in isolating and operating active and stable multienzyme complexes stable in vitro has still hampered broad technical implementation of plant enzymes. Based on up-to-date RNA-DNA techniques, dissection of the complex developmental process of flavor genesis becomes feasible. The identification of flavor related-genes and their corresponding proteins as well as the growing understanding of molecular regulation of gene expression now adds new tools to flavor biotechnology and thus establishes a basis for profitable exploitation.
The genes and peptides of an entire pathway toward the formation of volatile aliphatic and aromatic esters in strawberry fruit (Fragaria sp.) have been disclosed by Aharoni et al. (25). DNA sequences that encode strawberry fruit-specific aminotransferase, pyruvate decarboxylase, thiolase, alcohol dehydrogenase, or acyltransferase were cloned and characterized. Further acyltransferases and esterases involved in aroma genesis were isolated from apple (Malus domestica Borkh.), mango (Magnifera indica L.), and banana (Musa sp.). These nucleic acid or protein sequences may be used in expression systems or to modify plants with the goal of producing natural or synthetic flavors. In this context, a novel strawberry acyltransferase was identified by use of cDNA microarrays combined with appropriate statistical analyses. Such microarray assays allow systematic studies of the expression profiles of large subsets of genes in given tissues under specific physiologic and environmental conditions (26,27). Another approach to isolate ripening-related genes from strawberry fruit utilized the differential screening of a high-quality cDNA library, whereby altogether 26 ripening-related cDNAs were identified (28).
Complementary DNA clones encoding 4-coumarate-coenzymeA (CoA) ligase (4CL) (EC 18.104.22.168) and caffeic acid O-methyltransferase (EC 22.214.171.124) (29,30), key enzymes of phenylpropanoid metabolism, were isolated from a cDNA library constructed from messenger RNA (mRNA) of a kinetin-treated cell suspension culture of vanilla (Vanilla planifolia Andr.). Based on studies using inhibitors of 4CL, it was concluded that down-regulation of this enzyme by the antisense technique would result in a redirection of the flow of phenylpropanoid precursors from lignin biosynthesis into flavor compounds. These attempts hold much promise because vanillin not only is the impact compound of the world's most popular flavor, but also has antioxidant properties in food. The complex interrelations of phenylpropanoid biosynthesis were reviewed by Dixon et al. (31), who presented examples of genetic engineering of, e.g., tobacco (Nicotiana tabacum L.), alfalfa (Medicago sativa L.), and soybean (Glycine max (L.) Merr.) plants and cell cultures to alter pathway flux.
Several plant enzymes involved in the early steps of terpenoid biosynthesis have been characterized, including acetoacetyl-CoA thiolase (EC 126.96.36.199), mevalonate kinase (EC 188.8.131.52), isopentenyl diphosphate isomer-ase (EC 184.108.40.206), and 3-hydroxy-3-methylglutaryl-CoA synthase (EC 220.127.116.11) (32). Cyclases convert linear isoprenoid diphosphates such as geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate into a variety of mono- and polycyclic hydrocarbons and alcohols. A number of ter-pene cyclases from plants, representing soluble, magnesium-containing enzymes, have been cloned and expressed in Escherichia coli. Thus, there is now the potential to engineer plants to produce specific cyclic terpenes for use in the flavor and fragrance industries (33).
Cloning and expression of the monoterpene synthases (—)-4-S-limo-nene synthase from spearmint, ( + )-bornyl diphosphate synthase from sage (,Salvia officinalis L.), and ( — )-pinene synthase from grand fir (Abies gran-
dis) in E. coli enabled Schwab et al. (34) to gain insight into the mechanistic procedures of the reaction sequence toward cyclic monoterpenes. An overview of the enzymology and regulation of essential oil biosynthesis with detailed description of the reaction mechanisms leading to the basic iso-prenoid skeletons is given in Ref. 35.
Natural (Z)-3-hexenol (leaf alcohol), traditionally isolated from mint terpene fractions, is formed from linoleic and linolenic acid via the lipoxygenase pathway. Lipoxygenases have been isolated, cloned, and characterized from various plant sources (for a review see Ref. 36). The enzymatic genesis of the 13-hydroperoxy linoleic and linolenic acid is followed by the hydroperoxide lyase (HPO-lyase)-catalyzed cleavage to (Z)-3-hexenal, which is subsequently reduced to the corresponding alcohol. As in a reconstituted production system the activity of hydroperoxide lyase proved to be the rate-limiting factor, the gene coding for this enzyme was cloned from banana and heterologously expressed in yeast cells to yield a highly active lyase material (37). Another plant HOP-lyase was purified 300-fold from tomatoes (38). Whereas only 13-hydroperoxides from linoleic acid and a-linolenic acid were cleaved by the tomato enzyme, HOP-lyase from alfalfa (Medicago sativa L.) also accepted 9-hydroperoxides as substrates to form the respective volatile C9-aldehydes (39). The specific activity for 9-hydro-peroxy fatty acids was about 50% of the activity for the 13-isomers.
The characteristic flavor of onion occurs when the enzyme alliinase (EC 18.104.22.168) hydrolyzes S-alk(en)yl-L-cysteine sulfoxides (ACSOs) to form pyruvate, ammonia, and sulfur-containing volatiles. Physical characterization of alliinase and molecular analysis of the respective cDNA revealed that two genes and thus two protein subunits were expressed in onion bulb tissue. Although these genes for alliinase are highly homologous in their DNA sequence, there are differences in the proteins that they code for, probably due to varying degrees of glycosylation (40).
Flavorless glycosides represent one accumulation form of aroma substances in fruit and in many other plant tissues. In addition to developments in the analysis of these polar plant constituents, current attention is focused on biotechnological methods for flavor release and flavor enhancement through enzymatic hydrolysis of the glycosidically bound aroma precursors. The liberation of the "bound" aroma portion offers a tool for the production of natural flavors from otherwise waste materials such as peelings, skins, and stems. Because the practical application of endogenous and exogenous glycosidases is limited by their low activities at neutral pH values and strong inhibition by glucose, the construction of chimeric genes with improved hydrolytic properties and their overexpression in different hosts are the subjects of actual research (Refs. 13,14 and references therein).
An industrially relevant route for the production of a "natural" topnote flavor of concord grapes (Vitis labrusca "concord"), methylanthranilate (MA), is the peroxidase-catalyzed /V-demethylation of methyl iV-methyl-anthranilate (MNMA). Comparison of different commercial peroxidase preparations showed soybean peroxidase to be the most effective biocatalyst for the V-demethylation of MNMA to MA. Upon complete conversion of MNMA, the yield of soybean peroxidase-catalyzed MA amounted to 82% within 10 minutes at 70°C and pH 4 (41).
Current research is focused on the improvement of cacao plants (Theo-broma cacao L.) for the food industry. The efficiency of breeding programs could be increased if genetically based maps were available and markers associated with major quantitative trait loci for quality and productivity could be identified. Even though the target organism in this case is not genetically modified, genomics acts as an invaluable research tool (42).
III. PLANT CELL, TISSUE, AND ORGAN CULTURES
The term tissue culture is applied to any nondifferentiated cell culture grown on solid or, as suspension culture, in liquid medium. As the propagation of the cultures is strictly based on mitotic events, the entire genome and, thus, the full potential to form flavors and fragrances are maintained in each cultured cell. However, organized cultures often exhibit an enhanced capacity to form volatile flavors when compared with that of unorganized cell suspension cultures. Commonly used are the hairy root cultures induced by the transformation of aseptic plantlets with Agrobacterium rhizogenes. Independent of the type of cell culture used, usually simultaneous application of different strategies for the improvement of yields is necessary to reach time-dependent volumetric yields [mg product (L X day) '] sufficient for industrial commercialization. These strategies include the selection of stable, high-yielding cell lines, variation of medium components and gas phase composition, precursor feeding, use of elicitors, in situ product removal, and immobilization techniques. Although individual strategies may result in enhanced secondary metabolite formation, often several strategies have to be combined to give a synergistic response (43,44).
Numerous attempts to produce flavor and aroma compounds by plant cell, tissue, and organ cultures have been described (9,24,45,46). To avoid recapitulations, only references that were not cited in the preceding reviews were considered for the present section.
A. Tissue Cultures
Capsaicin, (Fig. 2), the major pungent principle of chilli pepper (Capsicum frutescens L.), may be extracted from callus cultures. By developing cell
Carvacrol y^ och3
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Our internal organs, the colon, liver and intestines, help our bodies eliminate toxic and harmful matter from our bloodstreams and tissues. Often, our systems become overloaded with waste. The very air we breathe, and all of its pollutants, build up in our bodies. Today’s over processed foods and environmental pollutants can easily overwhelm our delicate systems and cause toxic matter to build up in our bodies.