Control carotenoids

Little is known of how the biosynthetic metabolon is assembled, what mechanisms control the membrane-specific targeting, and how the conversions to apocarotenoids occur. Yet the current approach to drive import of bacterial or plant genes is to use transit sequences of a stromal protein that may limit the effectiveness of the transgene. In addition, for specific applications of controlling carotenoid composition, we need to better understand the interactions of the various enzymes,... 

IPP react with each other, releasiag pyrophosphate to form another allyl pyrophosphate containing 10 carbon atoms. The chain can successively build up by five-carbon units to yield polyisoprenes by head-to-tad condensations alternatively, tad-to-tad condensations of two units can yield squalene, a precursor of sterols. Similar condensation of two C2Q units yields phytoene, a precursor of carotenoids. This information is expected to help ia the development of genetic methods to control the hydrocarbon stmctures and yields. 

Carotenoids are also used as pigments and dietary supplements in animals and poultry feedstuffs. They are added to pharmaceutical products to provide a form of control during manufacturing and to distinguish one product from another. They also enhance the aesthetic aspects of the products (210). 

Carotenoids have two general characteristics of importance to the food iadustry they are not pH sensitive ia the normal 2—7 range found ia foods, and they are not affected by vitamin C, making them especially important for beverages. They are more expensive than synthetic food dyes and have a limited color range. In their natural environment they are quite stable, but they become more labile when heated or when they are ia solution. Under those conditions, there is a tendency for the trans-double bonds to isomerize to the cis-stmcture with a subsequent loss of color iatensity. The results of controlled tolerance and toxicity tests, usiag pure carotenoids, iadicate that they are perfecdy safe as food colors (132). 

A small but variable proportion of the carotenoids with one or two P-ionone rings (mainly P-carotene) are cleaved in the enterocytes to produce retinol (vitamin A). This process is very tightly controlled, so that too much vitamin A is not produced, although the control mechanism is not clear. Some cleavage of P-carotene can also occur in the liver, but this does not account for the turnover of P-carotene in the body. Small amounts of carotenoids are subject to enterohepatic circulation, but this does not account for losses. 

Clearly, the control of gene expression at the transcriptional level is a key regulatory mechanism controlling carotenogenesis in vivo. However, post-transcriptional regulation of carotenoid biosynthesis enzymes has been found in chromoplasts of the daffodil. The enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) are inactive in the soluble fraction of the plastid, but are active when membrane-bound (Al-Babili et al, 1996 Schledz et al, 1996). The presence of inactive proteins indicates that a post-translational regulation mechanism is present and is linked to the redox state of the membrane-bound electron acceptors. In addition, substrate specificity of the P- and e-lycopene cyclases may control the proportions of the p, P and P, e carotenoids in plants (Cunningham et al, 1996). 

CUNNINGHAM F X Jr, POGSON B, SUN Z, MCDONALD K A, DELLAPENNA D and GANTT E (1996) Functional analysis of the (3 and e lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation . Plant Cell, 8, 1613-26. 

Carotenoids and prostate cancer — Numerous epidemiological studies including prospective cohort and case-control studies have demonstrated the protective roles of lycopene, tomatoes, and tomato-derived products on prostate cancer risk other carotenoids showed no effects. " In two studies based on correlations between plasma levels or dietary intake of various carotenoids and prostate cancer risk, lycopene appeared inversely associated with prostate cancer but no association was reported for a-carotene, P-carotene, lutein, zeaxanthin, or p-cryptoxanthin. - Nevertheless, a protective role of all these carotenoids (provided by tomatoes, pumpkin, spinach, watermelon, and citrus fruits) against prostate cancer was recently reported by Jian et al. ... 

Carotenoids and breast cancer — Among seven case-control studies investigating the correlation between different carotenoid plasma levels or dietary intakes and breast cancer risk, five showed significant inverse associations with some carotenoids. - In most cases, this protective effect was due to 3-carotene and lutein. However, one (the Canadian National Breast Screening Study ) showed no association for all studied carotenoids including (I-carotene and lutein. More recently, another study even demonstrated a positive correlation between breast cancer risk and tissue and serum levels of P-carotenes and total carotenes. Nevertheless, these observational results must be confirmed by intervention studies to prove consistent. 

Carotenoids and urino-digestive cancers — On the whole, findings from epidemiological studies did not demonstrate a protective role of carotenoids against colorectal, gastric, and bladder cancers. Indeed, most prospective and case-control studies of colorectal cancer showed no association with dietary intake or plasma level of most carotenoids. - Only lycopene and lutein were shown to be protective against colorectal cancer. Otherwise, findings from the ATBC study s showed no effect of P-carotene supplementation on colorectal cancer. 

Carotenoids and cardiovascular diseases — Numerous epidemiological studies aimed to study the relationship of carotenoids and cardiovascular diseases (CVDs) including coronary accident risk and stroke. It appeared then that observational studies, namely prospective and case-control studies, pointed to a protective effect of carotenoids on myocardial infarct and stroke, but also on some atherosclerosis markers such as intima media thickness (IMT) of the common carotid artery (CCA) and atheromatous plaque formation. 

Among 27 prospective and case-control studies, 16 reported inverse associations between some carotenoids and CVDs, taking plasma or serum concentration as carotenoid biomarkers (11 of 16 studies), dietary intake (5 of 16 studies), or adipose tissue level (1 of 16 studies). With regard to the findings from the studies based on CVD risk, only two of seven presented significant inverse associations of carotenoids, particularly lycopene and P-carotene, whereas five studies of nine showed inverse correlations between myocardial infarcts and lycopene and/or P-carotene the others presented no associations. ... 

Some prospective and case-control studies also investigated the relationship of carotenoids and the evolution of CCA-IMT. Although the EVA study showed no association between total carotenoids and IMT, others like the ARIC study, the Los Angeles Atherosclerosis Study, " and the Kuopio Ischaemic Heart Disease Risk Factor Study demonstrated the protective role of isolated carotenoids such as lycopene, lutein, zeaxanthin, and P-cryptoxanthin on IMT. Thus, findings from prospective and case-control studies have suggested that some carotenoids such as lycopene and P-carotene may present protective effects against CVD and particularly myocardial infarcts and intima media thickness, a marker of atherosclerosis. 

Dorgan, J.F. et al.. Relationships of serum carotenoids, retinol, alpha-tocopherol, and selenium with breast cancer risk results from a prospective study in Columbia, Missouri (United States), Cancer Causes Control, 9, 89, 1998. 

Nkondjock, A. and Ghadirian, P, Dietary carotenoids and risk of colon cancer case-control study, Int. J. Cancer, 110, 110, 2004. 

Olmedilla, B. et al.. Serum status of carotenoids and tocopherols in patients with age-related cataracts a case-control study, J. Nutr. Health Aging, 6, 66, 2002. 

Insights into the mechanisms of carotenoid degradation can be followed in model systems that are more easily controlled than foods and the formation of initial, intermediate, and final products can also be more easily monitored. However, extrapolation to foods must be done with caution because simple model systems may not reflect the nature and complexity of a multicomponent food matrix and the interactions that can occur. In addition, even in model systems, one must keep in mind that carotenoid analysis and identification are not easy tasks. 

Since it is easier to control and change the conditions of carotenoid studies carried out in model systems, information on degradation kinetics (reaction order model, degradation rate, and activation energy) and products formed are often derived from such studies. 

Plant extracts rich in carotenoids, hydrolyzed with acetic and propionic aldehydes under controlled temperature and pressure... 

Stable dispersion of water-insoluble and/or hydrophobic natural pigment such as carotenoid, curcumin, porphyrin pigment, or vegetable carbon black in form of bodies of average size of 10 ram Addition of 0.5 ppm P-carotene to yogurt containing 200 ppm riboflavin color did not change after 40 days at 6°C compared with control (decoloration at 1 day)... 

Over-expression of bacterial phytoene synthase led to only modest increases in pigment accumulation (except in the case of chloroplast-contaiifing tissues). Attention turned to CrtI, one gene that might control flux through the entire four desaturation steps from phytoene to lycopene (discussed in Section 5.3.2.4). Only a modest increase in carotenoid content in tomatoes and a variety of changes in carotenoid composition including more P-carotene, accompanied by an overall decrease in total carotenoid content (no lycopene increase), resulted when CrtI was over-expressed under control of CaMV 35S. Apparently, the bacterial desaturase... 

Transgenic E. coli accumulate comparatively low levels of carotenoids " compared to microbial algae, yeasts, and bacteria. Many efforts ° have focused on increasing accumulation by manipulation of factors affecting metabolic flux and metabolite accnmnlation (listed and discnssed in Sections 5.3.1.1 and 5.3.1.3 A) and have been reviewed." - " In bacterial systems, approaches to control can be categorized as either infrastructural (plasmids, enzymes, strains) or ultrastructural (media and feeding, enviromnent, precursor pools, substrate flux). 

Understanding mechanisms controlling metabolon localization in plastids of different membrane architectures Little is known about metabolon structure, assembly, and membrane targeting. The carotenoid biosynthetic pathway exists on plastid membranes. However, plastids have different membrane architectures and therefore tissue- and plastid-specific differences in membrane targeting of the biosynthetic metabolon can be expected. Localization in chloroplasts that harbor both thylakoid and envelope membranes differs from the envelope membranes in endosperm amy-loplasts. In fact, localization on both thylakoid and envelope membranes implies that the carotenoid pathway is really not a single pathway, but a duplicated pathway that may very well have membrane-specific roles with regard to functions in primary and secondary metabolism. 

Analysis in diverse lines can facilitate identification of useful alleles that control expression of enzymes upstream of the carotenoid pathway, a feature that would not be evident from conventional end-product screening of breeding lines. Moreover, this characterization sets the stage for marker-assisted selection of superior endogenous alleles and facilitates selection of introduced transgenes that may be necessary to supplement the genotypic contribution required for a particular plant chemical outcome. 

SmoUce, C.D., Martin, V.J.J., and Keasling, J.D., Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization, Metabol. Eng. 3, 313, 2001. 

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