Alan Mackenzie

Regulation of carotenoid biosynthesis.

Publisher Summary This chapter discusses the regulation of carotenoid biosynthesis. The carotenoids constitute the most widespread group of pigments in nature. They are present in all photosynthetic organisms, where they form an essential part of the photosynthetic apparatus. They are also responsible for most of the yellow to red colors of flowers and fruits as they occur within chromoplasts. In addition, they are found in certain fungi and some nonphotosynthetic bacteria. The diversity of structures, and hence of biosynthetic pathways within the carotenoid group, together with their diverse locations and functions in complex organelles, such as the chloroplast or other photosynthetic membranes, clearly implies that efficient regulatory mechanisms are present within cells to control not only the rates of biosynthesis and metabolism of carotenoids in response to environmental and developmental changes but also the deposition at their functional locations within a cell. As carotenoids are terpenoids, they are related biosynthetically and share a common early pathway with other biologically important isoprenoids such as sterols, gibberellins, and terpenoid quinines.

Terpenoid biosynthesis in cell extracts of wild-type and mutant strains of Gibberella fujikuroi

Abstract Cell extracts from shake cultures of wild-type and mutant strains of Gibberella fujikuroi converted 3-(RS)-[2-14C]mevalonic acid into carotenes, squalene and precursors of gibberellins. Neurosporaxanthinand phytoene-superproducing strains (SG22 and SG78, respectively), produce in vitro more carotenes than the wild type or a torulene-superproducer (SG68). A more detailed study of strain SG22 established the dependence of carotenogenic activities on protein concentration and incubation time, as well as age of culture. In vitro activities correlated with the predominant activities in vivo. The cytosolic fraction of the cell extract was able to form some 45% of the phytoene produced in the crude cell extract, while the membrane fraction was unable to convert mevalonic acid into carotenes. The albino mutants, SG4, SG75 and SG76, were devoid of carotenoid-synthesising activities, and formed prephytoene pyrophophate from mevalonic acid, indicating that a mutation had occured in the gene for phytoene synthetase.

The photoregulation of carotenoid biosynthesis in Aspergillus giganteus mut. alba

Aspergillus giganteus mut. alba grown in darkness produced no carotenoids, but illuminated shake cultures accumulated 170 μg·g-1 dry weight of β-carotene. Maximum carotenoid production occurred in white light of energy fluence rate of 50 W·m-2. Blue light, but not red light, induced β-carotene formation. A light induction period of 10 h was required for maximum β-carotene synthesis, and this was attained 48 h after illumination. 5-Fluorouracil, actinomycin D and cycloheximide prevented photoinduction of carotenogenesis, indicating that photoregulation is at the transcriptional level. Comparisons of carotenogenic enzyme activities of light- and dark-grown cultures showed that phytoene synthetase, phytoene dehydrogenase and lycopene cyclase were totally photoinduced. Photoinduction of all three carotenogenic enzymes occurred after 12 h illumination. Squalene formation increased some four-fold upon illumination.

Phycomyces blakesleeanus car B mutants: Their use in assays of phytoene desaturase

Abstract Strains of car B (phytoene-accumulating) mutants of Phycomyces blakesleeanus have been characterized with respect to their carotene contents, in vitro formation of isoprenoids from [2-14C] mevalonic acid and their ability to produce [14C]phytoene in situ for use in coupled assays of phytoene desaturase activity. All strains produced predominantly (15-Z)-phytoene both in vivo and in vitro. Other isoprenoids were produced by cell extracts including squalene, sterols, prenyl diphosphates and prenyl alcohols. The addition of 1% Tween 60 to crude cell extracts of the mutants partially restored wild type carotenogenic activity and also altered the proportions of other isoprenoids formed. However, in a cytosolic fraction of the car B mutant, the addition of 1% Tween 60 did not result in the production of any carotenoid from phytoene. This fraction was the most effective source of [14C] phytoene for use in coupled assays of phytoene desaturase activity.

Characterization of ent-kaurene oxidase activity from Gibberella fujikuroi

Cell extracts of wild-type and mutant strains of Gibberella fujikuroi were assayed for kaurene oxidase activity, using ent-[3H]kaurene as the substrate. Extracts from strain SG78 exhibited the highest specific activity, and were used in subsequent experiments. The microsomal enzyme activity was solubilized with buffers or salt solutions at a concentration of 400 mM. Both the soluble and microsomal preparations showed characteristic cytochrome P-450 spectra, ligand binding spectra with the substrate and with the plant growth regulator, paclobutrazol, and inhibition of enzymic activity by carbon monoxide. The addition of 20% glycerol to the extraction buffer stabilized the activity to some extent. Loss of enzymic activity on storage was accompanied by conversion of P-450 to P-420. Michaelis-Menten kinetic parameters for the membrane-bound and soluble enzyme have been estimated, as have constants for the binding of ent-kaurene and paclobutrazol to the P-450 and P-420 forms of the protein.

Carotene biosynthesis by a cell extract of Aspergillus giganteus mut alba

Abstract A cell extract prepared from lyophilized mycelia of light-grown cultures of Aspergillus giganteus mut alba converted [2- 14 C]mevalonic acid into phytoene, lycopene, β-carotene and squalene, but from similar preparations from dark grown cultures formed only squalene. The carotenogenic activities of the cell extracts varied with the age of the cultures. Phytoene synthetase was located in the cytosolic fraction, whereas the dehydrogenation and cyclisation steps were catalysed by membrane-bound enzymes. Dithiothreitol, ATP, Mn 2+ , Mg 2+ , NAD and NADP were essential for the formation of carotenes from mevalonic acid, whilst FAD was required for phytoene metabolism. Oxygen enhanced the conversion of phytoene into other carotenes.

Changes in the sweet proteins (thaumatins) in thaumatococcus danielli fruits during development

Abstract The thaumatin content (forms TO, TI and TII) of fruits from Thaumatococcus danielli at various stages of maturation were examined. The amounts of all three forms of sweet protein increased during maturation to reach a total of about 50 mg/g in mature aril tissue in fruits from both the Ashanti and Kadjebe regions of Ghana. TO was a minor form in fruits at all stages of development from both regions. The major regional difference was that TII was absent from the Kadjebe fruits; however, the total level of sweet proteins was maintained by increased levels of TO and TI. Structural and immunological comparisons of the three thaumatin forms showed that TO is closely related to the other two forms which are known to differ at only five positions in their primary structure.

Secretion and biosynthesis of N-acetyl-β-D-hexosaminidases by human placental tissues in vitro

Chorionic villi selectively secrete the B isoenzyme and, to a lesser extent, the A isoenzyme of lysosomal beta-hexosaminidase when incubated in vitro, suggesting that this tissue could contribute to maternal serum beta-hexosaminidase B and A activities in vivo. The results make it unlikely, however, that the placenta is the source of the I2 isoenzyme found in maternal serum. Samples of amnion and term chorion laeve secreted predominantly the B isoenzyme whereas first-trimester chorion laeve secreted the A, B and I2 isoenzymes. Amnion and chorion laeve may, therefore, be a source of the A, B and I2 activities found in amniotic fluid. Chorionic villi supported the cycloheximide-sensitive incorporation of [3H]leucine into immunoreactive beta-hexosaminidase thereby providing direct evidence for enzyme biosynthesis. Newly synthesized beta-hexosaminidase was detectable in the medium only after extended incubation and the secretion of beta-hexosaminidase activity continued at normal rates for several hours in the absence of protein synthesis. These results indicate that enzyme synthesis is not a primary factor in the regulation of enzyme secretion.

Cloning in a cosmid vector of complete 37 kb and 25 kb ribosomal DNA repeat units from the chicken.

DNA fragments of up to 40 kb containing rRNA-coding sequences have been isolated from a chicken liver DNA library prepared in the cosmid pHC79. Characterization of the cloned DNA by R-loop and restriction mapping has shown that there are two size classes of repeat unit, one of 37 kb and one of 25 kb, the larger of which is a family of units which vary slightly in size. These two classes were shown to be present in the DNA of a single chicken. The size of the internal transcribed spacer in the chicken was measured to be 4.4 kb from analysis of R-loops and heteroduplexes between chicken and Xenopus laevis rDNAs. No introns were observed in either the 18 S or the 28 S coding sequences. The number of copies of the chicken rDNA unit was measured by titration against the cloned sequences to be 202 +/- 51 per haploid genome.