Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

Production of β-carotene by aRhodotorula strain

Summary The production of -carotene by the biomass ofRhodotorula strain var.glutinis, during the stationary phase of growth and in non-proliferating conditions was assayed. When the cells were transferred to distilled water, the fraction of -carotene produced increased from 130 to 630 g per gram of dried cells.     

Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Effective production of retinal from β-carotene using recombinant mouse β-carotene 15,15′-monooxygenase

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg⁻¹. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l⁻¹ β-carotene, 5% (w/v) Tween 40, and 0.2 U ml⁻¹ enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l⁻¹ retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported.     

Cyclization of lycopene in the biosynthesis of β-carotene

Mycobacterium marinum produces carotenoids when exposed to light or when antimycin A is added. Although the major pigment synthesized is β-carotene, lycopene is accumulated when the induced bacteria are incubated in the presence of nicotine (5 mM) or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) (50 μM). Both of these compounds inhibit β-carotene synthesis by blocking the cyclization of lycopene. When nicotine is removed by washing the cells, the accumulated lycopene is cyclized to form β-carotene. The cyclization of lycopene is not an energy-requiring reaction and, furthermore, does not require oxygen or any other electron acceptor. Chloramphenicol addition also does not inhibit the conversion of lycopene to β-carotene indicating that no de novo protein synthesis is involved. Nicotine appears to act by inhibiting the activity of the enzyme required for the cyclization of lycopene.Although the mode of action of CPTA is similar to nicotine, it cannot be removed by washing once the cells have been incubated in its presence, suggesting that the molecule is tightly bound to the enzyme. The possible active molecular sites of nicotine and CPTA are discussed.     

Kinetics of photoaccumulation of β-carotene in Phycomyces blakesleeanus

Upon carbon starvation the -carotene content of Phycomyces mycelium grown on minimal agar medium disappears with a time lag of about 90 min and a T_1/2 of 68–75 min. If continuous light is given 2 h after starvation, there is an increase in -carotene content with respect to the dark control. This increase has a time lag of 20–25 min. The fluence rate-response curve of wt is biphasic and two mutants in the gene madA (madA7, madA35) and in the gene madB (madB101, madB104) have higher thresholds than wt; madB mutants are blinder than madA mutants. Only blue light is effective and we suggest that it has an effect solely on the catabolism of -carotene.Abbreviations D dark - L light - wt wild type     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Production of deuterated β-carotene by metabolic labelling of Spirulina platensis

Method for production of deuterated -carotene for the bioavailability studies of vitamin A has been developed using Spirulina platensis in culture. Suspension cultures of Spirulina in heavy water (99.4% D₂O) medium produced maximum biomass and -carotene in 28 to 30 days. Of the total carotenoids, lutein constituted 30 to 35% while -carotene was about 24%. MS showed that 60 to 65% H atoms in -carotene were deuterated. 100% replacement of H atom with deuterium was achieved by preventing exchange with atmospheric moisture. The medium could be used in several cycles for metabolic labelling of carotenoids whereby the cost of production is reduced.     

Transcriptome profiling of β-carotene biosynthesis genes and β-carotene accumulation in leaf blades and petioles of celery cv. Jinnanshiqin

β-carotene is a kind of carotenoids and has many biological functions.Proper amount of β-carotene is beneficial to promote the synthesis of vitamin A [1].The unsaturated double bonds in β-carotene structure make it have strong antioxidant capacity.     

Over-production of β-carotene from metabolically engineered Escherichia coli

To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l⁻¹ and 240 mg l⁻¹, respectively, with overall productivities of 7.8 mg l⁻¹ h⁻¹ and 4.8 mg l⁻¹ h⁻¹.     

Development of β-Carotene Rich Maize Hybrids through Marker-Assisted Introgression of β-carotene hydroxylase Allele

Development of vitamin A-rich cereals can help in alleviating the widespread problem of vitamin A deficiency. We report here significant enhancement of kernel β-carotene in elite maize genotypes through accelerated marker-assisted backcross breeding. A favourable allele (543 bp) of the β-carotene hydroxylase (crtRB1) gene was introgressed in the seven elite inbred parents, which were low (1.4 µg/g) in kernel β-carotene, by using a crtRB1-specific DNA marker for foreground selection. About 90% of the recurrent parent genome was recovered in the selected progenies within two backcross generations. Concentration of β-carotene among the crtRB1-introgressed inbreds varied from 8.6 to 17.5 µg/g - a maximum increase up to 12.6-fold over recurrent parent. The reconstituted hybrids developed from improved parental inbreds also showed enhanced kernel β-carotene as high as 21.7 µg/g, compared to 2.6 µg/g in the original hybrid. The reconstituted hybrids evaluated at two locations possessed similar grain yield to that of original hybrids. These β-carotene enriched high yielding hybrids can be effectively utilized in the maize biofortification programs across the globe.     

Participation of β-carotene in reactivation of PSI of heptane-extracted spinach chloroplasts

A carotenoid requirement for photosystem I activity in spinach chloroplasts using extraction-reconstitution technique has been investigated. The transfer of electron from N,N,N,N-tetramethyl-p-phenylene diamine through the chloroplast photosystem to methyl viologen dye or to NADP⁺ was used as an assay of photosystem I activity. Extraction of lyophilized spinach chloroplasts with heptane at near 0°C removed almost all -carotene and reduced photochemical activities associated with photosystem I to a low level (about 15% of the original activity). Reconstitution of the extracted chloroplasts with -carotene completely restored photosystem I activity. The maximum rate of methyl viologen photoreduction in reconstituted chloroplasts occurred at an -carotene/chlorophyll molar ratio of 0.5. Cyclic phosphorylation mediated by phenazine methosulphate was partially restored. Xanthophylls (lutein, neoxanthin, violaxanthin), as components of chloroplast membranes, were not able to replace -carotene in reconstitution of chloroplasts and had essentially no effect on restoring photoreactions. On the basis of the P700/total chlorophyll ratio it can be assumed that extraction of lyophilized chloroplasts with heptane do not affect photosystem I reaction centre. Therefore it is possible that -carotene, removed during heptane extraction and belonging mainly to the antenna pigment pool of photosystem I, is effective in the restoration of photosystem I activity.Abbreviations chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - MV methyl viologen - PMS phenazine methosulphate - PQA plastoquinone A - PS I photosystem I - PS II photosystem II - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - Tricine N-tris(hydroxymethyl)methyglycine. D-1, D-10, D-50, D-144 represent chloroplast subfractions sedimented at 1000 × g, 10,000 g, 50,000 × g and 144,000 × g - s supernatant This paper is a partial fulfillment of the requirements for the Ph.D. degree of A.T. at Maria Curie-Skodowska University, Lublin.     

Deactivation mechanisms of triplet excited state hypericin by β-carotene

The deactivation mechanisms of the triplet excited state hypericin (HYP) by β-carotene (CAR) were studied employing quantum chemical calculations in the present study. The results suggest that CAR may deactivate the triplet excited state HYP through the following two pathways on thermodynamic grounds: (1) direct energy transfer from the triplet excited state HYP to CAR; (2) electron transfer from the triplet excited state CAR, which was formed through direct energy transfer pathway, to the triplet excited state HYP.     

Accumulation of a complex form of β-carotene byPhycomyces blakesleeanus cytoplasmic mutants

Phycomyces blakesleeanus colour mutants of a new class have been isolated. The mutants form red mycelia when grown in the dark and show vegetative segregation. The affected gene, amedcarE, behaves as an extranuclear genetic factor. The red phenotype ofcarE mutants is caused by the accumulation of -carotene in a form showing spectral shift to longer wavelengths. The spectral shift is abolished in vitro by heat or protease treatment and in vivo by the presence of light or retinol.Abbreviations SIV glucose-asparagine minimal medium - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Raman microspectroscopy of algal lipid bodies: β-carotene quantification

Advanced optical instruments can serve for analysis and manipulation of individual living cells and their internal structures. We have used Raman microspectroscopic analysis for assessment of β-carotene concentration in algal lipid bodies (LBs) in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have devised a simple method to measure the concentration of β-carotene in a mixture of algal storage lipids from the ratio of their Raman vibrations. This finding may allow fast acquisition of β-carotene concentration valuable, e.g., for Raman microspectroscopy assisted cell sorting for selection of the overproducing strains. Furthermore, we demonstrate that β-carotene concentration can be proportional to LB volume and light intensity during the cultivation. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on the spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.     

Marker-assisted backcross breeding for enhancing β-carotene of QPM inbreds

Molecular Breeding - Molecular breeding is capable of improving important agricultural crop traits by controlling functional genes, aiming to attain high yield, stability and quality. During this...     

Absolute configuration of β,γ-carotene and biosynthetic implications

Racemic γ-ionone, partly resolved via its menthydrazone, was used for total synthesis of β,γ-carotene enriched in the 6′R and 6′S enantiomers. By CD correlation with natural β,γ-carotene isolated from Caloscypha fulgens 6′S-chirality is demonstrated for the natural carotene. Biosynthetic implications regarding the cyclization reaction are discussed.