Biosynthesis of carotenoids by Phycomyces blakesleeanus mutants in the presence of nitrogenous heterocyclic compounds

Pyridine, imidazole and some of their derivatives stimulate lycopene and γ-carotene synthesis-simultaneously inhibiting β-carotene formation in Phycomyces blakesleeanus Strain C115. Isonicotinoly-hydrazine has a toxic effect on Strains C9 and C115 and 1-methylimidazole on Strain C115 in the concentrations of 1 g/l. Compounds which cause an accumulation of lycopene and γ-carotene usually cause an increase in phytoene synthesis and the disappearance of β-zeacarotene. The effect of succinimide, 4-hydroxypyridine, and isonicotinoylhydrazine on Strain C9 has also been studied. When β-picoline and 2-methylimidazole treated C115 mycelia were washed and resuspended in phosphate buffer at pH 5·6 β-zeacarotene reappeared and β-carotene increased with the simultaneous decrease in lycopene and γ-carotene. The sum of β-carotene, γ-carotene up to 3days of resuspension was almost equal to the total of these at zero time. These results show that the inhibitory action of these compounds is on the enzymes responsible for cyclization of acyclic carotenes. This inhibition varies with the nature of the substituent on the heterocyclic ring and pyridine derivatives having pK_a values of 6 ± 1 show the greatest degree of inhibition.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Carotene biosynthesis by a cell extract of Aspergillus giganteus mut alba

A cell extract prepared from lyophilized mycelia of light-grown cultures of Aspergillus giganteus mut alba converted [2-¹⁴C]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, Mn²⁺, Mg²⁺, 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.     

Effect of CPTA and cycocel on the biosynthesis of carotenoids by Phycomyces blakesleeanus mutants

CPTA and cycocel cause accumulation of lycopene and γ-carotene, simultaneously inhibiting the formation of β-carotene and β-zeacarotene in Phycomyces blakesleeanus mutant strain C115. Phytoene synthesis is enhanced. CPTA is more effective than cycocel. Kinetic studies show that with increasing concentrations of CPTA, lycopene and γ-carotene increase with the concomitant decrease in β-carotene, the total of these three carotenes being almost equal to β-carotene present in the control. When CPTA-treated mycelium is washed free of the chemical and resuspended in phosphate buffer solution containing 2·5% glucose (pH 5·6), β-carotene is formed at the expense of both γ-carotene and lycopene. β-Zeacarotene, which is not present in the mycelium, reappears upon resuspension. These results indicate that CPTA is inhibiting the enzymes causing cyclization both at neurosporene and lycopene levels. Studies on the effect of CPTA on the high lycopene mutant strain C9 reveal that with increasing concentrations of the compound, lycopene increases slightly and both β-carotene and γ-carotene decrease. Phytoene synthesis is stimulated up to a certain level of CPTA and then becomes steady. In the albino mutant strain C5, there is a slight increase in phytoene formation on the addition of CPTA to the medium. No other carotenoid is formed, suggesting that CPTA cannot remove the block caused by genetic mutation and exerts its influence in an already existing biosynthetic pathway.     

β-Carotene biosynthesis by extracts of the C115 mutant of Phycomyces blakesleeanus

A cell extract of the yellow C115 car-42 mad-107(?) mutant of Phycomyces blakesleeanus, capable of converting MVA-[2-¹⁴C] into isoprenoids, was used to investigate the formation of β-carotene. The incorporation of radioactivity into β-carotene was reduced by the addition of unlabelled carotenes, solubilised using detergent, to the incubation mixtures. On reisolation of these carotenes after anaerobic incubations, they were found to carry radioactivity. The relative efficiencies of these carotenes as trapping agents are discussed in relation to the pathways of carotene cyclisation and to the apparent operation of a system for the negative feedback control of carotene biosynthesis.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

Enzymatic synthesis of carotenes by cell-free preparations of fruit of several genetic selections of tomatoes

Several mutants of tomatoes are known in which the carotene content of the fruit is markedly altered qualitatively and quantitatively from that found in the standard red tomato variety. These selections are: rr (yellow flesh, low carotene); tt (tangerine, orange, proneurosporene and prolycopene); at at (apricot, low in acyclic carotenes); og^c og^c (crimson, high in lycopene); Verkerk 377-2αα (probably identical to vircscent orange vo vo, high in ζ-carotene); B B (H_i-β, high in β-carotene), and Del Del (Hi-δ, high in δ-carotenc). Studies of carotene synthesis from [1-¹⁴C]isopentenyl pyrophosphate, [¹⁴C]phytoene, and [¹⁴C]lycopcne by soluble enzyme systems obtained from fruits of these selections have shown unexpected enzyme activities. All selections evidence activity for the synthesis of phytoene. All mutants have also been found to contain an enzyme system for the synthesis of β-carotenefrom lycopene. Three of the selections analyzed (rr, at at, and og^c og^c) also contain an enzyme system for the conversion of lycopene to α-carotene and the variants rr and tt contain an enzyme for the synthesis of poly-cis-carotencs from isopentenyl pyrophosphate and phytocne.The reasons for the discrepancies that are observed between carotene composition of fruit of field-grown tomato selections and enzyme activities for carotene synthesis by cell-free preparations obtained from these fruits are not presently known. It is obvious, however, that either inhibitors are present, cofactors are missing, or there are permeability barriers to substrate or cofactor transport into plastids of selections in which enzyme activities are not expressed in field-grown fruit. Further investigations will be required for clarification of this problem.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to poly-cis-acyclic carotenes by a cell-free preparation of tangerine tomato fruit plastids

The conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C]phytofluene and the conversion of the latter to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from plastids of tangerine tomato fruits is reported. Each of these compounds is also converted to cis-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, and γ- and β-carotenes. [¹⁴C]Prolycopene was also incubated with the above enzyme system. No conversion of this compound to trans-lycopene or cyclic carotenes was observed. Proof for the formation of the above carotenes from each of the substrates mentioned above was obtained by cochromatography with authentic samples on an alumina column. A close correspondence between radioactivity and light absorbance of each carotene was observed. Further proof for the formation of acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed in each case.     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina: Possible Interrelationship with beta-Carotene Accumulation

The interrelationship between abscisic acid (ABA) production and β-carotene accumulation was investigated in salt-stressed cells of the halotolerant green alga Dunaliella salina var bardawil. Cells were supplied with either R-[2-¹⁴C]mevalonolactone or [¹⁴C] sodium bicarbonate for 20 hours and then exposed to increased salinity (1.5 to 3.0 molar NaCl) for various lengths of time. Incorporation of label into abscisic acid and phaseic acid and the distribution of [¹⁴C]ABA between the cells and incubation media were monitored. [¹⁴C]ABA and [¹⁴C]phaseic acid were identified as products of both R-[2-¹⁴C]mevalonolactone and [¹⁴C]sodium bicarbonate metabolism. ABA metabolism was enhanced by hypersalinity stress. Actinomycin D, chloramphenicol, and cycloheximide abolished the stress-induced production of ABA, suggesting a role for gene activation in the process. Kinetic analysis of both ABA and β-carotene production demonstrated two stages of accelerated β-carotene production. In addition, ABA levels increased rapidly, and this increase occurred coincident with the early period of accelerated β-carotene production. A possible role for ABA as a regulator of carotenogenesis in cells of D. salina is therefore discussed.     

The solubilization of carotenogenic enzymes of Phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-¹⁴C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(?) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(?) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at ?70°C.     

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.     

The effect of CPTA analogs and other nitrogenous compounds on the biosynthesis of carotenoids in Phycomyces blakesleeanus mutants

The inhibitory effect of a series of analogs of CPTA, 2-(4-chlorophenylthio)-triethylamine-HCl, and ammonia derivatives on carotenoid biosynthesis in Phycomyces blakesleeanus mutants was studied. The types of inhibition exhibited allowed no firm conclusions about the biosynthetic route to β-carotene from either β-zeacarotene or lycopene. However, the evidence suggests at present that both pathways are operative. It was found that a slight change in structure of inhibitor resulted in a different type of action. Conclusions based on a single inhibitor could be cited as “evidence” for a certain pathway.     

Ketocarotenoid biosynthesis by Haematococcus lacustris

Haematococcus lacustris incubated on a nutrient-depleted medium utilised acetate-[2-¹⁴C] from the medium and carbon fixed photosynthetically for the biosynthesis of ketocarotenoids. Conversion of β-carotene to astaxanthin occurs via the intermediates echinenone and canthaxanthin.     

The isolation, purification, and characterization of cis-zeta-carotene and the demonstration of its conversion to acyclic, monocyclic and dicyclic carotenes by a soluble enzyme system obtained from the plastids of tangerine tomato fruits

Cis-ζ-carotene was isolated, purified in several chromatographic systems, and then identified as an intermediate in the biosynthesis of poly-cis-carotenes. The structure of cis-ζ-carotene was tentatively established from its visible light-absorption spectrum, and by a comparison of the infrared spectrum with that of trans-ζ-carotene. Confirmation of the identity of this compound was obtained by high resolution mass spectroscopy. The presence of the cis-configuration was indicated by a bathochromic shift of 6–10 nm in the visible spectrum when the compound was subjected to iodinecatalyzed photoisomerization. The infrared spectrum also showed characteristic peaks for the cis-configuration. Proof of the conversion of cis-ζ-[¹⁴C]carotene to trans-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, β- and γ carotenes was obtained on incubation with soluble enzyme systems obtained from plastids of fruits of two different tangerine varieties of tomato. Proof for the formation of each of the carotenes was provided by column and thin-layer chromatography A close correspondence of radioactivity and optical density was observed for each carotene. Additional proof was obtained by gas-liquid chromatography of each hydrogenated carotene. A coincidence of mass and radioactivity was observed for each carotene.     

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.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.