ζ-Carotene and other carotenes in a Phycomyces mutant

Strain S442, a new mutant of the fungus Phycomyces blakesleeanus, has a greenish colour and a distinct green fluorescence under long-UV light. Carotene analyses reveal the presence of phytoene, ζ-carotene, phytofluene, an unidentified compound, and neurosporene (in descending order of abundance). Genetic analysis shows that the new mutation occurs at gene carB, whose protein product catalyses the four dehydrogenations of phytoene to lycopene via phytofluene, ζ-carotene, and neurosporene. S442 offers no indication of a specific ζ-carotene dehydrogenase. The residual dehydrogenase activity in S442 is inhibited by diphenylamine. The high ζ-carotene content makes S442 a good source of this compound.     

Functional properties of diapophytoene and related desaturases of C(30) and C(40) carotenoid biosynthetic pathways

The desaturation reactions of C(30) carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C(40) phytoene to some extend but exhibited a high affinity to zeta-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C(40) carotenes revealed that this desaturase can also convert C(30) diapophytoene. Other homologous bacterial C(40) carotene desaturases could also utilize C(30) substrates, including one type of zeta-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C(30) carotenogenic pathway is determined by this initial enzyme which is highly homologous to C(40) phytoene synthases.     

Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenase.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.     

An enzyme complex for the dehydrogenation of phytoene in Phycomyces.

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.     

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.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Accumulation of canthaxanthin in Chlorella emersonii

A strain of Chlorella emersonii grown under high light intensity and low nitrogen degrades its chlorophyll and synthesizes canthaxanthin as the major carotenoid. Nitrogen starvation or high light alone does not induce canthaxanthin production. Norflurazon, a carotene inhibitor at the level of phytoene desaturase, inhibits production of canthaxanthin in C. emersonii with no accumulation of phytoene. Chlorella vulgaris and C. sp. 993 exposed to similar conditions do not respond in the same way as C. emersonii .     

Current state of carotenoid biosynthesis in chloroplasts of eucaryotes

The author discusses the current state of biochemical and genetic aspects of carotenoid biosynthesis in chloroplasts of algae and higher plants. Two ways of biosynthesis of key C5-isoperene units have been considered: 1) from acetate (C2) via mevalonic acid (C6) and its enzymatic conversions up to isopenthenyl diphosphate (C5) and 2) from glucose (C6) to formation of glyceraldehyde-3-phosphate (C3), to piruvate and their condensation via intermediate products up to isopenthenyl diphosphate (C5). Further biosynthesis of carotenoids from isopenthenyl diphosphate (C5) and dimethylallyl diphosphate (C5) in every organism is effected by the common scheme with further conservation of them up to geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20) and synthesis of phytoene (C40). All stages of phytoene desaturation up to formation of acyclic compounds are discussed. It is shown how in the process of subsequent oxidation and formation of hydroxy-, epoxy- and oxo-groups cyclic xanthophylls in chloroplasts of plants and algae are formed. Genetic control over biosynthesis of carotenoids is discussed.     

Biosynthesis of carotenoids in plastids of plants

This review deals with various aspects of the biosynthesis of carotenoids in chromoplasts and chloroplasts of green algae and higher plants. Two pathways of biosynthesis of the key C5-isoprene units are considered: 1) from acetate via mevalonate (C6) followed by its enzymatic conversions to isopentenyl diphosphate (C5); 2) from glucose via formation of glyceraldehyde-3-phosphate (C3) and pyruvate and their condensation via intermediary products to isopentenyl diphosphate (C5). Subsequent biosynthesis of carotenoids from isopentenyl diphosphate (C5) and dimethylallyl diphosphate (C5) involves a common route including their conversion into geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20), and synthesis of phytoene (C40). All stages of phytoene desaturation accompanied by formation of acyclic compounds such as zeta-carotene, neurosporene, and lycopene and their cyclization to alpha-, beta-, and epsilon-carotenes are considered in detail. Formation of xanthophylls in chloroplasts and chromoplasts involves sequential oxidations yielding hydroxy, epoxy, and oxo groups. Genetic control of biosynthesis of carotenoids is considered.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.     

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.     

Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C(30), C(35), C(40), C(45), C(50) carotenoids

At least 700 natural carotenoids have been characterized; they can be classified into C(30), C(40) and C(50) subfamilies. The first step of C(40) pathway is the combination of two molecules of geranylgeranyl pyrophosphate to synthesize phytoene by phytoene synthase (CrtB or PSY). Most natural carotenoids originate from different types and levels of desaturation by phytoene desaturase (CrtI or PDS+ZDS), cyclization by lycopene cyclase (CrtY or LYC) and other modifications by different modifying enzyme (CrtA, CrtU, CrtZ or BCH, CrtX, CrtO, etc.) of this C(40) backbone. The first step of C(30) pathway is the combination of two molecules of FDP to synthesize diapophytoene by diapophytoene synthase (CrtM). But natural C(30) pathway only goes through a few steps of desaturation to form diaponeurosporene by diapophytoene desaturase (CrtN). Natural C(50) carotenoid decaprenoxanthin is synthesized starting from the C(40) carotenoid lycopene by the addition of 2 C(5) units. Concerned the importance of carotenoids, more and more attention has been concentrated on achieving novel carotenoids. The method being used successfully is to construct carotenoids biosynthesis pathways by metabolic engineering. The strategy of metabolic engineering is to engineer a small number of stringent upstream enzymes (CrtB, CrtI, CrtY, CrtM, or CrtN), then use a lot of promiscuous downstream enzymes to obtain large number of novel carotenoids. Two key enzymes phytoene desaturase (CrtI(m)) and lycopene cyclase (CrtY(m)) have been modified and used with a series of downstream modifying enzymes with broad substrate specificity, such as monooxygenase (CrtA), carotene desaturase (CrtU), carotene hydroxylase (CrtZ), zeaxanthin glycosylase (CrtX) and carotene ketolase (CrtO) to extend successfully natural C(30) and C(40) pathways in E. coli. Existing C(30) synthase CrtM to synthesize carotenoids with different chain length have been engineered and a series of novel carotenoids have been achieved using downstream modifying enzymes. C(35) carotenoid biosynthesis pathway has been constructed in E. coli as described. C(45) and C(50) carotenoid biosynthesis pathways have also been constructed in E. coli, but it is still necessary to extend these two pathways. Those novel acyclic or cyclic carotenoids have a potential ability to protect against photooxidation and radical-mediated peroxidation reactions which makes them interesting pharmaceutical candidates.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.     

Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

Phytoene (C₄₀H₆₄) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L⁻¹ medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

     

Molecular taxonomy of a soil actinomycete isolate, KCCM10454 showing neuroprotective activity by 16S rRNA and rpoB gene analysis

Epilepsy constitutes a significant public health problem, and even the newest drugs and neurosurgical techniques have proven unable to cure the disease. In order to select a group of isolates which could generate an active compound with neuroprotective or antiepileptic properties, we isolated 517 actinomycete strains from soil samples taken from Jeju Island, in South Korea. We then screened these strains for possible anti-apoptotic effects against serum deprivation-induced hippocampal cell death, using the 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay as an in vitro test. The excitotoxic glutamate analog, kainic acid (KA), was used to induce seizures in experimental mice in our in vivo tests. As a result of this testing, we located one strain which exhibited profound neuroprotective activity. This strain was identified as a Streptomyces species, and exhibited the rifampin-resistant genotype, Asn(AAC)442, according to the results of 16S rRNA and rpoB gene analyses.     

Nucleotide sequence of the cryptic plasmid pTT8 from Thermus thermophilus HB8 and isolation and characterization of its high-copy-number mutant

The complete nucleotide sequence of pTT8, a cryptic plasmid from Thermus thermophilus HB8, was determined. pTT8 was 9328bp long and its G+C content was 69%. pTT8 contained eight putative open reading frames, three of which showed extensive similarities to the plasmid addiction proteins PasA and PasB of pTC-F14 and pAM10.6, and the RepA protein of the ColE2-related plasmids, respectively. During the analysis of pTT8-based plasmid pPP442, which had been obtained during a promoter-screening experiment, we occasionally isolated a plasmid with a relatively high-copy-number. This plasmid, pPP442m, contained a 1025 bp fragment derived from the genome of the HB27 host strain immediately upstream of the putative repA gene. Using the ori region of pPP442m, we constructed an expression vector, pTEV131m, with an estimated high-copy-number of 30-40. This plasmid was stably maintained in T. thermophilus HB27 under nonselective conditions for at least 100 generations. Cloning of the alpha-amylase gene of Bacillus stearothermophilus DY-5 into pTEV131m gave more than twofold production of the enzyme compared with pTEV131, the parental plasmid.     

Hymenobacter negativus sp. nov., bacteria isolated from mountain soil collected in South Korea

Two novel Gram-negative bacterial strains BT442^T and BT584 were isolated from dry soil collected in mountains Busan and Guri, Korea during wintertime. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT442^T and BT584 both belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia). Strain BT442^T was closely related to Hymenobacter soli PB17^T (98.0% 16S rRNA gene similarity) and Hymenobacter terrae POA9^T (97.6%). No other recognized bacterial species showed more than 97% 16S rRNA gene sequence similarity to strains BT442^T. The genome size of strain BT442^T was 5,143,362 bp. Bacterial growth was observed at 10–30 °C (optimum 25 °C), pH 6.0–8.0 (optimum pH 6.0) in R2A agar and in the presence up to 1% NaCl. The major cellular fatty acids of strains BT442^T and BT584 were iso-C_15:0, anteiso-C_15:0 and summed feature 3 (C_16:1 ω6c / C_16:1 ω7c). In addition, their predominant respiratory quinone was MK-7. The major polar lipids of strains BT442^T and BT584 were identified to be phosphatidylethanolamine, aminophospholipid, and aminolipid. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, strains BT442^T and BT584 are novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter negativus. The strain type of Hymenobacter negativus is BT442^T (=?KCTC 72902^T?=?NBRC XXXX^T).

     

The carboxyl-terminal tyrosine residue of protein-tyrosine phosphatase alpha mediates association with focal adhesion plaques

The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.