Isolation of Carotenoid-deficient Mutant from Alkylated Dibenzothiophene Desulfurizing Nocardioform Bacteria, <Emphasis Type="Italic">Gordonia</Emphasis> sp. TM414

The dibenzothiophene-desulfurizing nocardioform bacteria, Gordonia sp. TM414, was isolated from oil-contaminated soil. To avoid coloration of the oil layer after the desulfurization reaction, which could decrease the quality of the oil, two colorless knock-out mutants, TPc and TPd, were isolated by using a broad-host-range transposon complex. Genomic sequence analysis revealed that the same gene was disrupted in these mutants and that the transposon-inserted gene was assigned as the gene for phytoene desaturase, crt I. The crt I mutants also showed desulfurization activity comparable to that of the parent strain in a model-oil/aqueous bi-phasic reaction, suggesting that the carotenoid production is not responsible for the bi-phasic desulfurization reaction that requires hydrophobic substrate incorporation from the organic phase.     

Cloning,sequencing and expressing the carotenoid biosynthesis genes,lycopene cyclase and phytoene desaturase,from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).     

A novel carotenoid biosynthesis gene coding for ζ-carotene desaturase: functional expression,sequence and phylogenetic origin

A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting -carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56 740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of -carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between -carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the -carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.     

Evaluation of colors in green mutants isolated from purple bacteria as a host for colorimetric whole-cell biosensors

The change in carotenoid-based bacterial color from yellow to red can be applied to whole-cell biosensors. We generated several green mutants to emphasize the color change in such biosensors. The blue-green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid precursor, phytoene. Green Rhodovulum sulfidophilum M31 accumulated neurosporene, a downstream product of phytoene. Another green mutant, Rhodobacter sphaeroides Ga, accumulated neurosporene and chloroxanthin, which are both downstream products of phytoene. All green mutants accumulated bacteriochlorophyll a. Photosynthetic membrane obtained from the green mutants all exhibited decreased absorption of wavelength range at 510–570 nm. Therefore, these indicate that the greenish bacterial colors were mainly caused by the existence of bacteriochlorophyll a and the changes in carotenoid composition in photosynthetic membrane. The colors of the green mutants and their wild-type strains were plotted in the CIE-L*a*b* color space, and the color difference (ΔE*ab) values between a green mutant and its wild type were calculated. ΔE*ab values were higher in the green mutants than in Rdv. sulfidophilum CDM2, the yellowish host strain of reported biosensors. These data indicate that change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors.     

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability

Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.     

Genetic modification of Methylobacterium extorquens G10 producer strain of polyhydroxybutyrate

The effect of the increased copy number of polyhydroxybutyrate (PHB) biosynthesis genes in pink-pigmented methylobacterium Methylobacterium extorquens G10 on properties of the biopolymer was studied. The activity of poly-3-hydroxybutyrate-synthase (PHB-synthase) was shown to increase and the molecular weight of synthesized PHB decreases twofold (150 → 79 kDa) after insertion of extra copies of phaC and phaCAB genes into cells of the producer strain, whereas the physicochemical properties of the plastic changed insignificantly. White mutant M. extorquens G10-W with disrupted synthesis of the carotenoid pigment (defect by the crtI gene, which codes for phytoene desaturase) was established to have the same rate of growth and level of PHB accumulation as the initial strain G10. The G10-W strain is a promising producer of PHB, with decreased expenses for purification and PHB biosynthesis.     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Structural and kinetics properties of a mutated phytoene desaturase from Rubrivivax gelatinosus with modified product specificity

The phytoene desaturase CrtI from Rubrivivax gelatinosus catalyzes simultaneously a three- and four-step desaturation producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively. Two different mutation libraries for the crtI gene from R. gelatinosus were constructed to screen for modified enzymes which synthesize almost exclusively either neurosporene or lycopene. The resulting mutants carried between one and four amino acid exchanges and at least one of them affected the secondary protein structure by shortening or extending one of the helices. A prominent amino acid which was exchanged in the neurosporene or lycopene-forming desaturase was leucine 208. Enzyme kinetic studies were carried out with the L208 modified desaturase and the specificities for phytoene and neurosporene as substrates determined. Higher and lower values correlate well with the higher or lower potential for the synthesis of lycopene from neurosporene. TopPred analysis of the mutations of L208 indicated that the location is in a highly hydrophobic membrane-integrated region which is a good candidate for the substrate-binding site of the desaturase.     

Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of β-carotene by S. cerevisiae.     

甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.     

Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice

Transgenics for the expression of β-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and β-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T₁ progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T₁ progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T₁ progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 μg/g of polished seeds. High variation in the carotenoid accumulation was observed within the T₁ progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the β-carotene accumulation.     

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.     

Nuclear—organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is ‘plastid autonomous’. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C₄₀ carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear—organelle interactions.