Sabine Steiger

Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli.

We have used combinatorial biosynthesis to synthesize novel lipophilic carotenoids that are powerful cellular antioxidants. By co-expressing three different carotenoid desaturases in combination with a carotenoid hydratase, a cyclase, and a hydroxylase on compatible plasmids in Escherichia coli, we synthesized four novel carotenoids not previously detected in biological material or chemically synthesized. Their identification was based on their relative retention times on HPLC, spectroscopic properties, molecular weights, number of hydroxy groups, and 1H-NMR spectra. The carotenoids were designated as 1-HO-3′, 4′-didehydrolycopene, 3, 1′-(HO)2-γ-carotene, 1,1′-(HO)2-3, 4, 3′, 4′-tetradehydrolycopene, and 1, 1′-(HO)2-3, 4-didehydrolycopene. These novel acyclic derivatives differ from structurally related compounds by extension of the conjugated polyene chain as well as additional hydroxy groups at position C-1′. We determined their antioxidative activity in a liposome-membrane model system, which showed that their ability to protect against photooxidation and radical-mediated peroxidation reactions was linked to the length of the conjugated double-bond system and the presence of a single hydroxy group. The protection of membrane degradation was superior to the related 1-HO and 1, 1′-(HO)2 lycopene derivatives, making them interesting pharmaceutical candidates.

High-light-dependent upregulation of carotenoids and their antioxidative properties in the cyanobacterium Synechocystis PCC 6803

Abstract Light-dependent carotenoid formation has been investigated in the cyanobacterium Synechocystis PCC 6803. Upon transfer of cultures from low- (35 μmol m −2 s −1 ) to high-intensity illumination (500 μmol m −2 s −1 ), a decrease of the endogenous carotenoids to 85% of the initial value is observed. Treatment with norflurazon, an inhibitor that blocks the synthesis of coloured carotenoids, demonstrates a much stronger degradation of the existing carotenoids, which was more pronounced at high light intensities. However, the amounts of the accumulating precursor phytoene reveal that de novo biosynthesis is enhanced under high-light conditions. Nevertheless, this upregulated synthesis cannot fully compensate for carotenoid loss by photodegradation. The potential of the carotenoids from Synechocystis to protect against photosensitized and radical peroxidation reactions has been evaluated. It is found that myxoxanthophyll, which is preferentially synthesized in high light, exhibits the strongest protection and that echinenone is the most stable carotenoid under photo-oxidative conditions.

A novel type of lycopene ε-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4

Chlorophyll-b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of α- and β-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, ε- and β-lycopene cyclase. All cyanobacteria studied so far possess a single β-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the β-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of ε- as well as of β-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of α- β-, δ-, and ε-carotene. Such an activity is in contrast to all lycopene ε-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either β- or ε-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.

Carotenoids found in Bacillus

Aims:  To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation.

Carotenoid biosynthesis in Gloeobacter violaceus PCC4721 involves a single crtI-type phytoene desaturase instead of typical cyanobacterial enzymes

Gloeobacter violaceus is a cyanobacterium isolated from other groups by lack of thylakoids and unique structural features of its photosynthetic protein complexes. Carotenoid biosynthesis has been investigated with respect to the carotenoids formed and the genes and enzymes involved. Carotenoid analysis identified ß-carotene as major carotenoid and echinenone as a minor component. This composition is quite unique and the cellular amounts are up to 10-fold lower than in other unicellular cyanobacteria. Carotenoid biosynthesis is up-regulated in a light-dependent manner. This enhanced biosynthesis partially compensates for photooxidation especially of ß-carotene. The sequenced genome of G. violaceus was analyzed and several gene candidates homologous to carotenogenic genes from other organisms obtained. Functional expression of all candidates and complementation in Escherichia coli led to the identification of all genes involved in the biosynthesis of the G. violaceus carotenoids with the exception of the lycopene cyclase gene. An additional diketolase gene was found that functioned in E. coli but is silent in G. violaceus cells. The biggest difference from all other cyanobacteria is the existence of a single bacterial-type 4-step desaturase instead of the poly cis cyanobacterial desaturation pathway catalyzed by two cyanobacterial-type desaturases and an isomerase. The genes for these three enzymes are absent in G. violaceus.

Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to β-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene β-cyclase. Intermediates of the pathway beyond β-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3′-HO group results in the formation of fucoxanthin.

Expression of a Ketolase Gene Mediates the Synthesis of Canthaxanthin in Synechococcus Leading to Tolerance Against Photoinhibition, Pigment Degradation and UV-B Sensitivity of Photosynthesis¶

Abstract The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign β-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous β-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 μmol m−2 s−1) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 μmol m−2 s−1 and UV-B radiation of 6.8 W m−2. Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 μmol m−2 s−1, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina

BackgroundXanthophyllomyces dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. dendrorhous for a phylogenomic investigation of the Basidiomycota.ResultsThe genome assembly of a haploid strain of Xanthophyllomyces dendrorhous revealed a genome of 19.50 Megabases with 6385 protein coding genes. Phylogenetic analyses were conducted including 48 fungal genomes. These revealed Ustilaginomycotina and Agaricomycotina as sister groups. In the latter a well-supported sister-group relationship of two major orders, Polyporales and Russulales, was inferred. Wallemia occupies a basal position within the Agaricomycotina and X. dendrorhous represents the basal lineage of the Tremellomycetes, highlighting that the typical tremelloid parenthesomes have either convergently evolved in Wallemia and the Tremellomycetes, or were lost in the Cystofilobasidiales lineage. A detailed characterization of the CoA-related pathways was done and all genes for fatty acid, sterol and carotenoid synthesis have been assigned.ConclusionsThe current study ascertains that Wallemia with tremelloid parenthesomes is the most basal agaricomycotinous lineage and that Cystofilobasidiales without tremelloid parenthesomes are deeply rooted within Tremellomycetes, suggesting that parenthesomes at septal pores might be the core synapomorphy for the Agaricomycotina. Apart from evolutionary insights the genome sequence of X. dendrorhous will facilitate genetic pathway engineering for optimized astaxanthin or oxidative alcohol production.

Combined transcript, proteome, and metabolite analysis of transgenic maize seeds engineered for enhanced carotenoid synthesis reveals pleotropic effects in core metabolism

Highlight Metabolomic, proteomic, and transcriptomic analysis of a maize line genetically engineered for enhanced seed carotenoid biosynthesis revealed how the sugar metabolism adapted to meet the additional precursor supply.

Biosynthesis of a novel C30 carotenoid in Bacillus firmus isolates

Pigmented Bacillus spp. with probiotic properties have been isolated. In the yellow‐/orange‐coloured strains, the carotenoid pigments present have been characterized. In contrast, the carotenoids present in the Bacillus isolates coloured red await identification. The present article reports progress on the elucidation of the pigment biosynthetic pathway in these red‐pigmented Bacillus firmus strains.