Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the β-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g⁻¹ of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.     

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

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.     

Expression of β-carotene hydroxylase gene (<Emphasis Type="Italic">crtR-B</Emphasis>) from the green alga <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> in chloroplasts of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding β-carotene hydroxylase that was able to catalyze the conversion of β-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RT-PCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.     

Elucidation of a Carotenoid Biosynthesis Gene Cluster Encoding a Novel Enzyme, 2,2′-β-Hydroxylase, from Brevundimonas sp. Strain SD212 and Combinatorial Biosynthesis of New or Rare Xanthophylls

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2′-β-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2′-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.     

Carotenoids in fish. XXVI. Pungitius pungitius (L.) and Gasterosteus aculeatus L. (Gasterosteidae)

The author investigated the presence of various carotenoids in the different parts of the body of Pungitius pungitius (L.) and Gasterosteus aculeatus L. by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

in Pungitius pungitius. α-carotene, β-carotene, β-cryptoxanthin, mutatochrome, zeaxanthin and astaxanthin;

in Gasterosteus aculeatus: β-carotene, β-cryptoxanthin, β-carotene epoxide, neothxanthin, canthaxanthin, mutatochrome, lutein, phoenicoxanthin, zeaxanthin, taraxanthin, tunaxanthin, astaxanthin, astaxanthin ester and α-doradexanthin. The total carotenoid content ranged from 2.229 to 138.504 µg/g wet weight.

     

Metabolism of carotenoids in sea-urchin Pseudocentrotus depressus

• 1.1. Feeding experiments with β,β-carotene, canthaxanthin and astaxanthin on the sea urchin Pseudocentrotus depressus were investigated.
• 2.2. In the case of β,β-carotene group, β-carotene was accumulated, β-isocryptoxanthin appeared and β-echinenone increased 6.8 times as much as the control group. On the other hand, in canthaxanthin and astaxanthin groups, canthaxanthin and astaxanthin increased significantly, respectively. The metabolic products of these carotenoids could not be found.
• 3.3. It was concluded that β,β-carotene was bioconverted to β-echinenone via β-isocryptoxanthin in P. depressus and could not be oxidatively metabolized beyond β-echinenone.

     

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T₁ seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.     

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.     

Carotenoids in fish IV. Salmonidae and Thymallidae from Polish waters

The author investigated the presence of various carotenoids in the Salmonidae and Thymallidae family by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

Abstract

- in the muscles of Salmo salar: astaxanthin (pure and ester), canthaxanthin, lutein and zeaxanthin.

- in the eggs of Salmo trutta m. trutta: β-carotene, iso- and zeaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Salmo trutta m. fario: β-carotene, canthaxanthin, 4-keto-4-hydroxy-β-carotene, astaxanthin (pure and ester), lutein, taraxanthin and astacene.

- in the eggs of Salmo gairdneri: β-carotene, γ-carotene (?), canthacanthin, isozeaxanthin, lutein and astaxanthin, and in the sperm Salmo gairdneri: β-carotene, γ-carotene (?), 4-keto-4-hydroxy-β-carotene, canthaxanthin, lutein and astaxanthin.

- in the eggs of Salvelinus fontinalis: ester astaxanthin, canthaxanthin, isozeaxanthin, lutein and astacene.

- in the eggs of Hucho hucho: β-carotene, tunaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Coregonus albula: β-carotene, 4-keto-4-hydroxy-β-carotene, ester astaxanthin, zeaxanthin, taraxanthin and astacene.

- in Coregonus lavaretus: a) in eggs: β-carotene, ester astaxanthin, canthaxanthin, iso- and zeaxanthin, lutein, taraxanthin and astacene b) in the sperm: canthaxanthin, 4-hydroxy-4-keto-β-carotene, isozeaxanthin and astaxanthin, and other organs: 4-hydroxy-α-carotene, canthaxanthin, tunaxanthin, monoepoxy lutein, lutein, iso- and zeaxanthin and astaxanthin.

- in the eggs of Coregonus peled: β-carotene, 4-keto-4-hydroxy-β-carotene, lutein, zeaxanthin, taraxanthin and astacene.

- in the eggs of Thymallus thymallus: β-carotene, tunaxanthin, lutein and astaxanthin.

     

High reliability transformation of the basal fungus Mucor circinelloides by electroporation

Molecular approaches to study the biology of the zygomycete Mucor circinelloides depend mainly on the existence of a polyethylene glycol-based transformation method, which is one of the most efficient in zygomycete fungi. However, the poor reliability and low transformation rates of this method are major obstacles in the molecular study of a number of biological processes. This paper describes an easy and reliable method to transform M. circinelloides protoplasts by electroporation. A high-voltage pulse of 25 μF capacitance, 400 Ω resistance, and 4 kV/cm field strength were seen to be the optimal electrical conditions for delivering DNA into M. circinelloides protoplasts. Under these electrical conditions, successful transformations were carried out with several self-replicative plasmid and strain combinations, producing up to more than 500 transformants per μg DNA. Targeted DNA integration of a transgene (atfA gene of Acinetobacter baylyi) in a particular locus (carRP) was also achieved. This transformation method will considerably facilitate in-depth molecular genetic studies of the biology of this fungus.     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Use of Transposon Promoter-Probe Vectors in the Metabolic Engineering of the Obligate Methanotroph Methylomonas sp. Strain 16a for Enhanced C40 Carotenoid Synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C₄₀ carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations—the fliCS, hsdM, ccp-3, cysH, and nirS regions—were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

过表达AtchyB基因提高洋桔梗光胁迫耐受性的研究

类胡萝卜素尤其是叶黄素循环类物质在植物抵抗由强光照引起的非生物胁迫中发挥着重要的作用,为了提高洋桔梗对强光照的抗性,从拟南芥中克隆了类胡萝卜素生物合成途径中参与叶黄素循环关键酶——β-胡萝卜素羟化酶基因(AtchyB),利用农杆菌介导法将其转入洋桔梗中,最终得到遗传转化植株2个株系,研究发现,转基因植株中总类胡萝卜素含量高于对照组,且叶黄素循环池被不同程度地放大。在不同光照强度下,转基因洋桔梗植株对光照耐受性明显强于对照组,且转基因植株生物量也明显提高。表明过表达AtchyB基因使洋桔梗光胁迫耐受性有所增强。     

Comparative biochemical studies of carotenoids in sea-urchins—II. The more primitive sea-urchins belonging to the orders Cidaroida,Echinothurioida, Diadematoida and Arbacioida

• 1.1. The carotenoids of seven species of more primitive sea-urchins, [orders Cidaroida (I), Echinothurioida (II), Diadematoida (III), and Arbacioida (IV)] were investigated from the comparative biochemical point of view.
• 2.2. β,β-carotene and β-echinenone have been isolated as major carotenoids in (I) and (III, IV), respectively. In (II), β,β-carotene, β-echinenone, canthaxanthin and (3S,3′S)-astaxanthin were foundto be predominant carotenoids.
• 3.3. The carotenoid patterns of (I) which is the most primitive sea-urchin from the phylogenetic point of view, and of (II) which is direct developers with non-feeding larvae, were quite different from those of the other sea-urchins showing typical development with feeding larvae.

     

Characterization of two β-carotene ketolases,CrtO and CrtW,by complementation analysis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The pathways from β-carotene to astaxanthin are crucial key steps for producing astaxanthin, one of industrially useful carotenoids, in heterologous hosts. Two β-carotene ketolases (β-carotene 4,4′-oxygenase), CrtO and CrtW, with different structure are known up to the present. In this paper, we compared the catalytic functions of a CrtO ketolase that was obtained from a marine bacterium Rhodococcus erythropolis strain PR4, CrtO derived from cyanobacterium Synechosistis sp. PCC6803, and CrtW derived from a marine bacterium Brevundimonas sp. SD212, by complementation analysis in Escherichia coli expressing the known crt genes. Results strongly suggested that a CrtO-type ketolase was unable to synthesize astaxanthin from zeaxanthin, i.e., only a CrtW-type ketolase could accept 3-hydroxy-β-ionone ring as the substrate. Their catalytic efficiency for synthesizing canthaxanthin from β-carotene was also examined. The results obtained up to the present clearly suggest that the bacterial crtW and crtZ genes are a combination of the most promising gene candidates for developing recombinant hosts that produce astaxanthin as the predominant carotenoid.     

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

Abstract High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora . Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from β-carotene is canthaxanthin (4,4'-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt . We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.