Norihiko Misawa

Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner.

Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.

Elevation of the provitamin A content of transgenic tomato plants

Tomato products are the principal dietary sources of lycopene and major source of β-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the β-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.

Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts

The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum. The functions of the individual genes have been identified. The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds. Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced. Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes. A gene capable of enhancing carotenoid levels in E. coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. This gene has been found to encode an isopentenyl pyrophophate isomerase. It has further been shown that the edible yeasts Candida utilis as well as Saccharomyces cerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators. It has been observed in the yeasts S. cerevisiae and C. utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively. It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis. Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme. A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C. utilis.

In Vitro Characterization of Astaxanthin Biosynthetic Enzymes

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from β-carotene via β-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from β-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from β-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3,(3′)-β-ionone ring hydroxylase and CrtW/BKT carotenoid 4,(4′)-β-ionone ring oxygenase.

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes β-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, β-carotene ketolase (β-carotene oxygenase), which converted β-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3′S), which was identified after purification by a variety of spectroscopic methods.

KaPPA-View. A Web-Based Analysis Tool for Integration of Transcript and Metabolite Data on Plant Metabolic Pathway Maps

The application of DNA array technology and chromatographic separation techniques coupled with mass spectrometry to transcriptomic and metabolomic analyses in plants has resulted in the generation of considerable quantitative data related to transcription and metabolism. The integration of “omic” data is one of the major concerns associated with research into identifying gene function. Thus, we developed a Web-based tool, KaPPA-View, for representing quantitative data for individual transcripts and/or metabolites on plant metabolic pathway maps. We prepared a set of comprehensive metabolic pathway maps for Arabidopsis (Arabidopsis thaliana) and depicted these graphically in Scalable Vector Graphics format. Individual transcripts assigned to a reaction are represented symbolically together with the symbols of the reaction and metabolites on metabolic pathway maps. Using quantitative values for transcripts and/or metabolites submitted by the user as Comma Separated Value-formatted text through the Internet, the KaPPA-View server inserts colored symbols corresponding to a defined metabolic process at that site on the maps and returns them to the users browser. The server also provides information on transcripts and metabolites in pop-up windows. To demonstrate the process, we describe the dataset obtained for transgenic plants that overexpress the PAP1 gene encoding a MYB transcription factor on metabolic pathway maps. The presentation of data in this manner is useful for viewing metabolic data in a way that facilitates the discussion of gene function.

Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation

Brassica napus (canola) plants were genetically manipulated to increase the amount and composition of carotenoids in seeds by using seven key enzyme genes involved in ketocarotenoid formation, which originated from a soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3), and marine bacteria Brevundimonas sp. strain SD212 and Paracoccus sp. strain N81106 (formerly called Agrobacterium aurantiacum). The seven key gene cassettes, in which each gene was surrounded by an appropriate promoter and terminator, were connected in a tandem manner, and the resulting constructs (17 kb) were inserted into a binary vector and used for transformation of B. napus. Surprisingly, 73-85% of the regenerated plants retained all seven genes, and formed orange- or pinkish orange-coloured seeds (embryos), while untransformed controls had light yellow-coloured seeds with predominant accumulation of lutein. Three of the transgenic lines were analysed further. The total amount of carotenoids in these seeds was 412-657 microg g(-1) fresh weight, which was a 19- to 30-fold increase compared with that of untransformed controls. The total amount of ketocarotenoids was 60-190 microg g(-1) fresh weight. beta-Carotene was the predominant carotenoid, with significant amounts of alpha-carotene, echinenone, phytoene, lutein, and canthaxanthin also detected in the transgenic seeds. The ratio of hydroxylated carotenoids to overall carotenoids was quite small relative to the ratio of ketocarotenoids to overall carotenoids. Interestingly, expression of many endogenous carotenogenic genes was also altered in the transgenic seeds, suggesting that their expression was affected by an increase in carotenoid biosynthesis.

Pathway engineering for functional isoprenoids

Pathway engineering is to engineer biosynthetic pathways for compounds of interests in heterologous organisms such as microbes and higher plants, which has also been one of the most important fields in metabolic engineering and synthetic biology. This review focuses on pathway engineering researches for the production of functional isoprenoids containing monoterpenes, sesquiterpenes, diterpenes, and triterpenes as well as carotenoids and for the elucidation of relevant biosynthesis genes and enzymes, which have been performed in the last two years. As microbial hosts, Escherichia coli and Saccharomyces cerevisiae have often been employed, since they, specifically the former, are fully amenable to genetic manipulations with extensive molecular resources. Various crops have also been used as the hosts for engineering pathways of functional isoprenoids of the plant origin, particularly carotenoids.

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering

SUMMARY The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (beta-carotene ketolase) and CrtZ (beta-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO2 concentrations of 1500 micromol photons m(-2) s(-1) light intensity.

Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of β-carotene

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo‐oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule β‐carotene. The gene was identified through the use of an experimental herbicide, 2‐(4‐methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically‐induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild‐type cells. A 1.5 kb genomic DNA fragment containing this MPTA‐resistance mutation was expressed in a lycopene‐accumulating strain of Escherichia coli. The conversion of lycopene to β‐carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produceβ‐carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.