Kanji Ohyama

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.

Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB

Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.

Divergent mRNA transcription in the chloroplast psbB operon.

The genes psbB, psbH, petB and petD for the components in photosystem II and the cytochrome b6/f complex are clustered and co‐transcribed in liverwort Marchantia polymorpha chloroplasts. On the opposite DNA strand in the spacer region between the genes psbB and psbH, we deduced an open reading frame consisting of 43 sense codons, and designated it as the ORF43 gene. The ORF43 gene was actively transcribed in liverwort chloroplasts. The ORF43 transcripts were entirely complementary to a part of the primary transcripts of the psbB operon. Heterogeneous Northern hybridization showed that the mRNA transcripts for the ORF43 gene increased with greening in pea seedlings. This is the first demonstration of divergent overlapping transcription in chloroplasts.

Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L.

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5′- and 3′-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.

Expression of the gene for sterol-biosynthesis enzyme squalene epoxidase in parenchyma cells of the oil plant, Euphorbia tirucalli

In plants, phytosterols and triterpenes are major secondary metabolites. In an attempt to reveal the mechanism for synthesis and storage of these compounds, we isolated and characterized cDNA clones for squalene epoxidase (SE), from a succulent shrub, Euphorbia tirucalli. Southern-blot analysis of total DNA using cDNA fragment as a probe showed that the E. tirucalli squalene epoxidase gene (EtSE) is single-copy type in terms of restriction fragment length polymorphism (RFLP). Deduced amino-acid sequence of the cDNA showed 83 and 75% identity to those of rice and ginseng, respectively, in an area excluding a less homologous putative transmembrane region in the N-terminal end. Functional characterization with heterologous expression using an erg1-disrupted yeast mutant KLN1 indicated that the EtSE recovered ergosterol auxotrophy of the mutant, and gave rise to an ergosterol accumulation in the EtSE transformant. RT-PCR analysis showed the EtSE transcripts in leaves and stem internodes accumulated in almost equal amounts, which were more abundant than those in roots. In situ hybridization using EtSE antisense probe revealed prominent EtSE expression on a parenchyma cell adjacent to primary laticifers that were located in a rosary orientation in the inner region of cortex. This is the first report of expression of a gene for a rate-limiting enzyme in mevalonate pathway in organs and tissues of a plant.

Isolation and functional characterization of fatty acid Δ5‐elongase gene from the liverwort Marchantia polymorpha L

Bryophyte Marchantia polymorpha L. produces C22 very‐long‐chain polyunsaturated fatty acid (VLCPUFA). Thus far, no enzyme that mediates elongation of C20 VLCPUFAs has been identified in land plants. Here, we report the isolation and characterization of the gene MpELO2, which encodes an ELO‐like fatty acid elongase in M. polymorpha. Heterologous expression in yeast demonstrated that MpELO2 encodes Δ5‐elongase, which mediates elongation of arachidonic (20:4) and eicosapentaenoic acids (20:5). Phylogenetic and gene structural analysis indicated that the MpELO2 gene is closely related to bryophyte Δ6‐elongase genes for C18 fatty acid elongation and diverged from them by local gene duplication.

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF2α in engineered Escherichiacoli

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7xa0mgxa0l−1 PGF2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF2α. Furthermore, these E. coli cells produced PGF2α in a medium supplemented with AA and secreted the PGF2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF2α at significantly reduced prices and greater purity.

Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli.

Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5- and 3-RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC-MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca(2+) ion in the oxygenation reaction.

Gene content, organization and molecular evolution of plant organellar genomes and sex chromosomes: insights from the case of the liverwort Marchantia polymorpha.

The complete nucleotide sequence of chloroplast DNA (121,025 base pairs, bp) from a liverwort, Marchantia polymorpha, has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. We also determined the complete nucleotide sequence of the liverwort mitochondrial DNA and deduced 96 possible genes in the sequence of 186,608 bp. The complete chloroplast genome encodes twenty introns (19 group II and 1 group I) in 18 different genes. One of the chloroplast group II introns separates a ribosomal protein gene in a trans-position. The mitochondrial genome contains thirty-two introns (25 group II and 7 group I) in the coding regions of 17 genes. From the evolutionary point of view, we describe the origin of organellar introns and give evidence for vertical and horizontal intron transfers and their intragenomic propagation. Furthermore, we describe the gene organization of the Y chromosome in the dioecious liverwort M. polymorpha, the first detailed view of a Y chromosome in a haploid organism. On the 10 megabase (Mb) Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome. These 14 genes are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

Molecular evolution of mitochondrial introns in the liverwort Marchantia polymorpha

We here describe in detail the characterization and molecular evolution of group II introns in the mitochondrial genome of the liverwort Marchantia polymorpha. We find that 18 introns of the 25 group II introns can be assigned by their similarities to six clusters, indicating an intra-genomic propagation of one ancestral intron each into the respective clusters in the liverwort mitochondrial genome. Interestingly, the intra-genomic propagation of some of these introns occurred only after the evolutionary separation of the bryophytes from the other clades of plants. Finally we report that the maturase-like sequences in the liverwort group II introns have further evolved by horizontal and independent transposition and substitution by analogous sequences from other fungal introns.