Matching is the Key Factor to Improve the Production of Patchoulol in the Plant Chassis of Marchantia paleacea.

The valuable terpenoids, such as artemisinin acid, have achieved bioproduction in the chassis of microbes recently. In this study, Marchantia paleacea L, a promising plant synthetic biology chassis, was used to explore the possibility of patchoulol production by constructing a synthetic biology pathway composed of FPS and PTS. The experiment results show that the maximum yields based on the cytoplasm and plastid pathway were 621.56 and 1006.45 µg/g, respectively. However, there is no statistically significant difference in the yield of patchoulol between transformant plants with different subcellular compartment-targeting pathways. However, it was found that the highest yield of patchoulol was achieved in transformant plants with similar transcription levels of FPS and PTS. Also, the optimized transcription ratio between PTS and FPS is determined at 1.12 based on statistical analysis and model simulation. Therefore, two kinds of new optimized pathway vectors were constructed. One is based on the fusion protein method, and the other is based on protein expression individually, in which the same promoter and terminator were used to derive the expression of both FPS and PTS. The effect of pathway optimization was tested by transient and stable transformation. The production of patchoulol in transient transformation was the same for the two abovementioned kinds of matching pathway and higher than that for the original pathway. Also, in stable transformation, the yield of patchoulol reached up to 3250.30 µg/g, being three times the maximum content before optimization. It is suggested that M. paleacea is a powerful plant chassis for terpenoid synthetic biology and the matching between enzymes may be the key factor in determining the metabolic flux of the pathway in the study of synthetic biology.

[Preparation of catalitaxol-deficient defective strains by using ultraviolet mutagenesis].

Secondary metabolites of endophytic fungi FSN002 from Juglans mandshurica Maxim have excellent liver cancer resistance. Preparation of mutant strains is an important means to study the biosynthesis mechanism of catalitaxol. Fungal spores germinating young hyphae with 4 to 6 cells after culturing for 13 hours were used as starting materials of ultraviolet (UV) mutagenesis. UV light intensity and irradiation time have a linear relationship with fungal mortality. The two factors had no obvious interactions. When UV light was 90 000 µJ/cm2 and irradiation time for 6 s, the mortality of fungi was around 95%. Under the optimization mutation condition, two mutant strains were obtained, of which one lost the synthesis ability of catalitaxol completely, and the another synthetized only 16% catalitaxol of the wild strain. Our findings may serve basis for further study on the biosynthesis mechanism and efficient production of catalitaxol.

Engineering isoprenoid biosynthesis in Artemisia annua L. for the production of taxadiene: a key intermediate of taxol.

Taxadiene is the first committed precursor to paclitaxel, marketed as Taxol, arguably the most important anticancer agent against ovarian and breast cancer. In Taxus, taxadiene is directly synthesized from geranylgeranyl diphosphate (GGPP) that is the common precursor for diterpenoids and is found in most plants and microbes. In this study, Artemisia annua L., a Chinese medicinal herb that grows fast and is rich in terpenoids, was used as a genetic engineering host to produce taxadiene. The TXS (taxadiene synthase) gene, cloned from Taxus and inserted into pCAMBIA1304, was transformed into Artemisia annua L. using the Agrobacterium tumefaciens-mediated method. Thirty independent transgenic plants were obtained, and GC-MS analysis was used to confirm that taxadiene was produced and accumulated up to 129.7 µg/g dry mass. However, the high expression of TXS did not affect plant growth or photosynthesis in transgenic Artemisia annua L. It is notable that artemisinin is produced and stored in leaves and most taxadiene accumulated in the stem of transgenic Artemisia annua L., suggesting a new way to produce two important compounds in one transgenic plant: leaves for artemisinin and stem for taxadiene. Overall, this study demonstrates that genetic engineering of the taxane biosynthetic pathway in Artemisia annua L. for the production of taxadiene is feasible.

Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

[Isolation, identification and anticancer activity of an endophytic fungi from Juglans mandshurica].

The endophytic fungus named FSN006 was isolated from the inner bark of Juglans mandshurica. It grew quickly and formed circular colony on PDA plate. The upper side of the colony was white, while the lower side of the colony and the conditioned medium were light yellow as a result of significant yellow pigment substances were produced and secreted by the fungi. Green elliptic conidia appeared when cultured on CMX plate. Based on the morphology identification and ITS sequence, it was clear that this fungus belonged to the Deuteromycotina, HyPhomycetes, Moniliales, Trichoderma longibrachiatum. The conditioned medium of FSN006 showed a high anti-tumor ability against liver cancer cell-HepG2, and reached its IC50 concentration after being diluted 20 times, while the IC50 concentration of curcumine was(11.49 +/- 0.12) mg x L(-1). In addition, there was preeminent selective inhibiting effect against the normal liver cell strain HL-7702 and its caner counter strain HepG2. The inhibiting effect against strain HL-7702 was only one quarter of that against HepG2 at the concentration of IC50. Therefore, the fermentation of FSN006 may provide a possible way to produce anticancer drug with higher efficiency and lower toxicity.

Silencing of PMT expression caused a surge of anatabine accumulation in tobacco.

Drastic increase of anatabine levels was observed in tobacco plants with markedly reduced nicotine concentrations through RNA silencing approaches. By down-regulation of PMT through three kinds of RNA silencing approaches, the nicotine levels decreased accordingly. In lines with slight and moderate reduction of nicotine levels, no anticipated negative linear correlation was found between anatabine and nicotine content. In lines with nicotine levels lower than 2.7 mg/g, drastic elevation of anatabine levels was found. Transcriptional levels of QPRT were unaffected in tobacco lines with surged anatabine levels. This report of an intriguing mutual relationship of nicotine and anatabine sheds new light on mechanisms between metabolic regulations in plants, and reconfirms complexity of metabolic networks.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense.

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of beta-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.

Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches.

Issues related to the nicotine content of tobacco have been public concerns. Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches. The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves. By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1-96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine,whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.

Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway).

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI).

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.

Molecular cloning and characterization of Crmdr1, a novel MDR-type ABC transporter gene from Catharanthus roseus.

A novel gene encoding a MDR-like ABC transporter protein was cloned from Catharanthus roseus, a medicinal plant with more than 120 kinds of secondary metabolites, through rapid amplification of cDNA ends (RACE). This gene (named as Crmdr1; GenBank accession no.: DQ660356) had a total length of 4395 bp with an open reading frame of 3801 bp, and encoded a predicted polypeptide of 1266 amino acids with a molecular weight of 137.1 kDa. The CrMDR1 protein shared 59.8, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAD31576), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Crmdr1 was a low-copy gene. Expression pattern analysis revealed that Crmdr1 constitutively expressed in the root, stem and leaf, but with lower expression in leaf. The domains analysis showed that CrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that CrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.

Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus x media.

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.

[Isolation and characterization of a 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media].

2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level.

Characterization and expression profile analysis of a new cDNA encoding taxadiene synthase from Taxus media.

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba.

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.

Isolation and characterization of a new mannose-binding lectin gene from Taxus media.

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species, Taxus media. The full-length cDNA of T. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and that tma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed that tma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.

Preference of simple sequence repeats in coding and non-coding regions of Arabidopsis thaliana.

MOTIVATION: Simple sequence repeats or microsatellites have been found abundantly in many genomes. However, the significance of distribution preference has not been completely understood. Completion of the Arabidopsis genome sequencing allows us to better understand and characterize microsatellites. RESULTS: Microsatellite distribution was more abundant in 5'-flanking regions of genes compared with that expected in the whole genome, with an over-representation of AG and AAG repeats; there were clear differences from distributions in 3'-flanks and coding fractions, where triplet frequencies evidently corresponded to codon usage. We identified 1140 full-length genes that contained at least one locus of AG or AAG repeats in their upstream sequences, and whose functional characteristics were significantly associated with the repeats. This observation indicates that selective pressure markedly differed in the three transcribed regions, with positive selection of AG and AAG repeats in 5'-flanks close to those genes whose products are preferentially involved in transcription.