Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Impact of the <Emphasis Type="Italic">MDM2</Emphasis> splice-variants <Emphasis Type="Italic">MDM2-A</Emphasis>, <Emphasis Type="Italic">MDM2-B</Emphasis> and <Emphasis Type="Italic">MDM2-C</Emphasis> on cytotoxic stress response in breast cancer cells

Background

The murine double minute 2 (MDM2) is an oncogene and a negative regulator of the tumor suppressor protein p53. MDM2 is known to be amplified in numerous human cancers, and upregulation of MDM2 is considered to be an alternative mechanism of p53 inactivation. The presence of many splice variants of MDM2 has been observed in both normal tissues and malignant cells; however their impact and functional properties in response to chemotherapy treatment are not fully understood.Here, we investigate the biological effects of three widely expressed alternatively spliced variants of MDM2; MDM2-A, MDM2-B and MDM2-C, both in unstressed MCF-7 breast cancer cells and in cells subjected to chemotherapy. We assessed protein stability, subcellular localization and induction of downstream genes known to be regulated by the MDM2-network, as well as impact on cellular endpoints, such as apoptosis, cell cycle arrest and senescence.

Results

We found both the splice variants MDM2-B and -C, to have a much longer half-life than MDM2 full-length (FL) protein after chemotherapy treatment indicating that, under stressed conditions, the regulation of degradation of these two variants differs from that of MDM2-FL. Interestingly, we observed all three splice variants to deviate from MDM2-FL protein with respect to subcellular distribution. Furthermore, while MDM2-A and -B induced the expression of the pro-apoptotic gene PUMA, this effect did not manifest in an increased level of apoptosis.

Conclusion

Although MDM2-B induced slight changes in the cell cycle profile, overall, we found the impact of the three MDM2 splice variants on potential cellular endpoints upon doxorubicin treatment to be limited.

     

Ribonuclease,deoxyribonuclease, and antiviral activity of <Emphasis Type="Italic">Escherichia coli</Emphasis>-expressed <Emphasis Type="Italic">Bougainvillea xbuttiana</Emphasis> antiviral protein 1

A full-length cDNA encoding ribosome-inactivating/antiviral protein from the leaves of Bougainvillea xbuttiana was recently isolated. The coding region of cDNA was cloned and expressed in Escherichia coli, and the protein product was designated as BBAP1 (Bougainvillea xbuttiana antiviral protein 1). BBAP1 showed ribonuclease activity against Torula yeast RNA. It also exhibited depurination activity against supercoiled pBlueScript SK+ plasmid DNA in a concentration dependent manner, and was found to convert nicked circular DNA into linear form only at higher concentration. On bioassay, BBAP1 exhibited antiviral activity against sunnhemp rosette virus infecting Cyamopsis tetragonoloba leaves in which 95% inhibition of local lesion formation was observed.     

Isolation and characterization of two <Emphasis Type="Italic">DREB1</Emphasis> genes encoding dehydration-responsive element binding proteins in chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis>)

Two novel DREB (dehydration-responsive element-binding protein) genes, designated as CiDREB1A and CiDREB1B, were cloned from chicory (Cichorium intybus). Both of these genes contained a conserved EREBP/AP2 domain and were classified into the A-1 subgroup of the DREB subfamily based on phylogenetic analysis. Quantitative real-time PCR analysis revealed that low temperature, but not ABA, greatly induced the expression of both CiDREB1 genes, suggesting that these genes are involved in ABA-independent stress signaling pathways. A subcellular localization assay revealed that both CiDREBs localized to the nucleus. In addition, we showed by yeast one-hybrid analysis that these two CiDREB proteins bind to the DRE motif of RD19A. These results suggest that CiDREB1A and CiDREB1B are important regulators of stress-responsive signaling in chicory.     

Quantification of Three Key Enzymes Involved in Artemisinin Biogenesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> by Polyclonal Antisera-Based ELISA

To elucidate the fine-tuned temporal and spatial modulation of artemisinin production in annual wormwood (Artemisia annua), we conducted enzyme-linked immunosorbent assay-based immunoquantification of three key enzymes involved in artemisinin biosynthesis, amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), and cytochrome P450 reductase (CPR), in various tissues and under different growth conditions. The field-grown plants accumulate abundant ADS and CYP71AV1 but a trace amount of CPR in all tested tissues. Furthermore, ADS and CYP71AV1 accumulations in leaves are 16- and eightfold higher than in roots, and ten- and fourfold higher than in stems, respectively, demonstrating a tissue-specific expression pattern. Interestingly, the flowering field plants and cold-acclimated cultural plants produce higher levels of ADS and CYP71AV1 than non-flowering field plants or untreated cultural plants, indicating the environmental and developmental induction on ADS and CYP71AV1 genes and providing possible explanation for the observation that elevation of artemisinin level occurs after flowering.     

Some peculiarities of steroid metabolism in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants bearing the <Emphasis Type="Italic">CYP11A1</Emphasis> cDNA of cytochrome P450<Subscript>SCC</Subscript> from the bovine adrenal cortex

In the mitochondria of animal steroidogenic tissues, cytochrome P450_SCC encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone—the general precursor of all steroid hormones. In this work we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA encoding cytochrome P450_SCC from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone—the product of a reaction catalyzed by cytochrome P450_SCC. The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450_SCC cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.     

A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

Immunolocalization of cholesterol side chain cleavage enzyme (P450scc) in <Emphasis Type="Italic">Mytilus </Emphasis><Emphasis Type="Italic">galloprovincialis</Emphasis> and its induction by nutritional levels

We have examined the localization of P450scc in different tissues of the mollusc Mytilus galloprovincialis along a gonadal cycle by using a polyclonal antibody against rat cholesterol side-chain cleavage enzyme (cytochrome P450scc). Immunoreactivity specific for P450scc was found only in the cytoplasm of basophilic cells from digestive gland; gonad, gills and kidney appeared to be devoid of P450scc immunoreactivity. SDS-gel electrophoresis and western blot analysis of different subcellular fractions revealed that this protein is mainly located in microsomes, has a molecular weight of 51.4 kDa and is recognized by the antibody against rat P450scc. These results suggest that the digestive gland of M. galloprovincialis could be considered a steroidogenic tissue where the first step in biosynthesis of steroid hormones occurs. Moreover, seasonal slot blot analysis of post-mitochondrial fractions showed a P450scc induction by available nutrients and phytoplankton blooms.     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

Molecular cloning of <Emphasis Type="Italic">BmTUDOR-SN</Emphasis> and analysis of its role in the RNAi pathway in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> (Lepidoptera: Bombycidae)

Tudor-sn, a conserved nuclease, was first isolated from RNA-induced silencing complex (RISC) and was subsequently implicated in the RNA interference (RNAi) pathway in humans, flies and nematodes. However, in the silkworm, Bombyx mori L, the RNAi mechanism and the components of RISC were quite unclear. Here, we cloned the full-length cDNA of TUDOR-SN (BmTUDOR-SN) from the silkworm. Phylogenetic analysis revealed that BmTudor-sn had a high homology with Tudor-sn proteins in other insects. Fluorescent microscopic observation indicated that the subcellular localization of enhanced green fluorescent protein fused BmTudor-sn was mainly in the cytoplasm of silkworm BmN4 cells. Knockdown of BmTUDOR-SN did not, however, affect the RNAi efficiency in BmN4 cells.     

Analysis of the expression of <Emphasis Type="Italic">BohLOL1</Emphasis>, which encodes an LSD1-like zinc finger protein in <Emphasis Type="Italic">Bambusa oldhamii</Emphasis>

A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger-Lesions Simulating Disease resistance 1) domains was cloned from growing bamboo (Bambusa oldhamii) shoots. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-one-like 1), which have been reported to act antagonistically in controlling cell death via the maintenance of reactive oxygen species homeostasis. The BohLOL1 gene was differentially expressed in various bamboo shoot tissues and was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, which were cultured in vitro, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid), and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear DNA-binding protein. BohLOL1 might function through protein-DNA interactions and thus affect the expression of its target genes. The results of this study extend the role of plant LSD1 and LOL1 proteins from the regulation of cell death to cell growth. The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant.     

Wound response in passion fruit (<Emphasis Type="Italic">Passiflora</Emphasis> f. <Emphasis Type="Italic">edulis flavicarpa</Emphasis>) plants: gene characterization of a novel chloroplast-targeted allene oxide synthase up-regulated by mechanical injury and methyl jasmonate

The induction of a chloroplast-localized 13-lipoxygenase (13-LOX) in passion fruit leaves in response to methyl jasmonate (MeJa) was previously reported. Since allene oxide synthase (AOS) is a key cytochrome P450 enzyme in the oxylipin pathway leading to AOS-derived jasmonates, the results above led in turn to an investigation of AOS in our model plant. Spectrophotometric assays showed that 24 h exposure of MeJa caused a high increase in 13-hydroperoxy linolenic acid (13-HPOT) metabolizing activity in leaf tissue. Western analysis using polyclonal antibodies against tomato AOS strongly indicate that, at least a part of the 13-HPOT metabolizing capacity can be attributed to AOS activity. We cloned the cDNA from a novel AOS encoding gene from passion fruit, named PfAOS. The 1,512 bp open reading frame of the AOS–cDNA codes a putative protein of 504 amino acid residues containing a chloroplast target sequence. Database comparisons of the deduced amino acid sequence showed highest similarity with dicot AOSs. Immunocytochemistry analysis showed the compartmentalization of AOS in chloroplasts of MeJa treated leaves, corroborating the predicted subcellular localization. Northern analysis showed that AOS gene expression is induced in leaf tissue in response to mechanical wounding and exposure to MeJa. In addition, such treatments caused an increase in papain inhibitor(s) in leaf tissue. Taken together, these results indicate that PfAOS may play an important role in systemic wound response against chewing insect attack. Furthermore, it can be useful as a tool for understanding the regulation of jasmonates biosynthesis in passion fruit.