Untransformed Cultures Research Articles

Alterations in the silymarin metabolism in transgenic Silybum marianum cultured cells by the heterologous expression of the Arabidopsis thaliana V-myb myeloblastosis viral oncogene homolog transcription factor MYB12 and Cicer arietinum chalcone synthase

Silymarin (Sm), the main bioactive principle of Silybum marianum (L.) Gaernt. fruits, has strong antihepatotoxic properties and also exhibits antitumor and antiviral activity. This flavonolignan complex is synthesized from the flavonoid precursor taxifolin, which undergoes an oxidative coupling with the monolignol coniferyl alcohol. Attempts to produce Sm in plant cell suspensions have met with limited success, probably due to insufficient flux into the flavonoid branch pathway of Sm biosynthesis.In this study, it was attempted to increase Sm production in in vitro cultures of S. marianum by genetically engineering the flavonoid pathway. Using Agrobacterium tumefaciens strain C58C1, S. marianum cell suspension cultures were transformed either with the Arabidopsis thaliana transcription factor AtMYB12 (V-myb myeloblastosis viral oncogene homolog) (MYB) or with a chalcone synthase (CHS) gene from Cicer arietinum. A double transformant was also generated by introducing the CaCHS gene in AtMYB12-expressing cultures.The ectopic expression of AtMYB12 in cultures activated some of the genes involved in the flavonoid pathway, notably chalcone isomerase (CHI) and flavonoid 3′-hydroxylase (F3’H), whereas the secondary metabolites altered by the inserted gene were chlorogenic acid glycoside derivatives. Expression of CaCHS in the cultures led to the production of the flavanone naringenin (Ng), which accumulated as glycosides. The simultaneous expression of AtMYB12-CaCHS resulted in the production of lower amounts of chlorogenic derivatives but similar levels of Ng glycosides compared with the CaCHS lines. Both CaCHS lines and the double transformant could be maintained over prolonged subcultures without Ng production losses, confirming long-term and stable CaCHS transgene expression.In no case did the inserted genes have a discernable effect on Sm production compared with the untransformed cultures, neither under control culture conditions or in cultures elicited with MeJA.

Raman spectroscopic detection of physiology changes in plasmid-bearing Escherichia coli with and without antibiotic treatment

Bacterial resistances against antibiotics are increasingly problematic for medical treatment of pathogenic bacteria, e.g., in hospitals. Resistances are, among other genes, often encoded on plasmids which can be transmitted between bacteria not only within one species, but also between different species, genera, and families. The plasmid pDrive is transformed into bacteria of the model strain Escherichia coli DH5α. Within this investigation, we applied micro-Raman spectroscopy with two different excitation wavelengths in combination with support vector machine (SVM) and linear discriminant analysis (LDA) to differentiate between bacterial cultures according to their cultural plasmid content. Recognition rates of about 92% and 90% are achieved by Raman excitation at 532 and 244 nm, respectively. The SVM loadings reveal that the pDrive transformed bacterial cultures exhibit a higher DNA content compared to the untransformed cultures. To elucidate the influence of the antibiotic, ampicillin-treated cultures are also comprised within this study and are classified with rates of about 97% and 100% for 532 and 244 nm Raman excitation, respectively. The Raman spectra recorded with 532 nm excitation wavelength show differences of the secondary protein structure and enhanced stress-related respiration rates for the ampicillin-treated cultures. Independent cultural replicates of either ampicillin-challenged or non-challenged cultures are successfully identified with identification rates of over 90%.

Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Linn.

Background:Glycyrrhizin, obtained from Abrus precatorius (Indian liquorice), is a phytoconstituent of importance for pharmaceutical and food industries.Materials and Methods:High producing and fast growing cell lines of A. precatorius were developed by transformation with Agrobacterium tumefaciens for glycyrrhizin production. Its maximum transformation efficiency of 85% was obtained by infecting leaves with A. tumefaciens MTCC-431 supplemented with 50 μM acetosyringone. Thorough culture growth kinetics with sugar consumption profiles was established.Results:A twofold increase in glycyrrhizin productivity was obtained in transformed A. precatorius cell suspension cultures over the untransformed cultures. The fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer were tested at different concentrations to enhance glycyrrhizin production in transformed cell suspension cultures of A. precatorius. Maximum enhancement of 4.9- and 3.8-fold in glycyrrhizin contents, were obtained with A. niger (7.5% v/v) and R. stolonifer (5.0% v/v), respectively, on the 5th day after elicitor treatment.Conclusion:This study indicates the prospective of the amalgamation of elicitation methodology with transformed cell cultures for the large-scale production of glycyrrhizin.

Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea

This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 × 10 8 cfu ml −1, 1 m m acetosyringone, 25-d-old mycelia at 0.2 g ml −1, and co-culture period of 6 d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88 % of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.

Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures.

Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells. Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks. Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days). Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots. However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture. These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable. A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA. The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium. This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.

Forskolin production in untransformed root culture of Coleus forskohlii

Untransformed cultures of Coleus forskohlii Briq. were initiated from in vitro micropropagated shoots derived from shoot tip culture. Effects of IAA, IBA and two IAA derivatives, 4-CI-IAA and 5,6-Cl 2-IAA were compared for growth and forskolin production in cultured roots grown on different basal media under different culture regimes. The roots grew well in the presence of all dosages of 4-CI-IAA and 5,6-Cl 2-IAA with 25% MS plus 1% sucrose and produced more forskolin as compared with IAA or IBA. The optimum concentrations needed for forskolin production were 1 μg 1 −1 for 5,6-Cl 2-IAA and 50,μg1 −1 for 4-CI-IAA. The highest amount of forskolin (0.09%) was raised at 50,μg1−1 4-CI-IAA after 60 days of culture. This enhanced level of forskolin was almost equal to the content in the wild plant. However, under similar conditions and with 500,μg 1 −1 IAA or IBA, the amount of forskolin produced was much smaller. IAA derivative-induced production of forskolin indicates the possibility for scaling up of the production of this compound in untransformed root cultur

Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78

A gene (ips) encoding the isopenicillin N synthase of Penicillium chrysogenum AS-P-78 was cloned in a 3.9 kb SalI fragment using a probe corresponding to the amino-terminal end of the enzyme. The SalI fragment was trimmed down to a 1.3 kb NcoI-BglII fragment that contained an open reading frame of 996 nucleotides encoding a polypeptide of 331 amino acids with an Mr of 38012 dalton. The predicted polypeptide encoded by the ips gene of strain AS-P-78 contains a tyrosine at position 195, whereas the gene of the high penicillin producing strain 23X-80-269-37-2 shows an isoleucine at the same position. The ips gene is expressed in Escherichia coli minicells using the lambda phage PL promoter. Some similar sequence motifs were found in the upstream region of the ips gene of P. chrysogenum when compared with the upstream sequences of the ips genes of Cephalosporium acremonium and Aspergillus nidulans. Primer extension studies indicated that the start of the mRNA coincides with a T in position -11 which is located in a conserved pyrimidine-rich sequence, near two CAAG boxes. Clones of P. chrysogenum Wis 54-1255 transformed with the ips gene showed a five-fold higher isopenicillin N synthase activity than the untransformed cultures.

Quinoline Alkaloid Production by Transformed Cultures ofCinchona ledgeriana

A culture of CINCHONA LEDGERIANA, transformed with AGROBACTERLUM TUMEFACIENS A6 capable of growing and producing quinoline alkaloids in medium free of exogenous phytohormones has been obtained. Unlike the untransformed culture of this species, addition of ZR in combination with either IAA or IBA to medium did not affect alkaloid production. Growing the transformed culture in the dark, however, produced a marked enhancement of alkaloid accumulation, up to 50 times that of cultures grown in the light. This dark-dependent accumulation was not confined to any particular time in the growth cycle, although the extent of the stimulatory effect increased the longer cultures were kept in darkness. Blue light was detrimental to alkaloid accumulation but in red or green light the level of accumulation was equivalent to that in the dark. Alternating cultures for several 28-day periods between light and dark conditions resulted in alternate periods of low and higfralkaloid productivity, indicating this was not an adaption effect. These findings correlate to previously reported differences in key enzymes from the biosynthetic pathway. Increasing the phosphate or nitrate concentration above that of Gamborg B5, increasing sucrose above 2% (w/v), replacing sucrose with glucose or adding tryptophan or casamino acids to the medium resuited in lower alkaloid yields by dark-grown cultures of the transformed line.

Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

Effect of cell transformation and iododeoxyuridine on the charge heterogeneity of large external transformation-sensitive proteins

Comparative two dimensional mapping of surface-iodinated WIRL epithelioid untransformed cells and their transformed counterparts, revealed in the latter a decreased labelling in the 220K region, particularly in cathodic components more evident in the untransformed cultures. A similar assay with phenotypically normal and morphologically transformed fibroblasts also revealed in the latter a preferentially decrease in cathodic 220K dalton species. Growth of the normal epithelioid cultures in the presence of iododeoxyuridine correlated with both an increased cell adherence and with an enhanced cathodic migration of the 220K surface iodinated species cross-reactive with antifibronectin serum. Our results suggest that a restricted expression of the transformed phenotype and an increased adherence mediated by iododeoxyuridine correlates with an increased cathodic migration of fibronectin-like transformation-sensitive surface components in rat epithelioid and fibroblastic cells.