Camilo Canel

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Abstract. Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L−1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Differential accumulation of isoprenoids in glanded and glandless Artemisia annua L

Abstract The relative abundance of steam-distillable isoprenoids and other volatile compounds in leaves of glanded and glandless Artemisia annua L. was investigated. Steam distillation of leaves bearing glandular trichomes yielded 0.24% essential oil on a fresh weight basis. Monoterpenes were predominant in this essential oil, of which α -pinene (26.7%), pinocarvone (15.8%), and artemisia ketone (11.0%) were the major constituents. The essential oil distilled from glandless leaves amounted to 0.06% of fresh weight, and consisted mostly of sesquiterpenes, of which germacrene- D (49.8%) and β -caryophyllene (25.1%) were the major components. Only one monoterpene, β -ocimene, present in trace amounts, was detected in the oil distilled from the glandless tissue. The sesquiterpene artemisinin, found only in the glanded biotype, is heat-labile and cannot be extracted by steam distillation, during which it decomposes into non-distillable products.

The American mayapple revisited : Podophyllum peltatum-still a potential cash crop?

Podophyllum peltatum, was reexamined for its potential use in the commercial production of podophyllotoxin, a lignan used in the semisynthesis of important anticancer drugs. A survey of the natural population of the American mayapple, Podophyllum peltatum, was conducted in order to identify high-yielding genotypes. Plants were collected from the eastern and central United States. The lignan content of leaf blades and rhizome material of the collected specimens was characterized by aqueous extraction followed by HPLC analysis. Podophyllotoxin and α-peltatin appeared most prominently among the lignans obtained. Leaf blades were generally richer in podophyllotoxin than rhizomes. Several high-yielding accessions were identified, the blades of which contained 4.0–5.6% podophyllotoxin. A negative correlation was observed between podophyllotoxin and peltatin content in the blades. The combination of high biosynthetic capacity and preferential accumulation of podophyllotoxin in leaves of mayapple makesthis plant an excellent candidate for agricultural production of podophyllotoxin.RésuméO potencial de utilização do Podophyllum peltatum foi rexaminado para a produção comercial de podofilotoxina, urn composto usado na semisìntese de importantes drogas anticancerìgenas. Urn levantamento da população norte-americana da mayapple, Podophyllum peltatum, foi conduzido com a finalidade de identificar genótipos de alta produtividade. As coletas foram feitas no centroeste dos Estados Unidos e o conteúdo de lignanas do limbo foliar e dos rizomas foi caracterizado por extração aquosa seguida da análise por HPLC. Podofilotoxina e a peltatin apareceram mais proemintes do que as demais lignanas. Geralmente, o limbo foliar apresentou um conteúdo de podofilotoxina mais rico do que os rizomas. Muitas accessões com alta produtividade foram identificadas e cujo o conteúdo de podophyllotoxina no limbo foliar variou entre 4.0–5.6%. Observou-se ainda uma correlação negativaentre apodofilotoxina e o conteúdo de peltatin no limbo foliar. Nas folhas, a combinaú ão entre a capacidade biosintética e o acúmulo preferencial da podofilotoxina, faz com que essa espécie seja considerada uma excelente candidata para a produção agrícola desta lignana.

Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the β-glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.

The lignans of Podophyllum

Abstract Lignans are a widely distributed class of dimeric phenylpropanoid derivatives, many of which have strong antimicrobial, antiviral, or antifeedant activity and thus play important roles in plant defense. Of more restricted taxonomic distribution, the aryltetralin lignans have been found in highest abundance in plants of the genus Podophyllum (Berberidaceae). Foremost among these lignans, podophyllotoxin is a particularly cytotoxic inhibitor of microtubule assembly and astrong antiviral agent. Semisynthetic epimeric derivatives of podophyllotoxin having inhibitory activity against DNA-topoisomerase II have been developed as effective antineoplastic drugs. Current work on Podophyllum lignans is focused on two fronts: 1) Structure optimization to generate derivatives with superior pharmacological profiles and broader therapeutic use, and 2) Development of alternative sources of podophyllotoxin. Numerous variations of the basicaryltetralin structure have been created. Some of the new compounds have shown promising activity profiles, but practically little has been achieved besides improvement in solubility. Interest in new derivatives remains strong, which, along with the formulation of existing drugs for new indications, is increasing the demand for podophyllotoxin. While intense collection has severely reduced the natural stocks of Indian Podophyllum , the primary source of podophyllotoxin, a North American species has emerged as a rich and renewable source of this compound.

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested.