Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Simple production of hydrophobin-fused domain III of dengue envelope protein and induction of neutralizing antibodies against the homotypic serotype of dengue virus.

Hydrophobin-fused domain III of dengue envelope proteins serotypes 1 and 2 were expressed in Rachiplusia nu larvae and purified by aqueous two-phase system. This biotechnological approach of hydrophobin-fused proteins, which allowed obtaining 97.7 µg/larva of fusion protein DomIII serotype 1 and 61.4 µg/larva of fusion protein DomIII serotype 2, represents an integrated strategy for simple production of recombinant antigens. Purified fusion proteins induced serotype-specific neutralizing antibodies without cross-reaction against other serotypes and arboviruses after mouse immunization. hydrophobin-fused domain III of dengue envelope protein could be a promising strategy for easy and low-cost production of components of a tetravalent sub-unit vaccine against dengue.

In situ removal of consensus dengue virus envelope protein domain III fused to hydrophobin in Pichia pastoris cultures.

This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.

Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 µM, respectively, and 111% with T4C added at 100 µM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 µM A2C and 100 µM T4C). Whereas A2C (50 µM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

Anisodamine production from natural sources: seedlings and hairy root cultures of Argentinean and Colombian Brugmansia candida plants.

The tropane alkaloid anisodamine ( 2) is obtained by 6 beta-hydroxylation of hyoscyamine ( 1). The application of this alkaloid in medicine is gaining attention due to the wide range of therapeutic applications described in addition to its anticholinergic activity. In this work, the production of anisodamine ( 2) by IN VITRO cultures of BRUGMANSIA CANDIDA (Argentinean and Colombian samples) was studied. This alkaloid was estimated in different organs of IN VITRO-germinated seedlings as well as in hairy roots obtained from seedlings from both sources. Colombian roots exhibited the highest content of tropane alkaloids, with anisodamine ( 2) being the main alkaloid measured. In the leaves, the main alkaloid was scopolamine ( 3) and no significant differences were observed between Argentinean and Colombian leaves. The tropane alkaloid content in Argentinean hairy roots was significantly higher than in Colombian ones. Also, in the Argentinean samples the main alkaloid detected was anisodamine ( 2). Argentinean and Colombian B. CANDIDA seedlings and hairy roots appear to be a promising system for the production of anisodamine ( 2).

Effect of shear stress on anthraquinones production by Rubia tinctorum suspension cultures.

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.